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Hufert)] TJ ET 0.271 0.267 0.267 rg BT 337.285 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 342.706 638.299 Td /F1 9.8 Tf [(Manfred Weidmann)] TJ ET 0.271 0.267 0.267 rg BT 26.250 626.394 Td /F1 9.8 Tf [(Abd El Wahed A, Patel P, Heidenreich D, Hufert FT, Weidmann M. Reverse Transcription Recombinase Polymerase )] TJ ET BT 26.250 614.490 Td /F1 9.8 Tf [(Amplification Assay for the Detection of Middle East Respiratory Syndrome Coronavirus. PLOS Currents Outbreaks. 2013 Dec )] TJ ET BT 26.250 602.585 Td /F1 9.8 Tf [(12 . Edition 1. doi: 10.1371/currents.outbreaks.62df1c7c75ffc96cd59034531e2e8364.)] TJ ET q 15.000 28.739 577.500 571.465 re W n 0.271 0.267 0.267 rg BT 26.250 573.482 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 553.528 Td /F1 9.8 Tf [(The emergence of Middle East Respiratory Syndrome Coronavirus \(MERS-CoV\) in the eastern Mediterranean and imported )] TJ ET BT 26.250 541.623 Td /F1 9.8 Tf [(cases to Europe has alerted public health authorities. Currently, detection of MERS-CoV in patient samples is done by real-time )] TJ ET BT 26.250 529.719 Td /F1 9.8 Tf [(RT-PCR. Samples collected from suspected cases are sent to highly-equipped centralized laboratories for screening. A rapid )] TJ ET BT 26.250 517.814 Td /F1 9.8 Tf [(point-of-care test is needed to allow more widespread mobile detection of the virus directly from patient material. In this study, )] TJ ET BT 26.250 505.909 Td /F1 9.8 Tf [(we describe the development of a reverse transcription isothermal Recombinase Polymerase Amplification \(RT-RPA\) assay for )] TJ ET BT 26.250 494.004 Td /F1 9.8 Tf [(the identification of MERS-CoV. A partial nucleocapsid gene RNA molecular standard of MERS-coronavirus was used to )] TJ ET BT 26.250 482.100 Td /F1 9.8 Tf [(determine the assay sensitivity. The isothermal \(42C\) MERS-CoV RT-RPA was as sensitive as real-time RT-PCR \(10 RNA )] TJ ET BT 26.250 470.195 Td /F1 9.8 Tf [(molecules\), rapid \(3-7 minutes\) and mobile \(using tubescanner weighing 1kg\). The MERS-CoV RT-RPA showed cross-)] TJ ET BT 26.250 458.290 Td /F1 9.8 Tf [(detection neither of any of the RNAs of several coronaviruses and respiratory viruses affecting humans nor of the human )] TJ ET BT 26.250 446.385 Td /F1 9.8 Tf [(genome. The developed isothermal real-time RT-RPA is ideal for rapid mobile molecular MERS-CoV monitoring in acute )] TJ ET BT 26.250 434.481 Td /F1 9.8 Tf [(patients and may also facilitate the search for the animal reservoir of MERS-CoV.)] TJ ET BT 26.250 397.878 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 377.924 Td /F1 9.8 Tf [(The study was funded by the Federal Ministry of Education and Research \(BMBF\) \(project name: RESCheck, ID: )] TJ ET BT 26.250 366.019 Td /F1 9.8 Tf [(FKN:16SV5436\).)] TJ ET BT 26.250 336.917 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 316.962 Td /F1 9.8 Tf [(The analysis of samples in Europe for a case of severe pulmonary syndrome and renal failure in Saudi Arabia led to the )] TJ ET BT 26.250 305.058 Td /F1 9.8 Tf [(isolation and characterization of a novel human betacoronavirus termed MERS-CoV )] TJ ET 0.267 0.267 0.267 rg BT 390.939 306.565 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 395.758 305.058 Td /F1 9.8 Tf [( . As of November 26)] TJ ET BT 487.339 308.946 Td /F1 8.7 Tf [(th )] TJ ET BT 496.977 305.058 Td /F1 9.8 Tf [(68 deaths of 160 )] TJ ET BT 26.250 293.153 Td /F1 9.8 Tf [(laboratory confirmed cases have been recorded by the WHO mainly from countries in the Middle East )] TJ ET 0.267 0.267 0.267 rg BT 466.794 294.660 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 471.613 293.153 Td /F1 9.8 Tf [(. The detection of MERS-)] TJ ET BT 26.250 281.248 Td /F1 9.8 Tf [(CoV infected cases depends primary on sensitive real-time RT-PCR )] TJ ET 0.267 0.267 0.267 rg BT 322.630 282.755 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 327.449 281.248 Td /F1 9.8 Tf [( . In developing countries, facilities for performing real-)] TJ ET BT 26.250 269.343 Td /F1 9.8 Tf [(time RT-PCR are limited to centralized laboratories. The time to result can therefore be long and in many cases last up to )] TJ ET BT 26.250 257.439 Td /F1 9.8 Tf [(several days. A portable, rapid, cheap, and sensitive test is needed to identify infected acute cases and would help to improve )] TJ ET BT 26.250 245.534 Td /F1 9.8 Tf [(the onsite search for animal reservoirs.)] TJ ET BT 26.250 226.129 Td /F1 9.8 Tf [(In recent years a variety of isothermal amplification methods have been developed which offer the possibility of developing rapid )] TJ ET BT 26.250 214.224 Td /F1 9.8 Tf [(nucleic acid detection assays for simple point-of-care systems. The recombinase polymerase amplification \(RPA\) assay is an )] TJ ET BT 26.250 202.320 Td /F1 9.8 Tf [(isothermal amplification technology )] TJ ET 0.267 0.267 0.267 rg BT 180.680 203.827 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 185.499 202.320 Td /F1 9.8 Tf [(. RPA assays run at 42C for 15 minutes )] TJ ET 0.267 0.267 0.267 rg BT 363.349 203.827 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 368.167 206.208 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 370.577 203.827 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 375.395 206.208 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 377.805 203.827 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 382.623 206.208 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 385.033 203.827 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 389.851 202.320 Td /F1 9.8 Tf [(. The amplification in the RPA reaction )] TJ ET BT 26.250 190.415 Td /F1 9.8 Tf [(depends on binding of the recombinase to oligonucleotide primers. The complex then scans the template DNA for the )] TJ ET BT 26.250 178.510 Td /F1 9.8 Tf [(corresponding sequence and initiates 5-strand invasion of the oligonucleotide at the site of homology. The strand invasion is )] TJ ET BT 26.250 166.605 Td /F1 9.8 Tf [(stabilized by the single strand binding protein. The extension of the primer ensues via a strand displacing DNA polymerase )] TJ ET 0.267 0.267 0.267 rg BT 557.313 168.113 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 562.132 170.494 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 564.541 168.113 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 569.360 170.494 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 571.769 168.113 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 26.250 154.701 Td /F1 9.8 Tf [(. Real-time detection is possible )] TJ ET BT 166.591 154.701 Td /F5 9.8 Tf [(via)] TJ ET BT 179.052 154.701 Td /F1 9.8 Tf [( an exo-probe system )] TJ ET 0.267 0.267 0.267 rg BT 275.509 156.208 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 280.327 158.589 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 282.737 156.208 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 287.555 158.589 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 289.965 156.208 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 294.783 158.589 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 297.193 156.208 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 302.011 154.701 Td /F1 9.8 Tf [(. In this study, MERS-CoV RT-RPA assay was developed to )] TJ ET BT 26.250 142.796 Td /F1 9.8 Tf [(amplify and detect in real-time a fragment of the nucleocapsid gene \(NC\) of MERS-CoV.)] TJ ET BT 26.250 106.193 Td /F4 12.0 Tf [(Materials and Methods)] TJ ET Q q 15.000 659.406 577.500 78.594 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Reverse Transcription Recombinase Polymerase Amplification )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(Assay for the Detection of Middle East Respiratory Syndrome )] TJ ET BT 15.000 668.146 Td /F2 21.0 Tf [(Coronavirus)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 650.140 Td /F3 9.8 Tf [(December 12, 2013)] TJ ET BT 96.500 650.140 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 101.375 650.140 Td /F3 9.8 Tf [(Research Article)] TJ ET BT 26.250 638.299 Td /F1 9.8 Tf [(Ahmed Abd El Wahed)] TJ ET 0.271 0.267 0.267 rg BT 122.171 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 127.592 638.299 Td /F1 9.8 Tf [(Pranav Patel)] TJ ET 0.271 0.267 0.267 rg BT 183.410 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 188.831 638.299 Td /F1 9.8 Tf [(Doris Heidenreich)] TJ ET 0.271 0.267 0.267 rg BT 266.305 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 271.726 638.299 Td /F1 9.8 Tf [(Frank T. Hufert)] TJ ET 0.271 0.267 0.267 rg BT 337.285 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 342.706 638.299 Td /F1 9.8 Tf [(Manfred Weidmann)] TJ ET 0.271 0.267 0.267 rg BT 26.250 626.394 Td /F1 9.8 Tf [(Abd El Wahed A, Patel P, Heidenreich D, Hufert FT, Weidmann M. Reverse Transcription Recombinase Polymerase )] TJ ET BT 26.250 614.490 Td /F1 9.8 Tf [(Amplification Assay for the Detection of Middle East Respiratory Syndrome Coronavirus. PLOS Currents Outbreaks. 2013 Dec )] TJ ET BT 26.250 602.585 Td /F1 9.8 Tf [(12 . Edition 1. doi: 10.1371/currents.outbreaks.62df1c7c75ffc96cd59034531e2e8364.)] TJ ET q 15.000 28.739 577.500 571.465 re W n 0.271 0.267 0.267 rg BT 26.250 573.482 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 553.528 Td /F1 9.8 Tf [(The emergence of Middle East Respiratory Syndrome Coronavirus \(MERS-CoV\) in the eastern Mediterranean and imported )] TJ ET BT 26.250 541.623 Td /F1 9.8 Tf [(cases to Europe has alerted public health authorities. Currently, detection of MERS-CoV in patient samples is done by real-time )] TJ ET BT 26.250 529.719 Td /F1 9.8 Tf [(RT-PCR. Samples collected from suspected cases are sent to highly-equipped centralized laboratories for screening. A rapid )] TJ ET BT 26.250 517.814 Td /F1 9.8 Tf [(point-of-care test is needed to allow more widespread mobile detection of the virus directly from patient material. In this study, )] TJ ET BT 26.250 505.909 Td /F1 9.8 Tf [(we describe the development of a reverse transcription isothermal Recombinase Polymerase Amplification \(RT-RPA\) assay for )] TJ ET BT 26.250 494.004 Td /F1 9.8 Tf [(the identification of MERS-CoV. A partial nucleocapsid gene RNA molecular standard of MERS-coronavirus was used to )] TJ ET BT 26.250 482.100 Td /F1 9.8 Tf [(determine the assay sensitivity. The isothermal \(42C\) MERS-CoV RT-RPA was as sensitive as real-time RT-PCR \(10 RNA )] TJ ET BT 26.250 470.195 Td /F1 9.8 Tf [(molecules\), rapid \(3-7 minutes\) and mobile \(using tubescanner weighing 1kg\). The MERS-CoV RT-RPA showed cross-)] TJ ET BT 26.250 458.290 Td /F1 9.8 Tf [(detection neither of any of the RNAs of several coronaviruses and respiratory viruses affecting humans nor of the human )] TJ ET BT 26.250 446.385 Td /F1 9.8 Tf [(genome. The developed isothermal real-time RT-RPA is ideal for rapid mobile molecular MERS-CoV monitoring in acute )] TJ ET BT 26.250 434.481 Td /F1 9.8 Tf [(patients and may also facilitate the search for the animal reservoir of MERS-CoV.)] TJ ET BT 26.250 397.878 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 377.924 Td /F1 9.8 Tf [(The study was funded by the Federal Ministry of Education and Research \(BMBF\) \(project name: RESCheck, ID: )] TJ ET BT 26.250 366.019 Td /F1 9.8 Tf [(FKN:16SV5436\).)] TJ ET BT 26.250 336.917 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 316.962 Td /F1 9.8 Tf [(The analysis of samples in Europe for a case of severe pulmonary syndrome and renal failure in Saudi Arabia led to the )] TJ ET BT 26.250 305.058 Td /F1 9.8 Tf [(isolation and characterization of a novel human betacoronavirus termed MERS-CoV )] TJ ET 0.267 0.267 0.267 rg BT 390.939 306.565 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 395.758 305.058 Td /F1 9.8 Tf [( . As of November 26)] TJ ET BT 487.339 308.946 Td /F1 8.7 Tf [(th )] TJ ET BT 496.977 305.058 Td /F1 9.8 Tf [(68 deaths of 160 )] TJ ET BT 26.250 293.153 Td /F1 9.8 Tf [(laboratory confirmed cases have been recorded by the WHO mainly from countries in the Middle East )] TJ ET 0.267 0.267 0.267 rg BT 466.794 294.660 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 471.613 293.153 Td /F1 9.8 Tf [(. The detection of MERS-)] TJ ET BT 26.250 281.248 Td /F1 9.8 Tf [(CoV infected cases depends primary on sensitive real-time RT-PCR )] TJ ET 0.267 0.267 0.267 rg BT 322.630 282.755 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 327.449 281.248 Td /F1 9.8 Tf [( . In developing countries, facilities for performing real-)] TJ ET BT 26.250 269.343 Td /F1 9.8 Tf [(time RT-PCR are limited to centralized laboratories. The time to result can therefore be long and in many cases last up to )] TJ ET BT 26.250 257.439 Td /F1 9.8 Tf [(several days. A portable, rapid, cheap, and sensitive test is needed to identify infected acute cases and would help to improve )] TJ ET BT 26.250 245.534 Td /F1 9.8 Tf [(the onsite search for animal reservoirs.)] TJ ET BT 26.250 226.129 Td /F1 9.8 Tf [(In recent years a variety of isothermal amplification methods have been developed which offer the possibility of developing rapid )] TJ ET BT 26.250 214.224 Td /F1 9.8 Tf [(nucleic acid detection assays for simple point-of-care systems. The recombinase polymerase amplification \(RPA\) assay is an )] TJ ET BT 26.250 202.320 Td /F1 9.8 Tf [(isothermal amplification technology )] TJ ET 0.267 0.267 0.267 rg BT 180.680 203.827 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 185.499 202.320 Td /F1 9.8 Tf [(. RPA assays run at 42C for 15 minutes )] TJ ET 0.267 0.267 0.267 rg BT 363.349 203.827 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 368.167 206.208 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 370.577 203.827 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 375.395 206.208 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 377.805 203.827 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 382.623 206.208 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 385.033 203.827 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 389.851 202.320 Td /F1 9.8 Tf [(. The amplification in the RPA reaction )] TJ ET BT 26.250 190.415 Td /F1 9.8 Tf [(depends on binding of the recombinase to oligonucleotide primers. The complex then scans the template DNA for the )] TJ ET BT 26.250 178.510 Td /F1 9.8 Tf [(corresponding sequence and initiates 5-strand invasion of the oligonucleotide at the site of homology. The strand invasion is )] TJ ET BT 26.250 166.605 Td /F1 9.8 Tf [(stabilized by the single strand binding protein. The extension of the primer ensues via a strand displacing DNA polymerase )] TJ ET 0.267 0.267 0.267 rg BT 557.313 168.113 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 562.132 170.494 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 564.541 168.113 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 569.360 170.494 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 571.769 168.113 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 26.250 154.701 Td /F1 9.8 Tf [(. Real-time detection is possible )] TJ ET BT 166.591 154.701 Td /F5 9.8 Tf [(via)] TJ ET BT 179.052 154.701 Td /F1 9.8 Tf [( an exo-probe system )] TJ ET 0.267 0.267 0.267 rg BT 275.509 156.208 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 280.327 158.589 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 282.737 156.208 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 287.555 158.589 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 289.965 156.208 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 294.783 158.589 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 297.193 156.208 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 302.011 154.701 Td /F1 9.8 Tf [(. In this study, MERS-CoV RT-RPA assay was developed to )] TJ ET BT 26.250 142.796 Td /F1 9.8 Tf [(amplify and detect in real-time a fragment of the nucleocapsid gene \(NC\) of MERS-CoV.)] TJ ET BT 26.250 106.193 Td /F4 12.0 Tf [(Materials and Methods)] TJ ET Q q 15.000 659.406 577.500 78.594 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Reverse Transcription Recombinase Polymerase Amplification )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(Assay for the Detection of Middle East Respiratory Syndrome )] TJ ET BT 15.000 668.146 Td /F2 21.0 Tf [(Coronavirus)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 650.140 Td /F3 9.8 Tf [(December 12, 2013)] TJ ET BT 96.500 650.140 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 101.375 650.140 Td /F3 9.8 Tf [(Research Article)] TJ ET BT 26.250 638.299 Td /F1 9.8 Tf [(Ahmed Abd El Wahed)] TJ ET 0.271 0.267 0.267 rg BT 122.171 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 127.592 638.299 Td /F1 9.8 Tf [(Pranav Patel)] TJ ET 0.271 0.267 0.267 rg BT 183.410 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 188.831 638.299 Td /F1 9.8 Tf [(Doris Heidenreich)] TJ ET 0.271 0.267 0.267 rg BT 266.305 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 271.726 638.299 Td /F1 9.8 Tf [(Frank T. Hufert)] TJ ET 0.271 0.267 0.267 rg BT 337.285 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 342.706 638.299 Td /F1 9.8 Tf [(Manfred Weidmann)] TJ ET 0.271 0.267 0.267 rg BT 26.250 626.394 Td /F1 9.8 Tf [(Abd El Wahed A, Patel P, Heidenreich D, Hufert FT, Weidmann M. Reverse Transcription Recombinase Polymerase )] TJ ET BT 26.250 614.490 Td /F1 9.8 Tf [(Amplification Assay for the Detection of Middle East Respiratory Syndrome Coronavirus. PLOS Currents Outbreaks. 2013 Dec )] TJ ET BT 26.250 602.585 Td /F1 9.8 Tf [(12 . Edition 1. doi: 10.1371/currents.outbreaks.62df1c7c75ffc96cd59034531e2e8364.)] TJ ET q 15.000 28.739 577.500 571.465 re W n 0.271 0.267 0.267 rg BT 26.250 573.482 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 553.528 Td /F1 9.8 Tf [(The emergence of Middle East Respiratory Syndrome Coronavirus \(MERS-CoV\) in the eastern Mediterranean and imported )] TJ ET BT 26.250 541.623 Td /F1 9.8 Tf [(cases to Europe has alerted public health authorities. Currently, detection of MERS-CoV in patient samples is done by real-time )] TJ ET BT 26.250 529.719 Td /F1 9.8 Tf [(RT-PCR. Samples collected from suspected cases are sent to highly-equipped centralized laboratories for screening. A rapid )] TJ ET BT 26.250 517.814 Td /F1 9.8 Tf [(point-of-care test is needed to allow more widespread mobile detection of the virus directly from patient material. In this study, )] TJ ET BT 26.250 505.909 Td /F1 9.8 Tf [(we describe the development of a reverse transcription isothermal Recombinase Polymerase Amplification \(RT-RPA\) assay for )] TJ ET BT 26.250 494.004 Td /F1 9.8 Tf [(the identification of MERS-CoV. A partial nucleocapsid gene RNA molecular standard of MERS-coronavirus was used to )] TJ ET BT 26.250 482.100 Td /F1 9.8 Tf [(determine the assay sensitivity. The isothermal \(42C\) MERS-CoV RT-RPA was as sensitive as real-time RT-PCR \(10 RNA )] TJ ET BT 26.250 470.195 Td /F1 9.8 Tf [(molecules\), rapid \(3-7 minutes\) and mobile \(using tubescanner weighing 1kg\). The MERS-CoV RT-RPA showed cross-)] TJ ET BT 26.250 458.290 Td /F1 9.8 Tf [(detection neither of any of the RNAs of several coronaviruses and respiratory viruses affecting humans nor of the human )] TJ ET BT 26.250 446.385 Td /F1 9.8 Tf [(genome. The developed isothermal real-time RT-RPA is ideal for rapid mobile molecular MERS-CoV monitoring in acute )] TJ ET BT 26.250 434.481 Td /F1 9.8 Tf [(patients and may also facilitate the search for the animal reservoir of MERS-CoV.)] TJ ET BT 26.250 397.878 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 377.924 Td /F1 9.8 Tf [(The study was funded by the Federal Ministry of Education and Research \(BMBF\) \(project name: RESCheck, ID: )] TJ ET BT 26.250 366.019 Td /F1 9.8 Tf [(FKN:16SV5436\).)] TJ ET BT 26.250 336.917 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 316.962 Td /F1 9.8 Tf [(The analysis of samples in Europe for a case of severe pulmonary syndrome and renal failure in Saudi Arabia led to the )] TJ ET BT 26.250 305.058 Td /F1 9.8 Tf [(isolation and characterization of a novel human betacoronavirus termed MERS-CoV )] TJ ET 0.267 0.267 0.267 rg BT 390.939 306.565 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 395.758 305.058 Td /F1 9.8 Tf [( . As of November 26)] TJ ET BT 487.339 308.946 Td /F1 8.7 Tf [(th )] TJ ET BT 496.977 305.058 Td /F1 9.8 Tf [(68 deaths of 160 )] TJ ET BT 26.250 293.153 Td /F1 9.8 Tf [(laboratory confirmed cases have been recorded by the WHO mainly from countries in the Middle East )] TJ ET 0.267 0.267 0.267 rg BT 466.794 294.660 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 471.613 293.153 Td /F1 9.8 Tf [(. The detection of MERS-)] TJ ET BT 26.250 281.248 Td /F1 9.8 Tf [(CoV infected cases depends primary on sensitive real-time RT-PCR )] TJ ET 0.267 0.267 0.267 rg BT 322.630 282.755 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 327.449 281.248 Td /F1 9.8 Tf [( . In developing countries, facilities for performing real-)] TJ ET BT 26.250 269.343 Td /F1 9.8 Tf [(time RT-PCR are limited to centralized laboratories. The time to result can therefore be long and in many cases last up to )] TJ ET BT 26.250 257.439 Td /F1 9.8 Tf [(several days. A portable, rapid, cheap, and sensitive test is needed to identify infected acute cases and would help to improve )] TJ ET BT 26.250 245.534 Td /F1 9.8 Tf [(the onsite search for animal reservoirs.)] TJ ET BT 26.250 226.129 Td /F1 9.8 Tf [(In recent years a variety of isothermal amplification methods have been developed which offer the possibility of developing rapid )] TJ ET BT 26.250 214.224 Td /F1 9.8 Tf [(nucleic acid detection assays for simple point-of-care systems. The recombinase polymerase amplification \(RPA\) assay is an )] TJ ET BT 26.250 202.320 Td /F1 9.8 Tf [(isothermal amplification technology )] TJ ET 0.267 0.267 0.267 rg BT 180.680 203.827 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 185.499 202.320 Td /F1 9.8 Tf [(. RPA assays run at 42C for 15 minutes )] TJ ET 0.267 0.267 0.267 rg BT 363.349 203.827 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 368.167 206.208 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 370.577 203.827 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 375.395 206.208 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 377.805 203.827 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 382.623 206.208 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 385.033 203.827 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 389.851 202.320 Td /F1 9.8 Tf [(. The amplification in the RPA reaction )] TJ ET BT 26.250 190.415 Td /F1 9.8 Tf [(depends on binding of the recombinase to oligonucleotide primers. The complex then scans the template DNA for the )] TJ ET BT 26.250 178.510 Td /F1 9.8 Tf [(corresponding sequence and initiates 5-strand invasion of the oligonucleotide at the site of homology. The strand invasion is )] TJ ET BT 26.250 166.605 Td /F1 9.8 Tf [(stabilized by the single strand binding protein. The extension of the primer ensues via a strand displacing DNA polymerase )] TJ ET 0.267 0.267 0.267 rg BT 557.313 168.113 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 562.132 170.494 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 564.541 168.113 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 569.360 170.494 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 571.769 168.113 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 26.250 154.701 Td /F1 9.8 Tf [(. Real-time detection is possible )] TJ ET BT 166.591 154.701 Td /F5 9.8 Tf [(via)] TJ ET BT 179.052 154.701 Td /F1 9.8 Tf [( an exo-probe system )] TJ ET 0.267 0.267 0.267 rg BT 275.509 156.208 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 280.327 158.589 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 282.737 156.208 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 287.555 158.589 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 289.965 156.208 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 294.783 158.589 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 297.193 156.208 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 302.011 154.701 Td /F1 9.8 Tf [(. In this study, MERS-CoV RT-RPA assay was developed to )] TJ ET BT 26.250 142.796 Td /F1 9.8 Tf [(amplify and detect in real-time a fragment of the nucleocapsid gene \(NC\) of MERS-CoV.)] TJ ET BT 26.250 106.193 Td /F4 12.0 Tf [(Materials and Methods)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(1)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Outbreaks)] TJ ET Q endstream endobj 8 0 obj << /Type /Font /Subtype /Type1 /Name /F1 /BaseFont /Helvetica /Encoding /WinAnsiEncoding >> endobj 9 0 obj << /Type /Font /Subtype /Type1 /Name /F2 /BaseFont /Times-Bold /Encoding /WinAnsiEncoding >> endobj 10 0 obj << /Type /Font /Subtype /Type1 /Name /F3 /BaseFont /Times-Italic /Encoding /WinAnsiEncoding >> endobj 11 0 obj << /Type /Font /Subtype /Type1 /Name /F4 /BaseFont /Helvetica-Bold /Encoding /WinAnsiEncoding >> endobj 12 0 obj << /Type /Font /Subtype /Type1 /Name /F5 /BaseFont /Helvetica-Oblique /Encoding /WinAnsiEncoding >> endobj 13 0 obj << /Type /XObject 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Viral RNA are listed in table 1. MERS-CoV was provided by Ron Fouchier, Erasmus University, Rotterdam, )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(Netherlands and the sample preparation was performed at the Robert Koch-Institut in Berlin, Germany under the ECDC )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(Framework Service Contract Ref. No. ECDC/2008/011. The respiratory viral RNA panel for cross reactivity was provided by )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(Landesgesundheitsamt Niedersachsen, Germany and Brunhilde Schweiger, National Reference Center for influenza viruses, )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(Robert Koch Institute, Berlin, Germany.)] TJ ET q 26.250 696.240 555.000 13.736 re W n 0.271 0.267 0.267 rg BT 26.250 698.987 Td /F1 9.8 Tf [(Table 1. Viral RNA tested.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 655.564 555.000 33.175 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 688.740 m 581.250 688.740 l 580.500 687.990 l 27.000 687.990 l f 581.250 688.740 m 581.250 655.564 l 580.500 655.564 l 580.500 687.990 l f 26.250 688.740 m 26.250 655.564 l 27.000 655.564 l 27.000 687.990 l f 0.271 0.267 0.267 rg BT 33.000 674.176 Td /F1 9.0 Tf [(The MERS-CoV RT-RPA assay only detected the MERS-CoV RNA, but not other viruses causing similar clinical picture. C)] TJ ET BT 520.143 671.660 Td /F1 8.0 Tf [(T)] TJ ET BT 525.031 674.176 Td /F1 9.0 Tf [(, cycle )] TJ ET BT 33.000 664.896 Td /F1 9.0 Tf [(threshold; TT, threshold time in minute.)] TJ ET 1.000 1.000 1.000 rg 26.250 249.845 555.000 405.719 re f 0.267 0.267 0.267 rg 26.625 654.439 182.219 0.750 re f 26.625 638.933 0.750 16.256 re f 26.625 630.618 0.750 8.316 re f 26.625 614.361 0.750 16.256 re f 0.271 0.267 0.267 rg BT 31.875 644.584 Td /F4 9.8 Tf [(Sample)] TJ ET 0.267 0.267 0.267 rg 208.094 654.439 144.446 0.750 re f 352.540 654.439 155.416 0.750 re f 208.094 638.558 0.750 16.631 re f 0.271 0.267 0.267 rg BT 213.344 644.584 Td /F4 9.8 Tf [(RNA real-time RT-PCR specific assay)] TJ ET 0.267 0.267 0.267 rg 507.206 654.439 73.669 0.750 re f 507.206 638.933 0.750 16.256 re f 580.125 638.933 0.750 16.256 re f 507.206 630.243 0.750 8.691 re f 580.125 630.243 0.750 8.691 re f 0.271 0.267 0.267 rg BT 512.456 644.584 Td /F4 9.8 Tf [(RT-RPA)] TJ ET 0.267 0.267 0.267 rg 208.094 638.558 144.821 0.750 re f 208.094 630.618 0.750 8.691 re f 208.094 614.361 0.750 16.256 re f 0.271 0.267 0.267 rg BT 213.344 628.703 Td /F4 9.8 Tf [(Reference)] TJ ET 0.267 0.267 0.267 rg 352.165 638.558 155.791 0.750 re f 352.165 630.618 0.750 8.691 re f 352.165 614.361 0.750 16.256 re f 0.271 0.267 0.267 rg BT 357.415 628.703 Td /F4 9.8 Tf [(Specific assays \()] TJ ET BT 436.000 628.703 Td /F4 9.8 Tf [(Mean Ct\))] TJ ET 0.267 0.267 0.267 rg 507.206 630.243 73.669 0.750 re f 507.206 614.361 0.750 16.631 re f 580.125 614.361 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 620.388 Td /F4 9.8 Tf [(TT [min])] TJ ET 0.267 0.267 0.267 rg 26.625 614.361 181.844 0.750 re f 208.469 614.361 144.071 0.750 re f 352.540 614.361 155.041 0.750 re f 507.581 614.361 73.294 0.750 re f 26.625 598.480 0.750 16.631 re f 580.125 598.480 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 604.506 Td /F4 9.8 Tf [(Coronaviruses)] TJ ET 0.267 0.267 0.267 rg 26.625 598.480 182.219 0.750 re f 26.625 579.648 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 588.625 Td /F1 9.8 Tf [(MERS-CoV)] TJ ET 0.267 0.267 0.267 rg 208.094 598.480 144.821 0.750 re f 208.094 579.648 0.750 19.582 re f BT 213.344 586.713 Td /F4 8.7 Tf [(13)] TJ ET 352.165 598.480 155.791 0.750 re f 352.165 579.648 0.750 19.582 re f 0.271 0.267 0.267 rg BT 357.415 588.625 Td /F1 9.8 Tf [(36.10)] TJ ET 0.267 0.267 0.267 rg 507.206 598.480 73.669 0.750 re f 507.206 579.648 0.750 19.582 re f 580.125 579.648 0.750 19.582 re f 0.271 0.267 0.267 rg BT 512.456 588.625 Td /F1 9.8 Tf [(6.70)] TJ ET 0.267 0.267 0.267 rg 26.625 579.648 182.219 0.750 re f 26.625 560.816 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 569.793 Td /F1 9.8 Tf [(229E)] TJ ET 0.267 0.267 0.267 rg 208.094 579.648 144.821 0.750 re f 208.094 560.816 0.750 19.582 re f BT 213.344 567.881 Td /F4 8.7 Tf [(14)] TJ ET 352.165 579.648 155.791 0.750 re f 352.165 560.816 0.750 19.582 re f 0.271 0.267 0.267 rg BT 357.415 569.793 Td /F1 9.8 Tf [(12.6)] TJ ET 0.267 0.267 0.267 rg 507.206 579.648 73.669 0.750 re f 507.206 560.816 0.750 19.582 re f 580.125 560.816 0.750 19.582 re f 0.271 0.267 0.267 rg BT 512.456 569.793 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 560.816 182.219 0.750 re f 26.625 541.984 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 550.961 Td /F1 9.8 Tf [(NL63)] TJ ET 0.267 0.267 0.267 rg 208.094 560.816 144.821 0.750 re f 208.094 541.984 0.750 19.582 re f BT 213.344 549.049 Td /F4 8.7 Tf [(15)] TJ ET 352.165 560.816 155.791 0.750 re f 352.165 541.984 0.750 19.582 re f 0.271 0.267 0.267 rg BT 357.415 550.961 Td /F1 9.8 Tf [(14.06)] TJ ET 0.267 0.267 0.267 rg 507.206 560.816 73.669 0.750 re f 507.206 541.984 0.750 19.582 re f 580.125 541.984 0.750 19.582 re f 0.271 0.267 0.267 rg BT 512.456 550.961 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 541.984 182.219 0.750 re f 26.625 523.152 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 532.129 Td /F1 9.8 Tf [(OC43)] TJ ET 0.267 0.267 0.267 rg 208.094 541.984 144.821 0.750 re f 208.094 523.152 0.750 19.582 re f BT 213.344 530.217 Td /F4 8.7 Tf [(14)] TJ ET 352.165 541.984 155.791 0.750 re f 352.165 523.152 0.750 19.582 re f 0.271 0.267 0.267 rg BT 357.415 532.129 Td /F1 9.8 Tf [(22.99)] TJ ET 0.267 0.267 0.267 rg 507.206 541.984 73.669 0.750 re f 507.206 523.152 0.750 19.582 re f 580.125 523.152 0.750 19.582 re f 0.271 0.267 0.267 rg BT 512.456 532.129 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 523.152 182.219 0.750 re f 26.625 504.320 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 513.297 Td /F1 9.8 Tf [(SARS)] TJ ET 0.267 0.267 0.267 rg 208.094 523.152 144.821 0.750 re f 208.094 504.320 0.750 19.582 re f BT 213.344 511.385 Td /F4 8.7 Tf [(16)] TJ ET 352.165 523.152 155.791 0.750 re f 352.165 504.320 0.750 19.582 re f 0.271 0.267 0.267 rg BT 357.415 513.297 Td /F1 9.8 Tf [(14.21)] TJ ET 0.267 0.267 0.267 rg 507.206 523.152 73.669 0.750 re f 507.206 504.320 0.750 19.582 re f 580.125 504.320 0.750 19.582 re f 0.271 0.267 0.267 rg BT 512.456 513.297 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 504.320 181.844 0.750 re f 208.469 504.320 144.071 0.750 re f 352.540 504.320 155.041 0.750 re f 507.581 504.320 73.294 0.750 re f 26.625 488.439 0.750 16.631 re f 580.125 488.439 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 494.465 Td /F4 9.8 Tf [(Respiratory Viruses)] TJ ET 0.267 0.267 0.267 rg 26.625 488.439 182.219 0.750 re f 26.625 472.558 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 478.584 Td /F1 9.8 Tf [(A/California/04/2009 H1N1)] TJ ET 0.267 0.267 0.267 rg 208.094 488.439 144.821 0.750 re f 208.094 472.933 0.750 16.256 re f 208.094 457.051 0.750 15.881 re f 208.094 441.170 0.750 15.881 re f 208.094 425.289 0.750 15.881 re f 208.094 409.033 0.750 16.256 re f BT 213.344 476.672 Td /F4 8.7 Tf [(18)] TJ ET 352.165 488.439 155.791 0.750 re f 352.165 472.558 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 478.584 Td /F1 9.8 Tf [(23.69)] TJ ET 0.267 0.267 0.267 rg 507.206 488.439 73.669 0.750 re f 507.206 472.558 0.750 16.631 re f 580.125 472.558 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 478.584 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 472.558 182.219 0.750 re f 26.625 456.676 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 462.703 Td /F1 9.8 Tf [(A/Wellington/1/04 H3N2)] TJ ET 0.267 0.267 0.267 rg 352.165 472.558 155.791 0.750 re f 352.165 456.676 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 462.703 Td /F1 9.8 Tf [(25.5)] TJ ET 0.267 0.267 0.267 rg 507.206 472.558 73.669 0.750 re f 507.206 456.676 0.750 16.631 re f 580.125 456.676 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 462.703 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 456.676 182.219 0.750 re f 26.625 440.795 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 446.821 Td /F1 9.8 Tf [(A/dk/Germany R603/06 H5N1)] TJ ET 0.267 0.267 0.267 rg 352.165 456.676 155.791 0.750 re f 352.165 440.795 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 446.821 Td /F1 9.8 Tf [(24.4)] TJ ET 0.267 0.267 0.267 rg 507.206 456.676 73.669 0.750 re f 507.206 440.795 0.750 16.631 re f 580.125 440.795 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 446.821 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 440.795 182.219 0.750 re f 26.625 424.914 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 430.940 Td /F1 9.8 Tf [(B/Malasiya/2506/04 \(Victoria lineage\))] TJ ET 0.267 0.267 0.267 rg 352.165 440.795 155.791 0.750 re f 352.165 424.914 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 430.940 Td /F1 9.8 Tf [(21.2)] TJ ET 0.267 0.267 0.267 rg 507.206 440.795 73.669 0.750 re f 507.206 424.914 0.750 16.631 re f 580.125 424.914 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 430.940 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 424.914 182.219 0.750 re f 26.625 409.033 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 415.059 Td /F1 9.8 Tf [(B/Iangsu/10/03 \(Yamagata lineage\))] TJ ET 0.267 0.267 0.267 rg 352.165 424.914 155.791 0.750 re f 352.165 409.033 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 415.059 Td /F1 9.8 Tf [(24.5)] TJ ET 0.267 0.267 0.267 rg 507.206 424.914 73.669 0.750 re f 507.206 409.033 0.750 16.631 re f 580.125 409.033 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 415.059 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 409.033 182.219 0.750 re f 26.625 393.151 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 399.178 Td /F1 9.8 Tf [(Parainfluenza virus 1 \(patient isolate\))] TJ ET 0.267 0.267 0.267 rg 208.094 409.033 144.821 0.750 re f 208.094 393.526 0.750 16.256 re f 208.094 377.645 0.750 15.881 re f 208.094 361.389 0.750 16.256 re f BT 213.344 397.266 Td /F4 8.7 Tf [(17)] TJ ET 352.165 409.033 155.791 0.750 re f 352.165 393.151 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 399.178 Td /F1 9.8 Tf [(28.09)] TJ ET 0.267 0.267 0.267 rg 507.206 409.033 73.669 0.750 re f 507.206 393.151 0.750 16.631 re f 580.125 393.151 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 399.178 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 393.151 182.219 0.750 re f 26.625 377.270 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 383.296 Td /F1 9.8 Tf [(Parainfluenza virus 2 \(patient isolate\))] TJ ET 0.267 0.267 0.267 rg 352.165 393.151 155.791 0.750 re f 352.165 377.270 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 383.296 Td /F1 9.8 Tf [(27.81)] TJ ET 0.267 0.267 0.267 rg 507.206 393.151 73.669 0.750 re f 507.206 377.270 0.750 16.631 re f 580.125 377.270 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 383.296 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 377.270 182.219 0.750 re f 26.625 361.389 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 367.415 Td /F1 9.8 Tf [(Parainfluenza virus 3 \(patient isolate\))] TJ ET 0.267 0.267 0.267 rg 352.165 377.270 155.791 0.750 re f 352.165 361.389 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 367.415 Td /F1 9.8 Tf [(29.13)] TJ ET 0.267 0.267 0.267 rg 507.206 377.270 73.669 0.750 re f 507.206 361.389 0.750 16.631 re f 580.125 361.389 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 367.415 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 361.389 182.219 0.750 re f 26.625 345.508 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 351.534 Td /F1 9.8 Tf [(Parainfluenza virus 4a \(patient isolate\))] TJ ET 0.267 0.267 0.267 rg 208.094 361.389 144.821 0.750 re f 208.094 345.883 0.750 16.256 re f 208.094 329.626 0.750 16.256 re f 0.271 0.267 0.267 rg BT 213.344 351.534 Td /F1 9.8 Tf [(In-house test)] TJ ET 0.267 0.267 0.267 rg 352.165 361.389 155.791 0.750 re f 352.165 345.508 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 351.534 Td /F1 9.8 Tf [(21.39)] TJ ET 0.267 0.267 0.267 rg 507.206 361.389 73.669 0.750 re f 507.206 345.508 0.750 16.631 re f 580.125 345.508 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 351.534 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 345.508 182.219 0.750 re f 26.625 329.626 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 335.653 Td /F1 9.8 Tf [(Parainfluenza virus 4b \(patient isolate\))] TJ ET 0.267 0.267 0.267 rg 352.165 345.508 155.791 0.750 re f 352.165 329.626 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 335.653 Td /F1 9.8 Tf [(28.05)] TJ ET 0.267 0.267 0.267 rg 507.206 345.508 73.669 0.750 re f 507.206 329.626 0.750 16.631 re f 580.125 329.626 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 335.653 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 329.626 182.219 0.750 re f 26.625 313.745 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 319.771 Td /F1 9.8 Tf [(Respiratory syncytial virus A)] TJ ET 0.267 0.267 0.267 rg 208.094 329.626 144.446 0.750 re f 208.094 250.220 144.446 0.750 re f 352.540 329.626 155.416 0.750 re f 352.540 250.220 155.416 0.750 re f 208.094 314.120 0.750 16.256 re f 208.094 298.239 0.750 15.881 re f 208.094 282.358 0.750 15.881 re f 208.094 266.476 0.750 15.881 re f 208.094 250.220 0.750 16.256 re f 0.271 0.267 0.267 rg BT 213.344 319.771 Td /F1 9.8 Tf [(Provided by National Reference Center for influenza viruses, )] TJ ET BT 213.344 312.140 Td /F1 9.8 Tf [(Robert Koch Institute, Germany)] TJ ET 0.267 0.267 0.267 rg 507.206 329.626 73.669 0.750 re f 507.206 313.745 0.750 16.631 re f 580.125 313.745 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 319.771 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 313.745 182.219 0.750 re f 26.625 297.864 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 303.890 Td /F1 9.8 Tf [(Respiratory syncytial virus B)] TJ ET 0.267 0.267 0.267 rg 507.206 313.745 73.669 0.750 re f 507.206 297.864 0.750 16.631 re f 580.125 297.864 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 303.890 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 297.864 182.219 0.750 re f 26.625 281.983 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 288.009 Td /F1 9.8 Tf [(Human rhinovirus A 89)] TJ ET 0.267 0.267 0.267 rg 507.206 297.864 73.669 0.750 re f 507.206 281.983 0.750 16.631 re f 580.125 281.983 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 288.009 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 281.983 182.219 0.750 re f 26.625 266.101 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 272.128 Td /F1 9.8 Tf [(Human rhinovirus A 1B)] TJ ET 0.267 0.267 0.267 rg 507.206 281.983 73.669 0.750 re f 507.206 266.101 0.750 16.631 re f 580.125 266.101 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 272.128 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 266.101 182.219 0.750 re f 26.625 250.220 182.219 0.750 re f 26.625 250.220 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 256.246 Td /F1 9.8 Tf [(Human rhinovirus B 37)] TJ ET 0.267 0.267 0.267 rg 507.206 266.101 73.669 0.750 re f 507.206 250.220 73.669 0.750 re f 507.206 250.220 0.750 16.631 re f 580.125 250.220 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 256.246 Td /F1 9.8 Tf [(Negative)] TJ ET BT 26.250 195.321 Td /F4 9.8 Tf [(Real-time RT-PCR and RT-RPA amplicon design. )] TJ ET BT 254.322 195.321 Td /F1 9.8 Tf [(Using the MERS-CoV sequence \(GenBank accession number: JX869059\), )] TJ ET BT 26.250 183.417 Td /F1 9.8 Tf [(we designed NC real-time RT-PCR primers and Taqman probe, and RT-RPA primers and an exo-probe \(Table 2\) to amplify the )] TJ ET BT 26.250 171.512 Td /F1 9.8 Tf [(NC gene. All oligonucleotides were produced by TIBMOLBIOL, Berlin, Germany.)] TJ ET Q q 15.000 89.515 577.500 687.485 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F4 9.8 Tf [(Viral RNA)] TJ ET BT 71.217 767.476 Td /F1 9.8 Tf [(. Viral RNA are listed in table 1. MERS-CoV was provided by Ron Fouchier, Erasmus University, Rotterdam, )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(Netherlands and the sample preparation was performed at the Robert Koch-Institut in Berlin, Germany under the ECDC )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(Framework Service Contract Ref. No. ECDC/2008/011. The respiratory viral RNA panel for cross reactivity was provided by )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(Landesgesundheitsamt Niedersachsen, Germany and Brunhilde Schweiger, National Reference Center for influenza viruses, )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(Robert Koch Institute, Berlin, Germany.)] TJ ET q 26.250 696.240 555.000 13.736 re W n 0.271 0.267 0.267 rg BT 26.250 698.987 Td /F1 9.8 Tf [(Table 1. Viral RNA tested.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 655.564 555.000 33.175 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 688.740 m 581.250 688.740 l 580.500 687.990 l 27.000 687.990 l f 581.250 688.740 m 581.250 655.564 l 580.500 655.564 l 580.500 687.990 l f 26.250 688.740 m 26.250 655.564 l 27.000 655.564 l 27.000 687.990 l f 0.271 0.267 0.267 rg BT 33.000 674.176 Td /F1 9.0 Tf [(The MERS-CoV RT-RPA assay only detected the MERS-CoV RNA, but not other viruses causing similar clinical picture. C)] TJ ET BT 520.143 671.660 Td /F1 8.0 Tf [(T)] TJ ET BT 525.031 674.176 Td /F1 9.0 Tf [(, cycle )] TJ ET BT 33.000 664.896 Td /F1 9.0 Tf [(threshold; TT, threshold time in minute.)] TJ ET 1.000 1.000 1.000 rg 26.250 249.845 555.000 405.719 re f 0.267 0.267 0.267 rg 26.625 654.439 182.219 0.750 re f 26.625 638.933 0.750 16.256 re f 26.625 630.618 0.750 8.316 re f 26.625 614.361 0.750 16.256 re f 0.271 0.267 0.267 rg BT 31.875 644.584 Td /F4 9.8 Tf [(Sample)] TJ ET 0.267 0.267 0.267 rg 208.094 654.439 144.446 0.750 re f 352.540 654.439 155.416 0.750 re f 208.094 638.558 0.750 16.631 re f 0.271 0.267 0.267 rg BT 213.344 644.584 Td /F4 9.8 Tf [(RNA real-time RT-PCR specific assay)] TJ ET 0.267 0.267 0.267 rg 507.206 654.439 73.669 0.750 re f 507.206 638.933 0.750 16.256 re f 580.125 638.933 0.750 16.256 re f 507.206 630.243 0.750 8.691 re f 580.125 630.243 0.750 8.691 re f 0.271 0.267 0.267 rg BT 512.456 644.584 Td /F4 9.8 Tf [(RT-RPA)] TJ ET 0.267 0.267 0.267 rg 208.094 638.558 144.821 0.750 re f 208.094 630.618 0.750 8.691 re f 208.094 614.361 0.750 16.256 re f 0.271 0.267 0.267 rg BT 213.344 628.703 Td /F4 9.8 Tf [(Reference)] TJ ET 0.267 0.267 0.267 rg 352.165 638.558 155.791 0.750 re f 352.165 630.618 0.750 8.691 re f 352.165 614.361 0.750 16.256 re f 0.271 0.267 0.267 rg BT 357.415 628.703 Td /F4 9.8 Tf [(Specific assays \()] TJ ET BT 436.000 628.703 Td /F4 9.8 Tf [(Mean Ct\))] TJ ET 0.267 0.267 0.267 rg 507.206 630.243 73.669 0.750 re f 507.206 614.361 0.750 16.631 re f 580.125 614.361 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 620.388 Td /F4 9.8 Tf [(TT [min])] TJ ET 0.267 0.267 0.267 rg 26.625 614.361 181.844 0.750 re f 208.469 614.361 144.071 0.750 re f 352.540 614.361 155.041 0.750 re f 507.581 614.361 73.294 0.750 re f 26.625 598.480 0.750 16.631 re f 580.125 598.480 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 604.506 Td /F4 9.8 Tf [(Coronaviruses)] TJ ET 0.267 0.267 0.267 rg 26.625 598.480 182.219 0.750 re f 26.625 579.648 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 588.625 Td /F1 9.8 Tf [(MERS-CoV)] TJ ET 0.267 0.267 0.267 rg 208.094 598.480 144.821 0.750 re f 208.094 579.648 0.750 19.582 re f BT 213.344 586.713 Td /F4 8.7 Tf [(13)] TJ ET 352.165 598.480 155.791 0.750 re f 352.165 579.648 0.750 19.582 re f 0.271 0.267 0.267 rg BT 357.415 588.625 Td /F1 9.8 Tf [(36.10)] TJ ET 0.267 0.267 0.267 rg 507.206 598.480 73.669 0.750 re f 507.206 579.648 0.750 19.582 re f 580.125 579.648 0.750 19.582 re f 0.271 0.267 0.267 rg BT 512.456 588.625 Td /F1 9.8 Tf [(6.70)] TJ ET 0.267 0.267 0.267 rg 26.625 579.648 182.219 0.750 re f 26.625 560.816 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 569.793 Td /F1 9.8 Tf [(229E)] TJ ET 0.267 0.267 0.267 rg 208.094 579.648 144.821 0.750 re f 208.094 560.816 0.750 19.582 re f BT 213.344 567.881 Td /F4 8.7 Tf [(14)] TJ ET 352.165 579.648 155.791 0.750 re f 352.165 560.816 0.750 19.582 re f 0.271 0.267 0.267 rg BT 357.415 569.793 Td /F1 9.8 Tf [(12.6)] TJ ET 0.267 0.267 0.267 rg 507.206 579.648 73.669 0.750 re f 507.206 560.816 0.750 19.582 re f 580.125 560.816 0.750 19.582 re f 0.271 0.267 0.267 rg BT 512.456 569.793 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 560.816 182.219 0.750 re f 26.625 541.984 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 550.961 Td /F1 9.8 Tf [(NL63)] TJ ET 0.267 0.267 0.267 rg 208.094 560.816 144.821 0.750 re f 208.094 541.984 0.750 19.582 re f BT 213.344 549.049 Td /F4 8.7 Tf [(15)] TJ ET 352.165 560.816 155.791 0.750 re f 352.165 541.984 0.750 19.582 re f 0.271 0.267 0.267 rg BT 357.415 550.961 Td /F1 9.8 Tf [(14.06)] TJ ET 0.267 0.267 0.267 rg 507.206 560.816 73.669 0.750 re f 507.206 541.984 0.750 19.582 re f 580.125 541.984 0.750 19.582 re f 0.271 0.267 0.267 rg BT 512.456 550.961 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 541.984 182.219 0.750 re f 26.625 523.152 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 532.129 Td /F1 9.8 Tf [(OC43)] TJ ET 0.267 0.267 0.267 rg 208.094 541.984 144.821 0.750 re f 208.094 523.152 0.750 19.582 re f BT 213.344 530.217 Td /F4 8.7 Tf [(14)] TJ ET 352.165 541.984 155.791 0.750 re f 352.165 523.152 0.750 19.582 re f 0.271 0.267 0.267 rg BT 357.415 532.129 Td /F1 9.8 Tf [(22.99)] TJ ET 0.267 0.267 0.267 rg 507.206 541.984 73.669 0.750 re f 507.206 523.152 0.750 19.582 re f 580.125 523.152 0.750 19.582 re f 0.271 0.267 0.267 rg BT 512.456 532.129 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 523.152 182.219 0.750 re f 26.625 504.320 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 513.297 Td /F1 9.8 Tf [(SARS)] TJ ET 0.267 0.267 0.267 rg 208.094 523.152 144.821 0.750 re f 208.094 504.320 0.750 19.582 re f BT 213.344 511.385 Td /F4 8.7 Tf [(16)] TJ ET 352.165 523.152 155.791 0.750 re f 352.165 504.320 0.750 19.582 re f 0.271 0.267 0.267 rg BT 357.415 513.297 Td /F1 9.8 Tf [(14.21)] TJ ET 0.267 0.267 0.267 rg 507.206 523.152 73.669 0.750 re f 507.206 504.320 0.750 19.582 re f 580.125 504.320 0.750 19.582 re f 0.271 0.267 0.267 rg BT 512.456 513.297 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 504.320 181.844 0.750 re f 208.469 504.320 144.071 0.750 re f 352.540 504.320 155.041 0.750 re f 507.581 504.320 73.294 0.750 re f 26.625 488.439 0.750 16.631 re f 580.125 488.439 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 494.465 Td /F4 9.8 Tf [(Respiratory Viruses)] TJ ET 0.267 0.267 0.267 rg 26.625 488.439 182.219 0.750 re f 26.625 472.558 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 478.584 Td /F1 9.8 Tf [(A/California/04/2009 H1N1)] TJ ET 0.267 0.267 0.267 rg 208.094 488.439 144.821 0.750 re f 208.094 472.933 0.750 16.256 re f 208.094 457.051 0.750 15.881 re f 208.094 441.170 0.750 15.881 re f 208.094 425.289 0.750 15.881 re f 208.094 409.033 0.750 16.256 re f BT 213.344 476.672 Td /F4 8.7 Tf [(18)] TJ ET 352.165 488.439 155.791 0.750 re f 352.165 472.558 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 478.584 Td /F1 9.8 Tf [(23.69)] TJ ET 0.267 0.267 0.267 rg 507.206 488.439 73.669 0.750 re f 507.206 472.558 0.750 16.631 re f 580.125 472.558 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 478.584 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 472.558 182.219 0.750 re f 26.625 456.676 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 462.703 Td /F1 9.8 Tf [(A/Wellington/1/04 H3N2)] TJ ET 0.267 0.267 0.267 rg 352.165 472.558 155.791 0.750 re f 352.165 456.676 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 462.703 Td /F1 9.8 Tf [(25.5)] TJ ET 0.267 0.267 0.267 rg 507.206 472.558 73.669 0.750 re f 507.206 456.676 0.750 16.631 re f 580.125 456.676 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 462.703 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 456.676 182.219 0.750 re f 26.625 440.795 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 446.821 Td /F1 9.8 Tf [(A/dk/Germany R603/06 H5N1)] TJ ET 0.267 0.267 0.267 rg 352.165 456.676 155.791 0.750 re f 352.165 440.795 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 446.821 Td /F1 9.8 Tf [(24.4)] TJ ET 0.267 0.267 0.267 rg 507.206 456.676 73.669 0.750 re f 507.206 440.795 0.750 16.631 re f 580.125 440.795 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 446.821 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 440.795 182.219 0.750 re f 26.625 424.914 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 430.940 Td /F1 9.8 Tf [(B/Malasiya/2506/04 \(Victoria lineage\))] TJ ET 0.267 0.267 0.267 rg 352.165 440.795 155.791 0.750 re f 352.165 424.914 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 430.940 Td /F1 9.8 Tf [(21.2)] TJ ET 0.267 0.267 0.267 rg 507.206 440.795 73.669 0.750 re f 507.206 424.914 0.750 16.631 re f 580.125 424.914 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 430.940 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 424.914 182.219 0.750 re f 26.625 409.033 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 415.059 Td /F1 9.8 Tf [(B/Iangsu/10/03 \(Yamagata lineage\))] TJ ET 0.267 0.267 0.267 rg 352.165 424.914 155.791 0.750 re f 352.165 409.033 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 415.059 Td /F1 9.8 Tf [(24.5)] TJ ET 0.267 0.267 0.267 rg 507.206 424.914 73.669 0.750 re f 507.206 409.033 0.750 16.631 re f 580.125 409.033 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 415.059 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 409.033 182.219 0.750 re f 26.625 393.151 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 399.178 Td /F1 9.8 Tf [(Parainfluenza virus 1 \(patient isolate\))] TJ ET 0.267 0.267 0.267 rg 208.094 409.033 144.821 0.750 re f 208.094 393.526 0.750 16.256 re f 208.094 377.645 0.750 15.881 re f 208.094 361.389 0.750 16.256 re f BT 213.344 397.266 Td /F4 8.7 Tf [(17)] TJ ET 352.165 409.033 155.791 0.750 re f 352.165 393.151 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 399.178 Td /F1 9.8 Tf [(28.09)] TJ ET 0.267 0.267 0.267 rg 507.206 409.033 73.669 0.750 re f 507.206 393.151 0.750 16.631 re f 580.125 393.151 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 399.178 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 393.151 182.219 0.750 re f 26.625 377.270 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 383.296 Td /F1 9.8 Tf [(Parainfluenza virus 2 \(patient isolate\))] TJ ET 0.267 0.267 0.267 rg 352.165 393.151 155.791 0.750 re f 352.165 377.270 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 383.296 Td /F1 9.8 Tf [(27.81)] TJ ET 0.267 0.267 0.267 rg 507.206 393.151 73.669 0.750 re f 507.206 377.270 0.750 16.631 re f 580.125 377.270 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 383.296 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 377.270 182.219 0.750 re f 26.625 361.389 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 367.415 Td /F1 9.8 Tf [(Parainfluenza virus 3 \(patient isolate\))] TJ ET 0.267 0.267 0.267 rg 352.165 377.270 155.791 0.750 re f 352.165 361.389 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 367.415 Td /F1 9.8 Tf [(29.13)] TJ ET 0.267 0.267 0.267 rg 507.206 377.270 73.669 0.750 re f 507.206 361.389 0.750 16.631 re f 580.125 361.389 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 367.415 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 361.389 182.219 0.750 re f 26.625 345.508 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 351.534 Td /F1 9.8 Tf [(Parainfluenza virus 4a \(patient isolate\))] TJ ET 0.267 0.267 0.267 rg 208.094 361.389 144.821 0.750 re f 208.094 345.883 0.750 16.256 re f 208.094 329.626 0.750 16.256 re f 0.271 0.267 0.267 rg BT 213.344 351.534 Td /F1 9.8 Tf [(In-house test)] TJ ET 0.267 0.267 0.267 rg 352.165 361.389 155.791 0.750 re f 352.165 345.508 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 351.534 Td /F1 9.8 Tf [(21.39)] TJ ET 0.267 0.267 0.267 rg 507.206 361.389 73.669 0.750 re f 507.206 345.508 0.750 16.631 re f 580.125 345.508 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 351.534 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 345.508 182.219 0.750 re f 26.625 329.626 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 335.653 Td /F1 9.8 Tf [(Parainfluenza virus 4b \(patient isolate\))] TJ ET 0.267 0.267 0.267 rg 352.165 345.508 155.791 0.750 re f 352.165 329.626 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 335.653 Td /F1 9.8 Tf [(28.05)] TJ ET 0.267 0.267 0.267 rg 507.206 345.508 73.669 0.750 re f 507.206 329.626 0.750 16.631 re f 580.125 329.626 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 335.653 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 329.626 182.219 0.750 re f 26.625 313.745 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 319.771 Td /F1 9.8 Tf [(Respiratory syncytial virus A)] TJ ET 0.267 0.267 0.267 rg 208.094 329.626 144.446 0.750 re f 208.094 250.220 144.446 0.750 re f 352.540 329.626 155.416 0.750 re f 352.540 250.220 155.416 0.750 re f 208.094 314.120 0.750 16.256 re f 208.094 298.239 0.750 15.881 re f 208.094 282.358 0.750 15.881 re f 208.094 266.476 0.750 15.881 re f 208.094 250.220 0.750 16.256 re f 0.271 0.267 0.267 rg BT 213.344 319.771 Td /F1 9.8 Tf [(Provided by National Reference Center for influenza viruses, )] TJ ET BT 213.344 312.140 Td /F1 9.8 Tf [(Robert Koch Institute, Germany)] TJ ET 0.267 0.267 0.267 rg 507.206 329.626 73.669 0.750 re f 507.206 313.745 0.750 16.631 re f 580.125 313.745 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 319.771 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 313.745 182.219 0.750 re f 26.625 297.864 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 303.890 Td /F1 9.8 Tf [(Respiratory syncytial virus B)] TJ ET 0.267 0.267 0.267 rg 507.206 313.745 73.669 0.750 re f 507.206 297.864 0.750 16.631 re f 580.125 297.864 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 303.890 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 297.864 182.219 0.750 re f 26.625 281.983 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 288.009 Td /F1 9.8 Tf [(Human rhinovirus A 89)] TJ ET 0.267 0.267 0.267 rg 507.206 297.864 73.669 0.750 re f 507.206 281.983 0.750 16.631 re f 580.125 281.983 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 288.009 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 281.983 182.219 0.750 re f 26.625 266.101 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 272.128 Td /F1 9.8 Tf [(Human rhinovirus A 1B)] TJ ET 0.267 0.267 0.267 rg 507.206 281.983 73.669 0.750 re f 507.206 266.101 0.750 16.631 re f 580.125 266.101 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 272.128 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 266.101 182.219 0.750 re f 26.625 250.220 182.219 0.750 re f 26.625 250.220 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 256.246 Td /F1 9.8 Tf [(Human rhinovirus B 37)] TJ ET 0.267 0.267 0.267 rg 507.206 266.101 73.669 0.750 re f 507.206 250.220 73.669 0.750 re f 507.206 250.220 0.750 16.631 re f 580.125 250.220 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 256.246 Td /F1 9.8 Tf [(Negative)] TJ ET BT 26.250 195.321 Td /F4 9.8 Tf [(Real-time RT-PCR and RT-RPA amplicon design. )] TJ ET BT 254.322 195.321 Td /F1 9.8 Tf [(Using the MERS-CoV sequence \(GenBank accession number: JX869059\), )] TJ ET BT 26.250 183.417 Td /F1 9.8 Tf [(we designed NC real-time RT-PCR primers and Taqman probe, and RT-RPA primers and an exo-probe \(Table 2\) to amplify the )] TJ ET BT 26.250 171.512 Td /F1 9.8 Tf [(NC gene. All oligonucleotides were produced by TIBMOLBIOL, Berlin, Germany.)] TJ ET Q q 15.000 89.515 577.500 687.485 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F4 9.8 Tf [(Viral RNA)] TJ ET BT 71.217 767.476 Td /F1 9.8 Tf [(. Viral RNA are listed in table 1. MERS-CoV was provided by Ron Fouchier, Erasmus University, Rotterdam, )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(Netherlands and the sample preparation was performed at the Robert Koch-Institut in Berlin, Germany under the ECDC )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(Framework Service Contract Ref. No. ECDC/2008/011. The respiratory viral RNA panel for cross reactivity was provided by )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(Landesgesundheitsamt Niedersachsen, Germany and Brunhilde Schweiger, National Reference Center for influenza viruses, )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(Robert Koch Institute, Berlin, Germany.)] TJ ET q 26.250 696.240 555.000 13.736 re W n 0.271 0.267 0.267 rg BT 26.250 698.987 Td /F1 9.8 Tf [(Table 1. Viral RNA tested.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 655.564 555.000 33.175 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 688.740 m 581.250 688.740 l 580.500 687.990 l 27.000 687.990 l f 581.250 688.740 m 581.250 655.564 l 580.500 655.564 l 580.500 687.990 l f 26.250 688.740 m 26.250 655.564 l 27.000 655.564 l 27.000 687.990 l f 0.271 0.267 0.267 rg BT 33.000 674.176 Td /F1 9.0 Tf [(The MERS-CoV RT-RPA assay only detected the MERS-CoV RNA, but not other viruses causing similar clinical picture. C)] TJ ET BT 520.143 671.660 Td /F1 8.0 Tf [(T)] TJ ET BT 525.031 674.176 Td /F1 9.0 Tf [(, cycle )] TJ ET BT 33.000 664.896 Td /F1 9.0 Tf [(threshold; TT, threshold time in minute.)] TJ ET 1.000 1.000 1.000 rg 26.250 249.845 555.000 405.719 re f 0.267 0.267 0.267 rg 26.625 654.439 182.219 0.750 re f 26.625 638.933 0.750 16.256 re f 26.625 630.618 0.750 8.316 re f 26.625 614.361 0.750 16.256 re f 0.271 0.267 0.267 rg BT 31.875 644.584 Td /F4 9.8 Tf [(Sample)] TJ ET 0.267 0.267 0.267 rg 208.094 654.439 144.446 0.750 re f 352.540 654.439 155.416 0.750 re f 208.094 638.558 0.750 16.631 re f 0.271 0.267 0.267 rg BT 213.344 644.584 Td /F4 9.8 Tf [(RNA real-time RT-PCR specific assay)] TJ ET 0.267 0.267 0.267 rg 507.206 654.439 73.669 0.750 re f 507.206 638.933 0.750 16.256 re f 580.125 638.933 0.750 16.256 re f 507.206 630.243 0.750 8.691 re f 580.125 630.243 0.750 8.691 re f 0.271 0.267 0.267 rg BT 512.456 644.584 Td /F4 9.8 Tf [(RT-RPA)] TJ ET 0.267 0.267 0.267 rg 208.094 638.558 144.821 0.750 re f 208.094 630.618 0.750 8.691 re f 208.094 614.361 0.750 16.256 re f 0.271 0.267 0.267 rg BT 213.344 628.703 Td /F4 9.8 Tf [(Reference)] TJ ET 0.267 0.267 0.267 rg 352.165 638.558 155.791 0.750 re f 352.165 630.618 0.750 8.691 re f 352.165 614.361 0.750 16.256 re f 0.271 0.267 0.267 rg BT 357.415 628.703 Td /F4 9.8 Tf [(Specific assays \()] TJ ET BT 436.000 628.703 Td /F4 9.8 Tf [(Mean Ct\))] TJ ET 0.267 0.267 0.267 rg 507.206 630.243 73.669 0.750 re f 507.206 614.361 0.750 16.631 re f 580.125 614.361 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 620.388 Td /F4 9.8 Tf [(TT [min])] TJ ET 0.267 0.267 0.267 rg 26.625 614.361 181.844 0.750 re f 208.469 614.361 144.071 0.750 re f 352.540 614.361 155.041 0.750 re f 507.581 614.361 73.294 0.750 re f 26.625 598.480 0.750 16.631 re f 580.125 598.480 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 604.506 Td /F4 9.8 Tf [(Coronaviruses)] TJ ET 0.267 0.267 0.267 rg 26.625 598.480 182.219 0.750 re f 26.625 579.648 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 588.625 Td /F1 9.8 Tf [(MERS-CoV)] TJ ET 0.267 0.267 0.267 rg 208.094 598.480 144.821 0.750 re f 208.094 579.648 0.750 19.582 re f BT 213.344 586.713 Td /F4 8.7 Tf [(13)] TJ ET 352.165 598.480 155.791 0.750 re f 352.165 579.648 0.750 19.582 re f 0.271 0.267 0.267 rg BT 357.415 588.625 Td /F1 9.8 Tf [(36.10)] TJ ET 0.267 0.267 0.267 rg 507.206 598.480 73.669 0.750 re f 507.206 579.648 0.750 19.582 re f 580.125 579.648 0.750 19.582 re f 0.271 0.267 0.267 rg BT 512.456 588.625 Td /F1 9.8 Tf [(6.70)] TJ ET 0.267 0.267 0.267 rg 26.625 579.648 182.219 0.750 re f 26.625 560.816 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 569.793 Td /F1 9.8 Tf [(229E)] TJ ET 0.267 0.267 0.267 rg 208.094 579.648 144.821 0.750 re f 208.094 560.816 0.750 19.582 re f BT 213.344 567.881 Td /F4 8.7 Tf [(14)] TJ ET 352.165 579.648 155.791 0.750 re f 352.165 560.816 0.750 19.582 re f 0.271 0.267 0.267 rg BT 357.415 569.793 Td /F1 9.8 Tf [(12.6)] TJ ET 0.267 0.267 0.267 rg 507.206 579.648 73.669 0.750 re f 507.206 560.816 0.750 19.582 re f 580.125 560.816 0.750 19.582 re f 0.271 0.267 0.267 rg BT 512.456 569.793 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 560.816 182.219 0.750 re f 26.625 541.984 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 550.961 Td /F1 9.8 Tf [(NL63)] TJ ET 0.267 0.267 0.267 rg 208.094 560.816 144.821 0.750 re f 208.094 541.984 0.750 19.582 re f BT 213.344 549.049 Td /F4 8.7 Tf [(15)] TJ ET 352.165 560.816 155.791 0.750 re f 352.165 541.984 0.750 19.582 re f 0.271 0.267 0.267 rg BT 357.415 550.961 Td /F1 9.8 Tf [(14.06)] TJ ET 0.267 0.267 0.267 rg 507.206 560.816 73.669 0.750 re f 507.206 541.984 0.750 19.582 re f 580.125 541.984 0.750 19.582 re f 0.271 0.267 0.267 rg BT 512.456 550.961 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 541.984 182.219 0.750 re f 26.625 523.152 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 532.129 Td /F1 9.8 Tf [(OC43)] TJ ET 0.267 0.267 0.267 rg 208.094 541.984 144.821 0.750 re f 208.094 523.152 0.750 19.582 re f BT 213.344 530.217 Td /F4 8.7 Tf [(14)] TJ ET 352.165 541.984 155.791 0.750 re f 352.165 523.152 0.750 19.582 re f 0.271 0.267 0.267 rg BT 357.415 532.129 Td /F1 9.8 Tf [(22.99)] TJ ET 0.267 0.267 0.267 rg 507.206 541.984 73.669 0.750 re f 507.206 523.152 0.750 19.582 re f 580.125 523.152 0.750 19.582 re f 0.271 0.267 0.267 rg BT 512.456 532.129 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 523.152 182.219 0.750 re f 26.625 504.320 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 513.297 Td /F1 9.8 Tf [(SARS)] TJ ET 0.267 0.267 0.267 rg 208.094 523.152 144.821 0.750 re f 208.094 504.320 0.750 19.582 re f BT 213.344 511.385 Td /F4 8.7 Tf [(16)] TJ ET 352.165 523.152 155.791 0.750 re f 352.165 504.320 0.750 19.582 re f 0.271 0.267 0.267 rg BT 357.415 513.297 Td /F1 9.8 Tf [(14.21)] TJ ET 0.267 0.267 0.267 rg 507.206 523.152 73.669 0.750 re f 507.206 504.320 0.750 19.582 re f 580.125 504.320 0.750 19.582 re f 0.271 0.267 0.267 rg BT 512.456 513.297 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 504.320 181.844 0.750 re f 208.469 504.320 144.071 0.750 re f 352.540 504.320 155.041 0.750 re f 507.581 504.320 73.294 0.750 re f 26.625 488.439 0.750 16.631 re f 580.125 488.439 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 494.465 Td /F4 9.8 Tf [(Respiratory Viruses)] TJ ET 0.267 0.267 0.267 rg 26.625 488.439 182.219 0.750 re f 26.625 472.558 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 478.584 Td /F1 9.8 Tf [(A/California/04/2009 H1N1)] TJ ET 0.267 0.267 0.267 rg 208.094 488.439 144.821 0.750 re f 208.094 472.933 0.750 16.256 re f 208.094 457.051 0.750 15.881 re f 208.094 441.170 0.750 15.881 re f 208.094 425.289 0.750 15.881 re f 208.094 409.033 0.750 16.256 re f BT 213.344 476.672 Td /F4 8.7 Tf [(18)] TJ ET 352.165 488.439 155.791 0.750 re f 352.165 472.558 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 478.584 Td /F1 9.8 Tf [(23.69)] TJ ET 0.267 0.267 0.267 rg 507.206 488.439 73.669 0.750 re f 507.206 472.558 0.750 16.631 re f 580.125 472.558 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 478.584 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 472.558 182.219 0.750 re f 26.625 456.676 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 462.703 Td /F1 9.8 Tf [(A/Wellington/1/04 H3N2)] TJ ET 0.267 0.267 0.267 rg 352.165 472.558 155.791 0.750 re f 352.165 456.676 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 462.703 Td /F1 9.8 Tf [(25.5)] TJ ET 0.267 0.267 0.267 rg 507.206 472.558 73.669 0.750 re f 507.206 456.676 0.750 16.631 re f 580.125 456.676 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 462.703 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 456.676 182.219 0.750 re f 26.625 440.795 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 446.821 Td /F1 9.8 Tf [(A/dk/Germany R603/06 H5N1)] TJ ET 0.267 0.267 0.267 rg 352.165 456.676 155.791 0.750 re f 352.165 440.795 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 446.821 Td /F1 9.8 Tf [(24.4)] TJ ET 0.267 0.267 0.267 rg 507.206 456.676 73.669 0.750 re f 507.206 440.795 0.750 16.631 re f 580.125 440.795 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 446.821 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 440.795 182.219 0.750 re f 26.625 424.914 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 430.940 Td /F1 9.8 Tf [(B/Malasiya/2506/04 \(Victoria lineage\))] TJ ET 0.267 0.267 0.267 rg 352.165 440.795 155.791 0.750 re f 352.165 424.914 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 430.940 Td /F1 9.8 Tf [(21.2)] TJ ET 0.267 0.267 0.267 rg 507.206 440.795 73.669 0.750 re f 507.206 424.914 0.750 16.631 re f 580.125 424.914 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 430.940 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 424.914 182.219 0.750 re f 26.625 409.033 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 415.059 Td /F1 9.8 Tf [(B/Iangsu/10/03 \(Yamagata lineage\))] TJ ET 0.267 0.267 0.267 rg 352.165 424.914 155.791 0.750 re f 352.165 409.033 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 415.059 Td /F1 9.8 Tf [(24.5)] TJ ET 0.267 0.267 0.267 rg 507.206 424.914 73.669 0.750 re f 507.206 409.033 0.750 16.631 re f 580.125 409.033 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 415.059 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 409.033 182.219 0.750 re f 26.625 393.151 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 399.178 Td /F1 9.8 Tf [(Parainfluenza virus 1 \(patient isolate\))] TJ ET 0.267 0.267 0.267 rg 208.094 409.033 144.821 0.750 re f 208.094 393.526 0.750 16.256 re f 208.094 377.645 0.750 15.881 re f 208.094 361.389 0.750 16.256 re f BT 213.344 397.266 Td /F4 8.7 Tf [(17)] TJ ET 352.165 409.033 155.791 0.750 re f 352.165 393.151 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 399.178 Td /F1 9.8 Tf [(28.09)] TJ ET 0.267 0.267 0.267 rg 507.206 409.033 73.669 0.750 re f 507.206 393.151 0.750 16.631 re f 580.125 393.151 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 399.178 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 393.151 182.219 0.750 re f 26.625 377.270 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 383.296 Td /F1 9.8 Tf [(Parainfluenza virus 2 \(patient isolate\))] TJ ET 0.267 0.267 0.267 rg 352.165 393.151 155.791 0.750 re f 352.165 377.270 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 383.296 Td /F1 9.8 Tf [(27.81)] TJ ET 0.267 0.267 0.267 rg 507.206 393.151 73.669 0.750 re f 507.206 377.270 0.750 16.631 re f 580.125 377.270 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 383.296 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 377.270 182.219 0.750 re f 26.625 361.389 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 367.415 Td /F1 9.8 Tf [(Parainfluenza virus 3 \(patient isolate\))] TJ ET 0.267 0.267 0.267 rg 352.165 377.270 155.791 0.750 re f 352.165 361.389 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 367.415 Td /F1 9.8 Tf [(29.13)] TJ ET 0.267 0.267 0.267 rg 507.206 377.270 73.669 0.750 re f 507.206 361.389 0.750 16.631 re f 580.125 361.389 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 367.415 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 361.389 182.219 0.750 re f 26.625 345.508 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 351.534 Td /F1 9.8 Tf [(Parainfluenza virus 4a \(patient isolate\))] TJ ET 0.267 0.267 0.267 rg 208.094 361.389 144.821 0.750 re f 208.094 345.883 0.750 16.256 re f 208.094 329.626 0.750 16.256 re f 0.271 0.267 0.267 rg BT 213.344 351.534 Td /F1 9.8 Tf [(In-house test)] TJ ET 0.267 0.267 0.267 rg 352.165 361.389 155.791 0.750 re f 352.165 345.508 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 351.534 Td /F1 9.8 Tf [(21.39)] TJ ET 0.267 0.267 0.267 rg 507.206 361.389 73.669 0.750 re f 507.206 345.508 0.750 16.631 re f 580.125 345.508 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 351.534 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 345.508 182.219 0.750 re f 26.625 329.626 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 335.653 Td /F1 9.8 Tf [(Parainfluenza virus 4b \(patient isolate\))] TJ ET 0.267 0.267 0.267 rg 352.165 345.508 155.791 0.750 re f 352.165 329.626 0.750 16.631 re f 0.271 0.267 0.267 rg BT 357.415 335.653 Td /F1 9.8 Tf [(28.05)] TJ ET 0.267 0.267 0.267 rg 507.206 345.508 73.669 0.750 re f 507.206 329.626 0.750 16.631 re f 580.125 329.626 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 335.653 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 329.626 182.219 0.750 re f 26.625 313.745 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 319.771 Td /F1 9.8 Tf [(Respiratory syncytial virus A)] TJ ET 0.267 0.267 0.267 rg 208.094 329.626 144.446 0.750 re f 208.094 250.220 144.446 0.750 re f 352.540 329.626 155.416 0.750 re f 352.540 250.220 155.416 0.750 re f 208.094 314.120 0.750 16.256 re f 208.094 298.239 0.750 15.881 re f 208.094 282.358 0.750 15.881 re f 208.094 266.476 0.750 15.881 re f 208.094 250.220 0.750 16.256 re f 0.271 0.267 0.267 rg BT 213.344 319.771 Td /F1 9.8 Tf [(Provided by National Reference Center for influenza viruses, )] TJ ET BT 213.344 312.140 Td /F1 9.8 Tf [(Robert Koch Institute, Germany)] TJ ET 0.267 0.267 0.267 rg 507.206 329.626 73.669 0.750 re f 507.206 313.745 0.750 16.631 re f 580.125 313.745 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 319.771 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 313.745 182.219 0.750 re f 26.625 297.864 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 303.890 Td /F1 9.8 Tf [(Respiratory syncytial virus B)] TJ ET 0.267 0.267 0.267 rg 507.206 313.745 73.669 0.750 re f 507.206 297.864 0.750 16.631 re f 580.125 297.864 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 303.890 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 297.864 182.219 0.750 re f 26.625 281.983 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 288.009 Td /F1 9.8 Tf [(Human rhinovirus A 89)] TJ ET 0.267 0.267 0.267 rg 507.206 297.864 73.669 0.750 re f 507.206 281.983 0.750 16.631 re f 580.125 281.983 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 288.009 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 281.983 182.219 0.750 re f 26.625 266.101 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 272.128 Td /F1 9.8 Tf [(Human rhinovirus A 1B)] TJ ET 0.267 0.267 0.267 rg 507.206 281.983 73.669 0.750 re f 507.206 266.101 0.750 16.631 re f 580.125 266.101 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 272.128 Td /F1 9.8 Tf [(Negative)] TJ ET 0.267 0.267 0.267 rg 26.625 266.101 182.219 0.750 re f 26.625 250.220 182.219 0.750 re f 26.625 250.220 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 256.246 Td /F1 9.8 Tf [(Human rhinovirus B 37)] TJ ET 0.267 0.267 0.267 rg 507.206 266.101 73.669 0.750 re f 507.206 250.220 73.669 0.750 re f 507.206 250.220 0.750 16.631 re f 580.125 250.220 0.750 16.631 re f 0.271 0.267 0.267 rg BT 512.456 256.246 Td /F1 9.8 Tf [(Negative)] TJ ET BT 26.250 195.321 Td /F4 9.8 Tf [(Real-time RT-PCR and RT-RPA amplicon design. )] TJ ET BT 254.322 195.321 Td /F1 9.8 Tf [(Using the MERS-CoV sequence \(GenBank accession number: JX869059\), )] TJ ET BT 26.250 183.417 Td /F1 9.8 Tf [(we designed NC real-time RT-PCR primers and Taqman probe, and RT-RPA primers and an exo-probe \(Table 2\) to amplify the )] TJ ET BT 26.250 171.512 Td /F1 9.8 Tf [(NC gene. All oligonucleotides were produced by TIBMOLBIOL, Berlin, Germany.)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(2)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Outbreaks)] TJ ET Q endstream endobj 161 0 obj << /Type /Annot /Subtype /Link /A 162 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 585.9118 222.9813 594.7301 ] >> endobj 162 0 obj << /Type /Action >> endobj 163 0 obj << /Type /Annot /Subtype /Link /A 164 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 567.0798 222.9813 575.8981 ] >> endobj 164 0 obj << /Type /Action >> endobj 165 0 obj << /Type /Annot /Subtype /Link /A 166 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 548.2478 222.9813 557.0661 ] >> endobj 166 0 obj << /Type /Action >> endobj 167 0 obj << /Type /Annot /Subtype /Link /A 168 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 529.4158 222.9813 538.2341 ] >> endobj 168 0 obj << /Type /Action >> endobj 169 0 obj << /Type /Annot /Subtype /Link /A 170 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 510.5838 222.9813 519.4021 ] >> endobj 170 0 obj << /Type /Action >> endobj 171 0 obj << /Type /Annot /Subtype /Link /A 172 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 475.8705 222.9813 484.6889 ] >> endobj 172 0 obj << /Type /Action >> endobj 173 0 obj << /Type /Annot /Subtype /Link /A 174 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 396.4643 222.9813 405.2826 ] >> endobj 174 0 obj << /Type /Action >> endobj 175 0 obj << /Type /Annot /Subtype /Link /A 176 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 585.9118 222.9813 594.7301 ] >> endobj 176 0 obj << /Type /Action >> endobj 177 0 obj << /Type /Annot /Subtype /Link /A 178 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 567.0798 222.9813 575.8981 ] >> endobj 178 0 obj << /Type /Action >> endobj 179 0 obj << /Type /Annot /Subtype /Link /A 180 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 548.2478 222.9813 557.0661 ] >> endobj 180 0 obj << /Type /Action >> endobj 181 0 obj << /Type /Annot /Subtype /Link /A 182 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 529.4158 222.9813 538.2341 ] >> endobj 182 0 obj << /Type /Action >> endobj 183 0 obj << /Type /Annot /Subtype /Link /A 184 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 510.5838 222.9813 519.4021 ] >> endobj 184 0 obj << /Type /Action >> endobj 185 0 obj << /Type /Annot /Subtype /Link /A 186 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 475.8705 222.9813 484.6889 ] >> endobj 186 0 obj << /Type /Action >> endobj 187 0 obj << /Type /Annot /Subtype /Link /A 188 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 396.4643 222.9813 405.2826 ] >> endobj 188 0 obj << /Type /Action >> endobj 189 0 obj << /Type /Annot /Subtype /Link /A 190 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 585.9118 222.9813 594.7301 ] >> endobj 190 0 obj << /Type /Action >> endobj 191 0 obj << /Type /Annot /Subtype /Link /A 192 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 567.0798 222.9813 575.8981 ] >> endobj 192 0 obj << /Type /Action >> endobj 193 0 obj << /Type /Annot /Subtype /Link /A 194 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 548.2478 222.9813 557.0661 ] >> endobj 194 0 obj << /Type /Action >> endobj 195 0 obj << /Type /Annot /Subtype /Link /A 196 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 529.4158 222.9813 538.2341 ] >> endobj 196 0 obj << /Type /Action >> endobj 197 0 obj << /Type /Annot /Subtype /Link /A 198 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 510.5838 222.9813 519.4021 ] >> endobj 198 0 obj << /Type /Action >> endobj 199 0 obj << /Type /Annot /Subtype /Link /A 200 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 475.8705 222.9813 484.6889 ] >> endobj 200 0 obj << /Type /Action >> endobj 201 0 obj << /Type /Annot /Subtype /Link /A 202 0 R /Border [0 0 0] /H /I /Rect [ 213.3440 396.4643 222.9813 405.2826 ] >> endobj 202 0 obj << /Type /Action >> endobj 203 0 obj << /Type /Page /Parent 3 0 R /Annots [ 205 0 R 207 0 R 209 0 R 211 0 R 213 0 R 215 0 R 217 0 R 219 0 R 221 0 R 223 0 R 225 0 R 227 0 R ] /Contents 204 0 R >> endobj 204 0 obj << /Length 31432 >> stream 0.271 0.267 0.267 rg 0.267 0.267 0.267 RG q 15.000 30.250 577.500 746.750 re W n q 26.250 763.264 555.000 13.736 re W n 0.271 0.267 0.267 rg BT 26.250 766.011 Td /F1 9.8 Tf [(Table 2. Primers and probes for the construction of molecular standard, NC real-time RT-PCR and RT-RPA assay)] TJ ET Q 0.965 0.965 0.965 rg 26.250 704.884 555.000 50.880 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 755.764 m 581.250 755.764 l 580.500 755.014 l 27.000 755.014 l f 581.250 755.764 m 581.250 704.884 l 580.500 704.884 l 580.500 755.014 l f 26.250 755.764 m 26.250 704.884 l 27.000 704.884 l 27.000 755.014 l f 0.271 0.267 0.267 rg BT 33.000 741.688 Td /F1 9.0 Tf [(COR12 UP/DP are NC gene amplification primers; COR12 NC FP/RP/P, NC real-time RT-PCR primers and Taqman probe )] TJ ET BT 33.000 732.530 Td /F1 9.0 Tf [(\(FAM/TAMRA\); COR12 RT-RPA FP/RP, RPA forward and reverse primer; COR12 RT-RPA P, exo-probe; BTF, B: thymidine nucleotide )] TJ ET BT 33.000 723.373 Td /F1 9.0 Tf [(carrying Blackhole quencher-1, T: tetrahydrofuran spacer, F: thymidine nucleotide carrying Fluorescein; Phosphate, 3phosphate to )] TJ ET BT 33.000 714.215 Td /F1 9.0 Tf [(block elongation.)] TJ ET 1.000 1.000 1.000 rg 26.250 456.330 555.000 248.554 re f 0.267 0.267 0.267 rg 26.625 703.759 101.365 0.750 re f 26.625 687.877 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 693.904 Td /F4 9.8 Tf [(Name)] TJ ET 0.267 0.267 0.267 rg 127.240 703.759 453.635 0.750 re f 127.240 687.877 0.750 16.631 re f 580.125 687.877 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 693.904 Td /F4 9.8 Tf [(Primers and probes )] TJ ET 0.267 0.267 0.267 rg 26.625 687.877 101.365 0.750 re f 26.625 567.874 0.750 120.754 re f 0.271 0.267 0.267 rg BT 31.875 678.022 Td /F1 9.8 Tf [(COR12 UP)] TJ ET 0.267 0.267 0.267 rg 127.240 687.877 453.635 0.750 re f 127.240 567.874 0.750 120.754 re f 580.125 567.874 0.750 120.754 re f 0.271 0.267 0.267 rg BT 132.490 678.022 Td /F1 9.8 Tf [(AATGATTCAGCTATTGTTACACAATTCG)] TJ ET 0.267 0.267 0.267 rg 26.625 567.874 101.365 0.750 re f 26.625 551.992 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 558.019 Td /F1 9.8 Tf [(COR12 DP)] TJ ET 0.267 0.267 0.267 rg 127.240 567.874 453.635 0.750 re f 127.240 551.992 0.750 16.631 re f 580.125 551.992 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 558.019 Td /F1 9.8 Tf [(ATCTTTCTTAGTGATTACTTTTGGCTGC)] TJ ET 0.267 0.267 0.267 rg 26.625 551.992 101.365 0.750 re f 26.625 536.111 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 542.137 Td /F1 9.8 Tf [(COR12 NC FP)] TJ ET 0.267 0.267 0.267 rg 127.240 551.992 453.635 0.750 re f 127.240 536.111 0.750 16.631 re f 580.125 536.111 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 542.137 Td /F1 9.8 Tf [(CAATAGTCAATCATCTTCAAGAGCCTC)] TJ ET 0.267 0.267 0.267 rg 26.625 536.111 101.365 0.750 re f 26.625 520.230 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 526.256 Td /F1 9.8 Tf [(COR12 NC RP)] TJ ET 0.267 0.267 0.267 rg 127.240 536.111 453.635 0.750 re f 127.240 520.230 0.750 16.631 re f 580.125 520.230 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 526.256 Td /F1 9.8 Tf [(GGAGAAGTGCCGCGGGTA)] TJ ET 0.267 0.267 0.267 rg 26.625 520.230 101.365 0.750 re f 26.625 504.349 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 510.375 Td /F1 9.8 Tf [(COR12 NC P)] TJ ET 0.267 0.267 0.267 rg 127.240 520.230 453.635 0.750 re f 127.240 504.349 0.750 16.631 re f 580.125 504.349 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 510.375 Td /F1 9.8 Tf [(AAACTCTTCCAGATCTAGTTCACAAGGTTCAAGATC)] TJ ET 0.267 0.267 0.267 rg 26.625 504.349 101.365 0.750 re f 26.625 488.467 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 494.494 Td /F1 9.8 Tf [(COR12 RT-RPA FP)] TJ ET 0.267 0.267 0.267 rg 127.240 504.349 453.635 0.750 re f 127.240 488.467 0.750 16.631 re f 580.125 488.467 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 494.494 Td /F1 9.8 Tf [(AACTTCCACATTGAGGGGACTGGAGGCAA)] TJ ET 0.267 0.267 0.267 rg 26.625 488.467 101.365 0.750 re f 26.625 472.586 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 478.612 Td /F1 9.8 Tf [(COR12 RT-RPA RP)] TJ ET 0.267 0.267 0.267 rg 127.240 488.467 453.635 0.750 re f 127.240 472.586 0.750 16.631 re f 580.125 472.586 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 478.612 Td /F1 9.8 Tf [(AGAGTTTCCTGATCTTGAACCTTGTGAACT)] TJ ET 0.267 0.267 0.267 rg 26.625 472.586 101.365 0.750 re f 26.625 456.705 101.365 0.750 re f 26.625 456.705 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 462.731 Td /F1 9.8 Tf [(COR12 RT-RPA P)] TJ ET 0.267 0.267 0.267 rg 127.240 472.586 453.635 0.750 re f 127.240 456.705 453.635 0.750 re f 127.240 456.705 0.750 16.631 re f 580.125 456.705 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 462.731 Td /F1 9.8 Tf [(TCTTCAAGAGCCTCTAGCTTAAGCAGAAAC-)] TJ ET BT 336.714 462.731 Td /F4 9.8 Tf [(BTF-)] TJ ET BT 358.914 462.731 Td /F1 9.8 Tf [(TCCAGATCTAGTTC-)] TJ ET BT 454.786 462.731 Td /F4 9.8 Tf [(P)] TJ ET BT 26.250 401.806 Td /F4 9.8 Tf [(RNA standard and RT-RPA conditions.)] TJ ET BT 205.553 401.806 Td /F1 9.8 Tf [( The NC gene was amplified using COR12 UP and COR12 DP \(Table 2\) \(nt 28989-)] TJ ET BT 26.250 389.901 Td /F1 9.8 Tf [(29291 of GenBank accession number: JX869059\). The amplificate was ligated into plasmid pCRII \(Invitrogen, Darmstadt, )] TJ ET BT 26.250 377.997 Td /F1 9.8 Tf [(Germany\). RNA was transcribed, quantified, diluted into a dilution range from 10)] TJ ET BT 371.975 381.885 Td /F1 8.7 Tf [(7)] TJ ET BT 376.794 377.997 Td /F1 9.8 Tf [( to 10)] TJ ET BT 401.188 381.885 Td /F1 8.7 Tf [(1)] TJ ET BT 406.007 377.997 Td /F1 9.8 Tf [( RNA molecules/l and tested by real-)] TJ ET BT 26.250 366.092 Td /F1 9.8 Tf [(time RT-PCR as described )] TJ ET 0.267 0.267 0.267 rg BT 144.898 367.599 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 154.535 366.092 Td /F1 9.8 Tf [(. RT-RPA was performed in a 50 l volume using the TwistAmp exo kit \(TwistDx, Cambridge, )] TJ ET BT 26.250 354.187 Td /F1 9.8 Tf [(UK\) 420 nM RPA primers, 120 nM exo-probe, 2 M DTT, 14 mM magnesium acetate, TwistAmp)] TJ ET BT 440.752 358.075 Td /F1 8.7 Tf [(TM)] TJ ET BT 453.266 354.187 Td /F1 9.8 Tf [( rehydration buffer, 2 U )] TJ ET BT 26.250 342.282 Td /F1 9.8 Tf [(Transcriptor \(Roche, Mannheim, Germany\), 20 U RiboLock RNase inhibitor \(Fisher, Schwerte, Germany\), and 19 mM DTT )] TJ ET BT 26.250 330.378 Td /F1 9.8 Tf [(\(Roche\). All reagents except for the template or sample RNA and magnesium acetate were prepared in a mastermix, which was )] TJ ET BT 26.250 318.473 Td /F1 9.8 Tf [(distributed into each tube of the 0,2 ml reaction eight-tube strip containing a dried enzyme pellet. Magnesium acetate was )] TJ ET BT 26.250 306.568 Td /F1 9.8 Tf [(pipetted into the tube lids. Subsequently, 5 l sample was added to the tubes. The lids were closed and the magnesium acetate )] TJ ET BT 26.250 294.663 Td /F1 9.8 Tf [(centrifuged into the tubes using a minispin centrifuge and the tubes immediately placed into the tubescanner device \(Qiagen )] TJ ET BT 26.250 282.759 Td /F1 9.8 Tf [(Lake Constance, Stockach, Germany\). Fluorescence measurements were performed in an ESEquant tubescanner at 42C for )] TJ ET BT 26.250 270.854 Td /F1 9.8 Tf [(10 minutes. A combined threshold and signal slope analysis were used for signal interpretation which can be confirmed by 2)] TJ ET BT 560.569 274.742 Td /F1 8.7 Tf [(nd)] TJ ET BT 26.250 258.949 Td /F1 9.8 Tf [(derivative analysis \(Tubescanner studio software, Qiagen Lake Constance, Stockach, Germany\).)] TJ ET BT 26.250 239.544 Td /F4 9.8 Tf [(Determination of sensitivity and specificity.)] TJ ET BT 227.266 239.544 Td /F1 9.8 Tf [(The sensitivity of the NC real-time RT-PCR assay and the RT-RPA assay was )] TJ ET BT 26.250 227.640 Td /F1 9.8 Tf [(tested using the quantitative RNA standard in 8 replicates, the threshold time was plotted against molecules detected and a )] TJ ET BT 26.250 215.735 Td /F1 9.8 Tf [(semi-log regression was calculated using the Prism software \(Graphpad Software Inc., San Diego, California\) and a probit )] TJ ET BT 26.250 203.830 Td /F1 9.8 Tf [(regression were calculated using the Statistica software \(StatSoft, Hamburg, Germany\).)] TJ ET BT 26.250 184.425 Td /F1 9.8 Tf [(Sensitivity was additionally evaluated using RNA extracts from dilutions of MERS-CoV virus culture supernatant. The )] TJ ET BT 26.250 172.521 Td /F1 9.8 Tf [(supernatant containing 6.3 x 10)] TJ ET BT 162.292 176.409 Td /F1 8.7 Tf [(8)] TJ ET BT 167.110 172.521 Td /F1 9.8 Tf [( genome equivalents/ml \(ge/ml\) as determined by the UpE real-time RT-PCR )] TJ ET 0.267 0.267 0.267 rg BT 501.418 174.028 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 511.056 172.521 Td /F1 9.8 Tf [( was diluted in )] TJ ET BT 26.250 160.616 Td /F1 9.8 Tf [(tenfold steps. RNA was extracted from these dilutions using the \(QIAamp viral RNA mini kit, Qiagen, Hilden, Germany\) and )] TJ ET BT 26.250 148.711 Td /F1 9.8 Tf [(eluted in 200 l nuclease free water. A volume of 2.5 l were tested simultaneously in three real-time RT-PCR assays \(UpE )] TJ ET 0.267 0.267 0.267 rg BT 559.994 150.218 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 569.632 148.711 Td /F1 9.8 Tf [( , )] TJ ET BT 26.250 136.806 Td /F1 9.8 Tf [(Orf1A )] TJ ET 0.267 0.267 0.267 rg BT 54.427 138.314 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 59.246 136.806 Td /F1 9.8 Tf [(, NC\) and the RT-RPA assay. For cross detection studies RNA of coronaviruses and other respiratory viruses listed in )] TJ ET BT 26.250 124.902 Td /F1 9.8 Tf [(table 1 were tested.)] TJ ET BT 26.250 88.299 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 68.345 Td /F1 9.8 Tf [(A 303 nt standard RNA was produced via in )] TJ ET BT 218.091 68.345 Td /F5 9.8 Tf [(vitro)] TJ ET BT 236.509 68.345 Td /F1 9.8 Tf [( transcription from a plasmid containing a NC gene fragment of MERS-CoV. )] TJ ET BT 26.250 56.440 Td /F1 9.8 Tf [(The sensitivity was determined using a dilution range of 10)] TJ ET BT 279.321 60.328 Td /F1 8.7 Tf [(7)] TJ ET BT 284.140 56.440 Td /F1 9.8 Tf [(-10)] TJ ET BT 298.228 60.328 Td /F1 8.7 Tf [(1)] TJ ET BT 303.047 56.440 Td /F1 9.8 Tf [( RNA molecules/l of the MERS-CoV RNA standard. The NC )] TJ ET BT 26.250 44.535 Td /F1 9.8 Tf [(real-time RT-PCR and the RT-RPA assay showed a sensitivity of 10)] TJ ET BT 319.920 48.424 Td /F1 8.7 Tf [(1)] TJ ET BT 324.739 44.535 Td /F1 9.8 Tf [( RNA molecules detected \(Figure 1 and 2\). The probit )] TJ ET Q q 15.000 30.250 577.500 746.750 re W n q 26.250 763.264 555.000 13.736 re W n 0.271 0.267 0.267 rg BT 26.250 766.011 Td /F1 9.8 Tf [(Table 2. Primers and probes for the construction of molecular standard, NC real-time RT-PCR and RT-RPA assay)] TJ ET Q 0.965 0.965 0.965 rg 26.250 704.884 555.000 50.880 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 755.764 m 581.250 755.764 l 580.500 755.014 l 27.000 755.014 l f 581.250 755.764 m 581.250 704.884 l 580.500 704.884 l 580.500 755.014 l f 26.250 755.764 m 26.250 704.884 l 27.000 704.884 l 27.000 755.014 l f 0.271 0.267 0.267 rg BT 33.000 741.688 Td /F1 9.0 Tf [(COR12 UP/DP are NC gene amplification primers; COR12 NC FP/RP/P, NC real-time RT-PCR primers and Taqman probe )] TJ ET BT 33.000 732.530 Td /F1 9.0 Tf [(\(FAM/TAMRA\); COR12 RT-RPA FP/RP, RPA forward and reverse primer; COR12 RT-RPA P, exo-probe; BTF, B: thymidine nucleotide )] TJ ET BT 33.000 723.373 Td /F1 9.0 Tf [(carrying Blackhole quencher-1, T: tetrahydrofuran spacer, F: thymidine nucleotide carrying Fluorescein; Phosphate, 3phosphate to )] TJ ET BT 33.000 714.215 Td /F1 9.0 Tf [(block elongation.)] TJ ET 1.000 1.000 1.000 rg 26.250 456.330 555.000 248.554 re f 0.267 0.267 0.267 rg 26.625 703.759 101.365 0.750 re f 26.625 687.877 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 693.904 Td /F4 9.8 Tf [(Name)] TJ ET 0.267 0.267 0.267 rg 127.240 703.759 453.635 0.750 re f 127.240 687.877 0.750 16.631 re f 580.125 687.877 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 693.904 Td /F4 9.8 Tf [(Primers and probes )] TJ ET 0.267 0.267 0.267 rg 26.625 687.877 101.365 0.750 re f 26.625 567.874 0.750 120.754 re f 0.271 0.267 0.267 rg BT 31.875 678.022 Td /F1 9.8 Tf [(COR12 UP)] TJ ET 0.267 0.267 0.267 rg 127.240 687.877 453.635 0.750 re f 127.240 567.874 0.750 120.754 re f 580.125 567.874 0.750 120.754 re f 0.271 0.267 0.267 rg BT 132.490 678.022 Td /F1 9.8 Tf [(AATGATTCAGCTATTGTTACACAATTCG)] TJ ET 0.267 0.267 0.267 rg 26.625 567.874 101.365 0.750 re f 26.625 551.992 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 558.019 Td /F1 9.8 Tf [(COR12 DP)] TJ ET 0.267 0.267 0.267 rg 127.240 567.874 453.635 0.750 re f 127.240 551.992 0.750 16.631 re f 580.125 551.992 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 558.019 Td /F1 9.8 Tf [(ATCTTTCTTAGTGATTACTTTTGGCTGC)] TJ ET 0.267 0.267 0.267 rg 26.625 551.992 101.365 0.750 re f 26.625 536.111 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 542.137 Td /F1 9.8 Tf [(COR12 NC FP)] TJ ET 0.267 0.267 0.267 rg 127.240 551.992 453.635 0.750 re f 127.240 536.111 0.750 16.631 re f 580.125 536.111 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 542.137 Td /F1 9.8 Tf [(CAATAGTCAATCATCTTCAAGAGCCTC)] TJ ET 0.267 0.267 0.267 rg 26.625 536.111 101.365 0.750 re f 26.625 520.230 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 526.256 Td /F1 9.8 Tf [(COR12 NC RP)] TJ ET 0.267 0.267 0.267 rg 127.240 536.111 453.635 0.750 re f 127.240 520.230 0.750 16.631 re f 580.125 520.230 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 526.256 Td /F1 9.8 Tf [(GGAGAAGTGCCGCGGGTA)] TJ ET 0.267 0.267 0.267 rg 26.625 520.230 101.365 0.750 re f 26.625 504.349 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 510.375 Td /F1 9.8 Tf [(COR12 NC P)] TJ ET 0.267 0.267 0.267 rg 127.240 520.230 453.635 0.750 re f 127.240 504.349 0.750 16.631 re f 580.125 504.349 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 510.375 Td /F1 9.8 Tf [(AAACTCTTCCAGATCTAGTTCACAAGGTTCAAGATC)] TJ ET 0.267 0.267 0.267 rg 26.625 504.349 101.365 0.750 re f 26.625 488.467 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 494.494 Td /F1 9.8 Tf [(COR12 RT-RPA FP)] TJ ET 0.267 0.267 0.267 rg 127.240 504.349 453.635 0.750 re f 127.240 488.467 0.750 16.631 re f 580.125 488.467 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 494.494 Td /F1 9.8 Tf [(AACTTCCACATTGAGGGGACTGGAGGCAA)] TJ ET 0.267 0.267 0.267 rg 26.625 488.467 101.365 0.750 re f 26.625 472.586 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 478.612 Td /F1 9.8 Tf [(COR12 RT-RPA RP)] TJ ET 0.267 0.267 0.267 rg 127.240 488.467 453.635 0.750 re f 127.240 472.586 0.750 16.631 re f 580.125 472.586 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 478.612 Td /F1 9.8 Tf [(AGAGTTTCCTGATCTTGAACCTTGTGAACT)] TJ ET 0.267 0.267 0.267 rg 26.625 472.586 101.365 0.750 re f 26.625 456.705 101.365 0.750 re f 26.625 456.705 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 462.731 Td /F1 9.8 Tf [(COR12 RT-RPA P)] TJ ET 0.267 0.267 0.267 rg 127.240 472.586 453.635 0.750 re f 127.240 456.705 453.635 0.750 re f 127.240 456.705 0.750 16.631 re f 580.125 456.705 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 462.731 Td /F1 9.8 Tf [(TCTTCAAGAGCCTCTAGCTTAAGCAGAAAC-)] TJ ET BT 336.714 462.731 Td /F4 9.8 Tf [(BTF-)] TJ ET BT 358.914 462.731 Td /F1 9.8 Tf [(TCCAGATCTAGTTC-)] TJ ET BT 454.786 462.731 Td /F4 9.8 Tf [(P)] TJ ET BT 26.250 401.806 Td /F4 9.8 Tf [(RNA standard and RT-RPA conditions.)] TJ ET BT 205.553 401.806 Td /F1 9.8 Tf [( The NC gene was amplified using COR12 UP and COR12 DP \(Table 2\) \(nt 28989-)] TJ ET BT 26.250 389.901 Td /F1 9.8 Tf [(29291 of GenBank accession number: JX869059\). The amplificate was ligated into plasmid pCRII \(Invitrogen, Darmstadt, )] TJ ET BT 26.250 377.997 Td /F1 9.8 Tf [(Germany\). RNA was transcribed, quantified, diluted into a dilution range from 10)] TJ ET BT 371.975 381.885 Td /F1 8.7 Tf [(7)] TJ ET BT 376.794 377.997 Td /F1 9.8 Tf [( to 10)] TJ ET BT 401.188 381.885 Td /F1 8.7 Tf [(1)] TJ ET BT 406.007 377.997 Td /F1 9.8 Tf [( RNA molecules/l and tested by real-)] TJ ET BT 26.250 366.092 Td /F1 9.8 Tf [(time RT-PCR as described )] TJ ET 0.267 0.267 0.267 rg BT 144.898 367.599 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 154.535 366.092 Td /F1 9.8 Tf [(. RT-RPA was performed in a 50 l volume using the TwistAmp exo kit \(TwistDx, Cambridge, )] TJ ET BT 26.250 354.187 Td /F1 9.8 Tf [(UK\) 420 nM RPA primers, 120 nM exo-probe, 2 M DTT, 14 mM magnesium acetate, TwistAmp)] TJ ET BT 440.752 358.075 Td /F1 8.7 Tf [(TM)] TJ ET BT 453.266 354.187 Td /F1 9.8 Tf [( rehydration buffer, 2 U )] TJ ET BT 26.250 342.282 Td /F1 9.8 Tf [(Transcriptor \(Roche, Mannheim, Germany\), 20 U RiboLock RNase inhibitor \(Fisher, Schwerte, Germany\), and 19 mM DTT )] TJ ET BT 26.250 330.378 Td /F1 9.8 Tf [(\(Roche\). All reagents except for the template or sample RNA and magnesium acetate were prepared in a mastermix, which was )] TJ ET BT 26.250 318.473 Td /F1 9.8 Tf [(distributed into each tube of the 0,2 ml reaction eight-tube strip containing a dried enzyme pellet. Magnesium acetate was )] TJ ET BT 26.250 306.568 Td /F1 9.8 Tf [(pipetted into the tube lids. Subsequently, 5 l sample was added to the tubes. The lids were closed and the magnesium acetate )] TJ ET BT 26.250 294.663 Td /F1 9.8 Tf [(centrifuged into the tubes using a minispin centrifuge and the tubes immediately placed into the tubescanner device \(Qiagen )] TJ ET BT 26.250 282.759 Td /F1 9.8 Tf [(Lake Constance, Stockach, Germany\). Fluorescence measurements were performed in an ESEquant tubescanner at 42C for )] TJ ET BT 26.250 270.854 Td /F1 9.8 Tf [(10 minutes. A combined threshold and signal slope analysis were used for signal interpretation which can be confirmed by 2)] TJ ET BT 560.569 274.742 Td /F1 8.7 Tf [(nd)] TJ ET BT 26.250 258.949 Td /F1 9.8 Tf [(derivative analysis \(Tubescanner studio software, Qiagen Lake Constance, Stockach, Germany\).)] TJ ET BT 26.250 239.544 Td /F4 9.8 Tf [(Determination of sensitivity and specificity.)] TJ ET BT 227.266 239.544 Td /F1 9.8 Tf [(The sensitivity of the NC real-time RT-PCR assay and the RT-RPA assay was )] TJ ET BT 26.250 227.640 Td /F1 9.8 Tf [(tested using the quantitative RNA standard in 8 replicates, the threshold time was plotted against molecules detected and a )] TJ ET BT 26.250 215.735 Td /F1 9.8 Tf [(semi-log regression was calculated using the Prism software \(Graphpad Software Inc., San Diego, California\) and a probit )] TJ ET BT 26.250 203.830 Td /F1 9.8 Tf [(regression were calculated using the Statistica software \(StatSoft, Hamburg, Germany\).)] TJ ET BT 26.250 184.425 Td /F1 9.8 Tf [(Sensitivity was additionally evaluated using RNA extracts from dilutions of MERS-CoV virus culture supernatant. The )] TJ ET BT 26.250 172.521 Td /F1 9.8 Tf [(supernatant containing 6.3 x 10)] TJ ET BT 162.292 176.409 Td /F1 8.7 Tf [(8)] TJ ET BT 167.110 172.521 Td /F1 9.8 Tf [( genome equivalents/ml \(ge/ml\) as determined by the UpE real-time RT-PCR )] TJ ET 0.267 0.267 0.267 rg BT 501.418 174.028 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 511.056 172.521 Td /F1 9.8 Tf [( was diluted in )] TJ ET BT 26.250 160.616 Td /F1 9.8 Tf [(tenfold steps. RNA was extracted from these dilutions using the \(QIAamp viral RNA mini kit, Qiagen, Hilden, Germany\) and )] TJ ET BT 26.250 148.711 Td /F1 9.8 Tf [(eluted in 200 l nuclease free water. A volume of 2.5 l were tested simultaneously in three real-time RT-PCR assays \(UpE )] TJ ET 0.267 0.267 0.267 rg BT 559.994 150.218 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 569.632 148.711 Td /F1 9.8 Tf [( , )] TJ ET BT 26.250 136.806 Td /F1 9.8 Tf [(Orf1A )] TJ ET 0.267 0.267 0.267 rg BT 54.427 138.314 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 59.246 136.806 Td /F1 9.8 Tf [(, NC\) and the RT-RPA assay. For cross detection studies RNA of coronaviruses and other respiratory viruses listed in )] TJ ET BT 26.250 124.902 Td /F1 9.8 Tf [(table 1 were tested.)] TJ ET BT 26.250 88.299 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 68.345 Td /F1 9.8 Tf [(A 303 nt standard RNA was produced via in )] TJ ET BT 218.091 68.345 Td /F5 9.8 Tf [(vitro)] TJ ET BT 236.509 68.345 Td /F1 9.8 Tf [( transcription from a plasmid containing a NC gene fragment of MERS-CoV. )] TJ ET BT 26.250 56.440 Td /F1 9.8 Tf [(The sensitivity was determined using a dilution range of 10)] TJ ET BT 279.321 60.328 Td /F1 8.7 Tf [(7)] TJ ET BT 284.140 56.440 Td /F1 9.8 Tf [(-10)] TJ ET BT 298.228 60.328 Td /F1 8.7 Tf [(1)] TJ ET BT 303.047 56.440 Td /F1 9.8 Tf [( RNA molecules/l of the MERS-CoV RNA standard. The NC )] TJ ET BT 26.250 44.535 Td /F1 9.8 Tf [(real-time RT-PCR and the RT-RPA assay showed a sensitivity of 10)] TJ ET BT 319.920 48.424 Td /F1 8.7 Tf [(1)] TJ ET BT 324.739 44.535 Td /F1 9.8 Tf [( RNA molecules detected \(Figure 1 and 2\). The probit )] TJ ET Q q 15.000 30.250 577.500 746.750 re W n q 26.250 763.264 555.000 13.736 re W n 0.271 0.267 0.267 rg BT 26.250 766.011 Td /F1 9.8 Tf [(Table 2. Primers and probes for the construction of molecular standard, NC real-time RT-PCR and RT-RPA assay)] TJ ET Q 0.965 0.965 0.965 rg 26.250 704.884 555.000 50.880 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 755.764 m 581.250 755.764 l 580.500 755.014 l 27.000 755.014 l f 581.250 755.764 m 581.250 704.884 l 580.500 704.884 l 580.500 755.014 l f 26.250 755.764 m 26.250 704.884 l 27.000 704.884 l 27.000 755.014 l f 0.271 0.267 0.267 rg BT 33.000 741.688 Td /F1 9.0 Tf [(COR12 UP/DP are NC gene amplification primers; COR12 NC FP/RP/P, NC real-time RT-PCR primers and Taqman probe )] TJ ET BT 33.000 732.530 Td /F1 9.0 Tf [(\(FAM/TAMRA\); COR12 RT-RPA FP/RP, RPA forward and reverse primer; COR12 RT-RPA P, exo-probe; BTF, B: thymidine nucleotide )] TJ ET BT 33.000 723.373 Td /F1 9.0 Tf [(carrying Blackhole quencher-1, T: tetrahydrofuran spacer, F: thymidine nucleotide carrying Fluorescein; Phosphate, 3phosphate to )] TJ ET BT 33.000 714.215 Td /F1 9.0 Tf [(block elongation.)] TJ ET 1.000 1.000 1.000 rg 26.250 456.330 555.000 248.554 re f 0.267 0.267 0.267 rg 26.625 703.759 101.365 0.750 re f 26.625 687.877 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 693.904 Td /F4 9.8 Tf [(Name)] TJ ET 0.267 0.267 0.267 rg 127.240 703.759 453.635 0.750 re f 127.240 687.877 0.750 16.631 re f 580.125 687.877 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 693.904 Td /F4 9.8 Tf [(Primers and probes )] TJ ET 0.267 0.267 0.267 rg 26.625 687.877 101.365 0.750 re f 26.625 567.874 0.750 120.754 re f 0.271 0.267 0.267 rg BT 31.875 678.022 Td /F1 9.8 Tf [(COR12 UP)] TJ ET 0.267 0.267 0.267 rg 127.240 687.877 453.635 0.750 re f 127.240 567.874 0.750 120.754 re f 580.125 567.874 0.750 120.754 re f 0.271 0.267 0.267 rg BT 132.490 678.022 Td /F1 9.8 Tf [(AATGATTCAGCTATTGTTACACAATTCG)] TJ ET 0.267 0.267 0.267 rg 26.625 567.874 101.365 0.750 re f 26.625 551.992 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 558.019 Td /F1 9.8 Tf [(COR12 DP)] TJ ET 0.267 0.267 0.267 rg 127.240 567.874 453.635 0.750 re f 127.240 551.992 0.750 16.631 re f 580.125 551.992 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 558.019 Td /F1 9.8 Tf [(ATCTTTCTTAGTGATTACTTTTGGCTGC)] TJ ET 0.267 0.267 0.267 rg 26.625 551.992 101.365 0.750 re f 26.625 536.111 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 542.137 Td /F1 9.8 Tf [(COR12 NC FP)] TJ ET 0.267 0.267 0.267 rg 127.240 551.992 453.635 0.750 re f 127.240 536.111 0.750 16.631 re f 580.125 536.111 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 542.137 Td /F1 9.8 Tf [(CAATAGTCAATCATCTTCAAGAGCCTC)] TJ ET 0.267 0.267 0.267 rg 26.625 536.111 101.365 0.750 re f 26.625 520.230 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 526.256 Td /F1 9.8 Tf [(COR12 NC RP)] TJ ET 0.267 0.267 0.267 rg 127.240 536.111 453.635 0.750 re f 127.240 520.230 0.750 16.631 re f 580.125 520.230 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 526.256 Td /F1 9.8 Tf [(GGAGAAGTGCCGCGGGTA)] TJ ET 0.267 0.267 0.267 rg 26.625 520.230 101.365 0.750 re f 26.625 504.349 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 510.375 Td /F1 9.8 Tf [(COR12 NC P)] TJ ET 0.267 0.267 0.267 rg 127.240 520.230 453.635 0.750 re f 127.240 504.349 0.750 16.631 re f 580.125 504.349 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 510.375 Td /F1 9.8 Tf [(AAACTCTTCCAGATCTAGTTCACAAGGTTCAAGATC)] TJ ET 0.267 0.267 0.267 rg 26.625 504.349 101.365 0.750 re f 26.625 488.467 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 494.494 Td /F1 9.8 Tf [(COR12 RT-RPA FP)] TJ ET 0.267 0.267 0.267 rg 127.240 504.349 453.635 0.750 re f 127.240 488.467 0.750 16.631 re f 580.125 488.467 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 494.494 Td /F1 9.8 Tf [(AACTTCCACATTGAGGGGACTGGAGGCAA)] TJ ET 0.267 0.267 0.267 rg 26.625 488.467 101.365 0.750 re f 26.625 472.586 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 478.612 Td /F1 9.8 Tf [(COR12 RT-RPA RP)] TJ ET 0.267 0.267 0.267 rg 127.240 488.467 453.635 0.750 re f 127.240 472.586 0.750 16.631 re f 580.125 472.586 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 478.612 Td /F1 9.8 Tf [(AGAGTTTCCTGATCTTGAACCTTGTGAACT)] TJ ET 0.267 0.267 0.267 rg 26.625 472.586 101.365 0.750 re f 26.625 456.705 101.365 0.750 re f 26.625 456.705 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 462.731 Td /F1 9.8 Tf [(COR12 RT-RPA P)] TJ ET 0.267 0.267 0.267 rg 127.240 472.586 453.635 0.750 re f 127.240 456.705 453.635 0.750 re f 127.240 456.705 0.750 16.631 re f 580.125 456.705 0.750 16.631 re f 0.271 0.267 0.267 rg BT 132.490 462.731 Td /F1 9.8 Tf [(TCTTCAAGAGCCTCTAGCTTAAGCAGAAAC-)] TJ ET BT 336.714 462.731 Td /F4 9.8 Tf [(BTF-)] TJ ET BT 358.914 462.731 Td /F1 9.8 Tf [(TCCAGATCTAGTTC-)] TJ ET BT 454.786 462.731 Td /F4 9.8 Tf [(P)] TJ ET BT 26.250 401.806 Td /F4 9.8 Tf [(RNA standard and RT-RPA conditions.)] TJ ET BT 205.553 401.806 Td /F1 9.8 Tf [( The NC gene was amplified using COR12 UP and COR12 DP \(Table 2\) \(nt 28989-)] TJ ET BT 26.250 389.901 Td /F1 9.8 Tf [(29291 of GenBank accession number: JX869059\). The amplificate was ligated into plasmid pCRII \(Invitrogen, Darmstadt, )] TJ ET BT 26.250 377.997 Td /F1 9.8 Tf [(Germany\). RNA was transcribed, quantified, diluted into a dilution range from 10)] TJ ET BT 371.975 381.885 Td /F1 8.7 Tf [(7)] TJ ET BT 376.794 377.997 Td /F1 9.8 Tf [( to 10)] TJ ET BT 401.188 381.885 Td /F1 8.7 Tf [(1)] TJ ET BT 406.007 377.997 Td /F1 9.8 Tf [( RNA molecules/l and tested by real-)] TJ ET BT 26.250 366.092 Td /F1 9.8 Tf [(time RT-PCR as described )] TJ ET 0.267 0.267 0.267 rg BT 144.898 367.599 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 154.535 366.092 Td /F1 9.8 Tf [(. RT-RPA was performed in a 50 l volume using the TwistAmp exo kit \(TwistDx, Cambridge, )] TJ ET BT 26.250 354.187 Td /F1 9.8 Tf [(UK\) 420 nM RPA primers, 120 nM exo-probe, 2 M DTT, 14 mM magnesium acetate, TwistAmp)] TJ ET BT 440.752 358.075 Td /F1 8.7 Tf [(TM)] TJ ET BT 453.266 354.187 Td /F1 9.8 Tf [( rehydration buffer, 2 U )] TJ ET BT 26.250 342.282 Td /F1 9.8 Tf [(Transcriptor \(Roche, Mannheim, Germany\), 20 U RiboLock RNase inhibitor \(Fisher, Schwerte, Germany\), and 19 mM DTT )] TJ ET BT 26.250 330.378 Td /F1 9.8 Tf [(\(Roche\). All reagents except for the template or sample RNA and magnesium acetate were prepared in a mastermix, which was )] TJ ET BT 26.250 318.473 Td /F1 9.8 Tf [(distributed into each tube of the 0,2 ml reaction eight-tube strip containing a dried enzyme pellet. Magnesium acetate was )] TJ ET BT 26.250 306.568 Td /F1 9.8 Tf [(pipetted into the tube lids. Subsequently, 5 l sample was added to the tubes. The lids were closed and the magnesium acetate )] TJ ET BT 26.250 294.663 Td /F1 9.8 Tf [(centrifuged into the tubes using a minispin centrifuge and the tubes immediately placed into the tubescanner device \(Qiagen )] TJ ET BT 26.250 282.759 Td /F1 9.8 Tf [(Lake Constance, Stockach, Germany\). Fluorescence measurements were performed in an ESEquant tubescanner at 42C for )] TJ ET BT 26.250 270.854 Td /F1 9.8 Tf [(10 minutes. A combined threshold and signal slope analysis were used for signal interpretation which can be confirmed by 2)] TJ ET BT 560.569 274.742 Td /F1 8.7 Tf [(nd)] TJ ET BT 26.250 258.949 Td /F1 9.8 Tf [(derivative analysis \(Tubescanner studio software, Qiagen Lake Constance, Stockach, Germany\).)] TJ ET BT 26.250 239.544 Td /F4 9.8 Tf [(Determination of sensitivity and specificity.)] TJ ET BT 227.266 239.544 Td /F1 9.8 Tf [(The sensitivity of the NC real-time RT-PCR assay and the RT-RPA assay was )] TJ ET BT 26.250 227.640 Td /F1 9.8 Tf [(tested using the quantitative RNA standard in 8 replicates, the threshold time was plotted against molecules detected and a )] TJ ET BT 26.250 215.735 Td /F1 9.8 Tf [(semi-log regression was calculated using the Prism software \(Graphpad Software Inc., San Diego, California\) and a probit )] TJ ET BT 26.250 203.830 Td /F1 9.8 Tf [(regression were calculated using the Statistica software \(StatSoft, Hamburg, Germany\).)] TJ ET BT 26.250 184.425 Td /F1 9.8 Tf [(Sensitivity was additionally evaluated using RNA extracts from dilutions of MERS-CoV virus culture supernatant. The )] TJ ET BT 26.250 172.521 Td /F1 9.8 Tf [(supernatant containing 6.3 x 10)] TJ ET BT 162.292 176.409 Td /F1 8.7 Tf [(8)] TJ ET BT 167.110 172.521 Td /F1 9.8 Tf [( genome equivalents/ml \(ge/ml\) as determined by the UpE real-time RT-PCR )] TJ ET 0.267 0.267 0.267 rg BT 501.418 174.028 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 511.056 172.521 Td /F1 9.8 Tf [( was diluted in )] TJ ET BT 26.250 160.616 Td /F1 9.8 Tf [(tenfold steps. RNA was extracted from these dilutions using the \(QIAamp viral RNA mini kit, Qiagen, Hilden, Germany\) and )] TJ ET BT 26.250 148.711 Td /F1 9.8 Tf [(eluted in 200 l nuclease free water. A volume of 2.5 l were tested simultaneously in three real-time RT-PCR assays \(UpE )] TJ ET 0.267 0.267 0.267 rg BT 559.994 150.218 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 569.632 148.711 Td /F1 9.8 Tf [( , )] TJ ET BT 26.250 136.806 Td /F1 9.8 Tf [(Orf1A )] TJ ET 0.267 0.267 0.267 rg BT 54.427 138.314 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 59.246 136.806 Td /F1 9.8 Tf [(, NC\) and the RT-RPA assay. For cross detection studies RNA of coronaviruses and other respiratory viruses listed in )] TJ ET BT 26.250 124.902 Td /F1 9.8 Tf [(table 1 were tested.)] TJ ET BT 26.250 88.299 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 68.345 Td /F1 9.8 Tf [(A 303 nt standard RNA was produced via in )] TJ ET BT 218.091 68.345 Td /F5 9.8 Tf [(vitro)] TJ ET BT 236.509 68.345 Td /F1 9.8 Tf [( transcription from a plasmid containing a NC gene fragment of MERS-CoV. )] TJ ET BT 26.250 56.440 Td /F1 9.8 Tf [(The sensitivity was determined using a dilution range of 10)] TJ ET BT 279.321 60.328 Td /F1 8.7 Tf [(7)] TJ ET BT 284.140 56.440 Td /F1 9.8 Tf [(-10)] TJ ET BT 298.228 60.328 Td /F1 8.7 Tf [(1)] TJ ET BT 303.047 56.440 Td /F1 9.8 Tf [( RNA molecules/l of the MERS-CoV RNA standard. The NC )] TJ ET BT 26.250 44.535 Td /F1 9.8 Tf [(real-time RT-PCR and the RT-RPA assay showed a sensitivity of 10)] TJ ET BT 319.920 48.424 Td /F1 8.7 Tf [(1)] TJ ET BT 324.739 44.535 Td /F1 9.8 Tf [( RNA molecules detected \(Figure 1 and 2\). The probit )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(3)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Outbreaks)] TJ ET Q endstream endobj 205 0 obj << /Type /Annot /Subtype /Link /A 206 0 R /Border [0 0 0] /H /I /Rect [ 144.8978 366.7974 154.5351 375.6157 ] >> endobj 206 0 obj << /Type /Action >> endobj 207 0 obj << /Type /Annot /Subtype /Link /A 208 0 R /Border [0 0 0] /H /I /Rect [ 501.4184 173.2262 511.0557 182.0445 ] >> endobj 208 0 obj << /Type /Action >> endobj 209 0 obj << /Type /Annot /Subtype /Link /A 210 0 R /Border [0 0 0] /H /I /Rect [ 559.9942 149.4167 569.6316 158.2350 ] >> endobj 210 0 obj << /Type /Action >> endobj 211 0 obj << /Type /Annot /Subtype /Link /A 212 0 R /Border [0 0 0] /H /I /Rect [ 54.4275 137.5119 59.2462 146.3302 ] >> endobj 212 0 obj << /Type /Action >> endobj 213 0 obj << /Type /Annot /Subtype /Link /A 214 0 R /Border [0 0 0] /H /I /Rect [ 144.8978 366.7974 154.5351 375.6157 ] >> endobj 214 0 obj << /Type /Action >> endobj 215 0 obj << /Type /Annot /Subtype /Link /A 216 0 R /Border [0 0 0] /H /I /Rect [ 501.4184 173.2262 511.0557 182.0445 ] >> endobj 216 0 obj << /Type /Action >> endobj 217 0 obj << /Type /Annot /Subtype /Link /A 218 0 R /Border [0 0 0] /H /I /Rect [ 559.9942 149.4167 569.6316 158.2350 ] >> endobj 218 0 obj << /Type /Action >> endobj 219 0 obj << /Type /Annot /Subtype /Link /A 220 0 R /Border [0 0 0] /H /I /Rect [ 54.4275 137.5119 59.2462 146.3302 ] >> endobj 220 0 obj << /Type /Action >> endobj 221 0 obj << /Type /Annot /Subtype /Link /A 222 0 R /Border [0 0 0] /H /I /Rect [ 144.8978 366.7974 154.5351 375.6157 ] >> endobj 222 0 obj << /Type /Action >> endobj 223 0 obj << /Type /Annot /Subtype /Link /A 224 0 R /Border [0 0 0] /H /I /Rect [ 501.4184 173.2262 511.0557 182.0445 ] >> endobj 224 0 obj << /Type /Action >> endobj 225 0 obj << /Type /Annot /Subtype /Link /A 226 0 R /Border [0 0 0] /H /I /Rect [ 559.9942 149.4167 569.6316 158.2350 ] >> endobj 226 0 obj << /Type /Action >> endobj 227 0 obj << /Type /Annot /Subtype /Link /A 228 0 R /Border [0 0 0] /H /I /Rect [ 54.4275 137.5119 59.2462 146.3302 ] >> endobj 228 0 obj << /Type /Action >> endobj 229 0 obj << /Type /Page /Parent 3 0 R /Annots [ 231 0 R 234 0 R 238 0 R 241 0 R 243 0 R 247 0 R 249 0 R 251 0 R 255 0 R ] /Contents 230 0 R >> endobj 230 0 obj << /Length 11505 >> stream 0.271 0.267 0.267 rg 0.267 0.267 0.267 RG q 15.000 -112.841 577.500 889.841 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(regression predicted a sensitivity of 21 and 10 RNA molecules respectively for RT-RPA \(figure 3\) and NC RT-PCR assay.)] TJ ET 0.965 0.965 0.965 rg 26.250 494.009 555.000 263.586 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 757.595 m 581.250 757.595 l 581.250 756.845 l 26.250 756.845 l f 26.250 494.009 m 581.250 494.009 l 581.250 494.759 l 26.250 494.759 l f q 225.000 0 0 142.500 35.250 605.345 cm /I3 Do Q q 35.250 505.259 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 589.821 Td /F4 9.8 Tf [(Fig. 1: MERS-CoV RT-RPA assay.)] TJ ET BT 35.250 570.451 Td /F1 9.8 Tf [(Over time development of fluorescence using a dilution range of 10)] TJ ET BT 324.640 576.171 Td /F1 8.7 Tf [(7)] TJ ET BT 329.458 570.451 Td /F1 9.8 Tf [(-10)] TJ ET BT 343.547 576.171 Td /F1 8.7 Tf [(1)] TJ ET BT 348.366 570.451 Td /F1 9.8 Tf [( molecules/l of the RNA molecular standard )] TJ ET BT 35.250 556.715 Td /F1 9.8 Tf [(\(Tubescanner Studio Software, Qiagen, Germany\). MERS-CoV RT-RPA assay sensitivity is 10 copies and yielded results in )] TJ ET BT 35.250 542.979 Td /F1 9.8 Tf [(maximum 7 minutes. 10)] TJ ET BT 138.737 548.699 Td /F1 8.7 Tf [(7)] TJ ET BT 143.555 542.979 Td /F1 9.8 Tf [( represented by black line; 10)] TJ ET BT 270.910 548.699 Td /F1 8.7 Tf [(6)] TJ ET BT 275.728 542.979 Td /F1 9.8 Tf [(, gray; 10)] TJ ET BT 316.376 548.699 Td /F1 8.7 Tf [(5)] TJ ET BT 321.195 542.979 Td /F1 9.8 Tf [(, red; 10)] TJ ET BT 356.967 548.699 Td /F1 8.7 Tf [(4)] TJ ET BT 361.786 542.979 Td /F1 9.8 Tf [(, blue; 10)] TJ ET BT 401.898 548.699 Td /F1 8.7 Tf [(3)] TJ ET BT 406.716 542.979 Td /F1 9.8 Tf [(, green; 10)] TJ ET BT 453.331 548.699 Td /F1 8.7 Tf [(2)] TJ ET BT 458.150 542.979 Td /F1 9.8 Tf [(, cyan; 10)] TJ ET BT 500.426 548.699 Td /F1 8.7 Tf [(1)] TJ ET BT 505.244 542.979 Td /F1 9.8 Tf [(, dark khaki; )] TJ ET BT 35.250 529.243 Td /F1 9.8 Tf [(negative control, orange. No fluorescence signals was measured for one minute \(after 3 minutes of the start of the reaction\) )] TJ ET BT 35.250 515.506 Td /F1 9.8 Tf [(because of the need of mixing the content.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 88.909 555.000 397.600 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 486.509 m 581.250 486.509 l 581.250 485.759 l 26.250 485.759 l f 26.250 88.909 m 581.250 88.909 l 581.250 89.659 l 26.250 89.659 l f q 225.000 0 0 290.250 35.250 186.509 cm /I5 Do Q q 35.250 100.159 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 170.985 Td /F4 9.8 Tf [(Fig. 2: Analytical sensitivity of MERS-CoV NC real-time RT-PCR and RT-RPA.)] TJ ET BT 35.250 151.615 Td /F1 9.8 Tf [(The analytical sensitivity was determined on RNA molecular standard \(8 runs\) for real-time RT-PCR \(A\) and real-time RT-)] TJ ET BT 35.250 137.879 Td /F1 9.8 Tf [(RPA \(B\). Both assays have a sensitivity of 10 RNA molecules. The RT-RPA assay was much faster than the RT-PCR as )] TJ ET BT 35.250 124.143 Td /F1 9.8 Tf [(the run time of the RT-RPA is between 3-7 minutes for 10)] TJ ET BT 283.436 129.863 Td /F1 8.7 Tf [(7 )] TJ ET BT 290.664 124.143 Td /F1 9.8 Tf [(and 10)] TJ ET BT 320.480 129.863 Td /F1 8.7 Tf [(1)] TJ ET BT 325.298 124.143 Td /F1 9.8 Tf [(molecules, respectively. While the RT-PCR needed up )] TJ ET BT 35.250 110.407 Td /F1 9.8 Tf [(to 2 hours. Consequently, RT-PCR results is linear, while RT-RPA not.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 -112.841 555.000 194.250 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 81.409 m 581.250 81.409 l 581.250 80.659 l 26.250 80.659 l f q 225.000 0 0 178.500 35.250 -106.841 cm /I6 Do Q q 35.250 -112.841 537.000 0.000 re W n Q Q q 15.000 -112.841 577.500 889.841 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(regression predicted a sensitivity of 21 and 10 RNA molecules respectively for RT-RPA \(figure 3\) and NC RT-PCR assay.)] TJ ET 0.965 0.965 0.965 rg 26.250 494.009 555.000 263.586 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 757.595 m 581.250 757.595 l 581.250 756.845 l 26.250 756.845 l f 26.250 494.009 m 581.250 494.009 l 581.250 494.759 l 26.250 494.759 l f q 225.000 0 0 142.500 35.250 605.345 cm /I3 Do Q q 35.250 505.259 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 589.821 Td /F4 9.8 Tf [(Fig. 1: MERS-CoV RT-RPA assay.)] TJ ET BT 35.250 570.451 Td /F1 9.8 Tf [(Over time development of fluorescence using a dilution range of 10)] TJ ET BT 324.640 576.171 Td /F1 8.7 Tf [(7)] TJ ET BT 329.458 570.451 Td /F1 9.8 Tf [(-10)] TJ ET BT 343.547 576.171 Td /F1 8.7 Tf [(1)] TJ ET BT 348.366 570.451 Td /F1 9.8 Tf [( molecules/l of the RNA molecular standard )] TJ ET BT 35.250 556.715 Td /F1 9.8 Tf [(\(Tubescanner Studio Software, Qiagen, Germany\). MERS-CoV RT-RPA assay sensitivity is 10 copies and yielded results in )] TJ ET BT 35.250 542.979 Td /F1 9.8 Tf [(maximum 7 minutes. 10)] TJ ET BT 138.737 548.699 Td /F1 8.7 Tf [(7)] TJ ET BT 143.555 542.979 Td /F1 9.8 Tf [( represented by black line; 10)] TJ ET BT 270.910 548.699 Td /F1 8.7 Tf [(6)] TJ ET BT 275.728 542.979 Td /F1 9.8 Tf [(, gray; 10)] TJ ET BT 316.376 548.699 Td /F1 8.7 Tf [(5)] TJ ET BT 321.195 542.979 Td /F1 9.8 Tf [(, red; 10)] TJ ET BT 356.967 548.699 Td /F1 8.7 Tf [(4)] TJ ET BT 361.786 542.979 Td /F1 9.8 Tf [(, blue; 10)] TJ ET BT 401.898 548.699 Td /F1 8.7 Tf [(3)] TJ ET BT 406.716 542.979 Td /F1 9.8 Tf [(, green; 10)] TJ ET BT 453.331 548.699 Td /F1 8.7 Tf [(2)] TJ ET BT 458.150 542.979 Td /F1 9.8 Tf [(, cyan; 10)] TJ ET BT 500.426 548.699 Td /F1 8.7 Tf [(1)] TJ ET BT 505.244 542.979 Td /F1 9.8 Tf [(, dark khaki; )] TJ ET BT 35.250 529.243 Td /F1 9.8 Tf [(negative control, orange. No fluorescence signals was measured for one minute \(after 3 minutes of the start of the reaction\) )] TJ ET BT 35.250 515.506 Td /F1 9.8 Tf [(because of the need of mixing the content.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 88.909 555.000 397.600 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 486.509 m 581.250 486.509 l 581.250 485.759 l 26.250 485.759 l f 26.250 88.909 m 581.250 88.909 l 581.250 89.659 l 26.250 89.659 l f q 225.000 0 0 290.250 35.250 186.509 cm /I8 Do Q q 35.250 100.159 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 170.985 Td /F4 9.8 Tf [(Fig. 2: Analytical sensitivity of MERS-CoV NC real-time RT-PCR and RT-RPA.)] TJ ET BT 35.250 151.615 Td /F1 9.8 Tf [(The analytical sensitivity was determined on RNA molecular standard \(8 runs\) for real-time RT-PCR \(A\) and real-time RT-)] TJ ET BT 35.250 137.879 Td /F1 9.8 Tf [(RPA \(B\). Both assays have a sensitivity of 10 RNA molecules. The RT-RPA assay was much faster than the RT-PCR as )] TJ ET BT 35.250 124.143 Td /F1 9.8 Tf [(the run time of the RT-RPA is between 3-7 minutes for 10)] TJ ET BT 283.436 129.863 Td /F1 8.7 Tf [(7 )] TJ ET BT 290.664 124.143 Td /F1 9.8 Tf [(and 10)] TJ ET BT 320.480 129.863 Td /F1 8.7 Tf [(1)] TJ ET BT 325.298 124.143 Td /F1 9.8 Tf [(molecules, respectively. While the RT-PCR needed up )] TJ ET BT 35.250 110.407 Td /F1 9.8 Tf [(to 2 hours. Consequently, RT-PCR results is linear, while RT-RPA not.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 -112.841 555.000 194.250 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 81.409 m 581.250 81.409 l 581.250 80.659 l 26.250 80.659 l f q 225.000 0 0 178.500 35.250 -106.841 cm /I6 Do Q q 35.250 -112.841 537.000 0.000 re W n Q Q q 15.000 -112.841 577.500 889.841 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(regression predicted a sensitivity of 21 and 10 RNA molecules respectively for RT-RPA \(figure 3\) and NC RT-PCR assay.)] TJ ET 0.965 0.965 0.965 rg 26.250 494.009 555.000 263.586 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 757.595 m 581.250 757.595 l 581.250 756.845 l 26.250 756.845 l f 26.250 494.009 m 581.250 494.009 l 581.250 494.759 l 26.250 494.759 l f q 225.000 0 0 142.500 35.250 605.345 cm /I3 Do Q q 35.250 505.259 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 589.821 Td /F4 9.8 Tf [(Fig. 1: MERS-CoV RT-RPA assay.)] TJ ET BT 35.250 570.451 Td /F1 9.8 Tf [(Over time development of fluorescence using a dilution range of 10)] TJ ET BT 324.640 576.171 Td /F1 8.7 Tf [(7)] TJ ET BT 329.458 570.451 Td /F1 9.8 Tf [(-10)] TJ ET BT 343.547 576.171 Td /F1 8.7 Tf [(1)] TJ ET BT 348.366 570.451 Td /F1 9.8 Tf [( molecules/l of the RNA molecular standard )] TJ ET BT 35.250 556.715 Td /F1 9.8 Tf [(\(Tubescanner Studio Software, Qiagen, Germany\). MERS-CoV RT-RPA assay sensitivity is 10 copies and yielded results in )] TJ ET BT 35.250 542.979 Td /F1 9.8 Tf [(maximum 7 minutes. 10)] TJ ET BT 138.737 548.699 Td /F1 8.7 Tf [(7)] TJ ET BT 143.555 542.979 Td /F1 9.8 Tf [( represented by black line; 10)] TJ ET BT 270.910 548.699 Td /F1 8.7 Tf [(6)] TJ ET BT 275.728 542.979 Td /F1 9.8 Tf [(, gray; 10)] TJ ET BT 316.376 548.699 Td /F1 8.7 Tf [(5)] TJ ET BT 321.195 542.979 Td /F1 9.8 Tf [(, red; 10)] TJ ET BT 356.967 548.699 Td /F1 8.7 Tf [(4)] TJ ET BT 361.786 542.979 Td /F1 9.8 Tf [(, blue; 10)] TJ ET BT 401.898 548.699 Td /F1 8.7 Tf [(3)] TJ ET BT 406.716 542.979 Td /F1 9.8 Tf [(, green; 10)] TJ ET BT 453.331 548.699 Td /F1 8.7 Tf [(2)] TJ ET BT 458.150 542.979 Td /F1 9.8 Tf [(, cyan; 10)] TJ ET BT 500.426 548.699 Td /F1 8.7 Tf [(1)] TJ ET BT 505.244 542.979 Td /F1 9.8 Tf [(, dark khaki; )] TJ ET BT 35.250 529.243 Td /F1 9.8 Tf [(negative control, orange. No fluorescence signals was measured for one minute \(after 3 minutes of the start of the reaction\) )] TJ ET BT 35.250 515.506 Td /F1 9.8 Tf [(because of the need of mixing the content.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 88.909 555.000 397.600 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 486.509 m 581.250 486.509 l 581.250 485.759 l 26.250 485.759 l f 26.250 88.909 m 581.250 88.909 l 581.250 89.659 l 26.250 89.659 l f q 225.000 0 0 290.250 35.250 186.509 cm /I10 Do Q q 35.250 100.159 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 170.985 Td /F4 9.8 Tf [(Fig. 2: Analytical sensitivity of MERS-CoV NC real-time RT-PCR and RT-RPA.)] TJ ET BT 35.250 151.615 Td /F1 9.8 Tf [(The analytical sensitivity was determined on RNA molecular standard \(8 runs\) for real-time RT-PCR \(A\) and real-time RT-)] TJ ET BT 35.250 137.879 Td /F1 9.8 Tf [(RPA \(B\). Both assays have a sensitivity of 10 RNA molecules. The RT-RPA assay was much faster than the RT-PCR as )] TJ ET BT 35.250 124.143 Td /F1 9.8 Tf [(the run time of the RT-RPA is between 3-7 minutes for 10)] TJ ET BT 283.436 129.863 Td /F1 8.7 Tf [(7 )] TJ ET BT 290.664 124.143 Td /F1 9.8 Tf [(and 10)] TJ ET BT 320.480 129.863 Td /F1 8.7 Tf [(1)] TJ ET BT 325.298 124.143 Td /F1 9.8 Tf [(molecules, respectively. While the RT-PCR needed up )] TJ ET BT 35.250 110.407 Td /F1 9.8 Tf [(to 2 hours. 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9'tt?B1NG1BU} U,Ə¤?tQRkQjw5J6TqGڗR=FeFb"nkMʀw9STqɣ>?_GD0t#W Ij"H`aj1Xr@DF_+JO4б@Q=#Ho_Jn! endstream endobj 259 0 obj << /Type /Page /Parent 3 0 R /Annots [ 261 0 R 263 0 R 265 0 R 267 0 R 269 0 R 271 0 R 273 0 R 275 0 R 277 0 R 279 0 R 281 0 R 283 0 R 285 0 R 287 0 R 289 0 R 291 0 R 293 0 R 295 0 R 297 0 R 299 0 R 301 0 R 303 0 R 305 0 R 307 0 R 309 0 R 311 0 R 313 0 R 315 0 R 317 0 R 319 0 R 321 0 R 323 0 R 325 0 R ] /Contents 260 0 R >> endobj 260 0 obj << /Length 44158 >> stream q 15.000 25.347 577.500 751.653 re W n 0.965 0.965 0.965 rg 26.250 712.873 555.000 64.127 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 712.873 m 581.250 712.873 l 581.250 713.623 l 26.250 713.623 l f q 35.250 724.123 537.000 52.877 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: Probit regression analysis of MERS-CoV RT-RPA assay)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(The probit analysis was performed using Statistica software on data of the eight runs of 10)] TJ ET BT 424.353 753.826 Td /F1 8.7 Tf [(7)] TJ ET BT 429.172 748.106 Td /F1 9.8 Tf [(-10)] TJ ET BT 443.260 753.826 Td /F1 8.7 Tf [(1 )] TJ ET BT 450.488 748.106 Td /F1 9.8 Tf [(RNA molecular standard. )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(The limit of detection at 95% probability \(21 RNA molecules\) is depicted by a Rhomboid.)] TJ ET Q BT 26.250 695.849 Td /F1 9.8 Tf [(Using a ten-fold serial dilution of RNA extracted from MERS-CoV tissue culture supernatant, the UpE real-time RT-PCR assays )] TJ ET 0.267 0.267 0.267 rg BT 26.250 685.451 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 35.887 683.944 Td /F1 9.8 Tf [( detected down to a dilution of 10)] TJ ET BT 178.978 687.832 Td /F1 8.7 Tf [(-6)] TJ ET BT 186.683 683.944 Td /F1 9.8 Tf [(, whereas the Orf1A )] TJ ET 0.267 0.267 0.267 rg BT 276.100 685.451 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 280.919 683.944 Td /F1 9.8 Tf [( and NC real-time RT-PCR assays as well as the RT-RPA assay )] TJ ET BT 26.250 672.039 Td /F1 9.8 Tf [(detected RNA down to a dilution of 10)] TJ ET BT 189.923 675.928 Td /F1 8.7 Tf [(-5)] TJ ET BT 197.628 672.039 Td /F1 9.8 Tf [(, corresponding to 7.5 genome equivalents/reaction \(3000 genome equivalents/ml\) )] TJ ET BT 26.250 660.135 Td /F1 9.8 Tf [(\(Table 3\). The RT-RPA assay specificity was determined using several coronaviruses and respiratory viruses \(Table 1\) as well )] TJ ET BT 26.250 648.230 Td /F1 9.8 Tf [(as human genome. None of them were detected.)] TJ ET q 26.250 624.613 555.000 13.736 re W n 0.271 0.267 0.267 rg BT 26.250 627.360 Td /F1 9.8 Tf [(Table 3. Sensitivity of RT-RPA assay in comparison with three real-time RT-PCR assays)] TJ ET Q 0.965 0.965 0.965 rg 26.250 583.937 555.000 33.175 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 617.113 m 581.250 617.113 l 580.500 616.363 l 27.000 616.363 l f 581.250 617.113 m 581.250 583.937 l 580.500 583.937 l 580.500 616.363 l f 26.250 617.113 m 26.250 583.937 l 27.000 583.937 l 27.000 616.363 l f 0.271 0.267 0.267 rg BT 33.000 602.548 Td /F1 9.0 Tf [(Ten-fold serial dilution of RNA extracted from MERS-CoV culture supernatant was used. UD is undetected; C)] TJ ET BT 467.160 600.032 Td /F1 8.0 Tf [(T)] TJ ET BT 472.048 602.548 Td /F1 9.0 Tf [(, cycle threshold; TT, )] TJ ET BT 33.000 593.269 Td /F1 9.0 Tf [(threshold time in minute)] TJ ET 1.000 1.000 1.000 rg 26.250 436.555 555.000 147.382 re f 0.267 0.267 0.267 rg 26.625 582.812 238.447 0.750 re f 26.625 567.306 0.750 16.256 re f 26.625 548.474 0.750 18.832 re f 26.625 532.218 0.750 16.256 re f 0.271 0.267 0.267 rg BT 31.875 572.957 Td /F4 9.8 Tf [(Genome equivalence/reaction)] TJ ET 0.267 0.267 0.267 rg 264.322 582.812 79.021 0.750 re f 343.343 582.812 80.810 0.750 re f 424.153 582.812 79.021 0.750 re f 264.322 566.931 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 572.957 Td /F4 9.8 Tf [(Real-time RT-PCR)] TJ ET 0.267 0.267 0.267 rg 502.424 582.812 78.451 0.750 re f 502.424 567.306 0.750 16.256 re f 580.125 567.306 0.750 16.256 re f 502.424 548.099 0.750 19.207 re f 580.125 548.099 0.750 19.207 re f 0.271 0.267 0.267 rg BT 507.674 572.957 Td /F4 9.8 Tf [(RT-RPA)] TJ ET 0.267 0.267 0.267 rg 264.322 566.931 79.396 0.750 re f 264.322 548.099 0.750 19.582 re f 0.271 0.267 0.267 rg BT 269.572 554.715 Td /F4 9.8 Tf [(UpE )] TJ ET 0.267 0.267 0.267 rg BT 291.783 555.164 Td /F4 8.7 Tf [(13)] TJ ET 342.968 566.931 81.560 0.750 re f 342.968 548.099 0.750 19.582 re f 0.271 0.267 0.267 rg BT 348.218 554.715 Td /F4 9.8 Tf [(Orf1A )] TJ ET 0.267 0.267 0.267 rg BT 378.014 555.164 Td /F4 8.7 Tf [(3)] TJ ET 423.778 566.931 79.396 0.750 re f 423.778 548.099 0.750 19.582 re f 0.271 0.267 0.267 rg BT 429.028 557.076 Td /F4 9.8 Tf [(NC)] TJ ET 0.267 0.267 0.267 rg 264.322 548.099 79.396 0.750 re f 264.322 532.218 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 538.244 Td /F4 9.8 Tf [(C? Mean)] TJ ET 0.267 0.267 0.267 rg 342.968 548.099 81.560 0.750 re f 342.968 532.218 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 538.244 Td /F4 9.8 Tf [(C? Mean)] TJ ET 0.267 0.267 0.267 rg 423.778 548.099 79.396 0.750 re f 423.778 532.218 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 538.244 Td /F4 9.8 Tf [(C? Mean)] TJ ET 0.267 0.267 0.267 rg 502.424 548.099 78.451 0.750 re f 502.424 532.218 0.750 16.631 re f 580.125 532.218 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 538.244 Td /F4 9.8 Tf [(TT Mean)] TJ ET 0.267 0.267 0.267 rg 26.625 532.218 238.447 0.750 re f 26.625 516.336 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 522.363 Td /F1 9.8 Tf [(9.92E+04)] TJ ET 0.267 0.267 0.267 rg 264.322 532.218 79.396 0.750 re f 264.322 516.336 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 522.363 Td /F1 9.8 Tf [(21.97)] TJ ET 0.267 0.267 0.267 rg 342.968 532.218 81.560 0.750 re f 342.968 516.336 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 522.363 Td /F1 9.8 Tf [(23.93)] TJ ET 0.267 0.267 0.267 rg 423.778 532.218 79.396 0.750 re f 423.778 516.336 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 522.363 Td /F1 9.8 Tf [(22.02)] TJ ET 0.267 0.267 0.267 rg 502.424 532.218 78.451 0.750 re f 502.424 516.336 0.750 16.631 re f 580.125 516.336 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 522.363 Td /F1 9.8 Tf [(4.30)] TJ ET 0.267 0.267 0.267 rg 26.625 516.336 238.447 0.750 re f 26.625 500.455 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 506.481 Td /F1 9.8 Tf [(1.23E+04)] TJ ET 0.267 0.267 0.267 rg 264.322 516.336 79.396 0.750 re f 264.322 500.455 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 506.481 Td /F1 9.8 Tf [(25.10)] TJ ET 0.267 0.267 0.267 rg 342.968 516.336 81.560 0.750 re f 342.968 500.455 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 506.481 Td /F1 9.8 Tf [(27.14)] TJ ET 0.267 0.267 0.267 rg 423.778 516.336 79.396 0.750 re f 423.778 500.455 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 506.481 Td /F1 9.8 Tf [(24.84)] TJ ET 0.267 0.267 0.267 rg 502.424 516.336 78.451 0.750 re f 502.424 500.455 0.750 16.631 re f 580.125 500.455 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 506.481 Td /F1 9.8 Tf [(4.70)] TJ ET 0.267 0.267 0.267 rg 26.625 500.455 238.447 0.750 re f 26.625 484.574 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 490.600 Td /F1 9.8 Tf [(1.30E+03)] TJ ET 0.267 0.267 0.267 rg 264.322 500.455 79.396 0.750 re f 264.322 484.574 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 490.600 Td /F1 9.8 Tf [(28.58)] TJ ET 0.267 0.267 0.267 rg 342.968 500.455 81.560 0.750 re f 342.968 484.574 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 490.600 Td /F1 9.8 Tf [(31.32)] TJ ET 0.267 0.267 0.267 rg 423.778 500.455 79.396 0.750 re f 423.778 484.574 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 490.600 Td /F1 9.8 Tf [(28.37)] TJ ET 0.267 0.267 0.267 rg 502.424 500.455 78.451 0.750 re f 502.424 484.574 0.750 16.631 re f 580.125 484.574 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 490.600 Td /F1 9.8 Tf [(5.00)] TJ ET 0.267 0.267 0.267 rg 26.625 484.574 238.447 0.750 re f 26.625 468.693 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 474.719 Td /F1 9.8 Tf [(9.66E+01)] TJ ET 0.267 0.267 0.267 rg 264.322 484.574 79.396 0.750 re f 264.322 468.693 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 474.719 Td /F1 9.8 Tf [(32.50)] TJ ET 0.267 0.267 0.267 rg 342.968 484.574 81.560 0.750 re f 342.968 468.693 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 474.719 Td /F1 9.8 Tf [(36.49)] TJ ET 0.267 0.267 0.267 rg 423.778 484.574 79.396 0.750 re f 423.778 468.693 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 474.719 Td /F1 9.8 Tf [(31.63)] TJ ET 0.267 0.267 0.267 rg 502.424 484.574 78.451 0.750 re f 502.424 468.693 0.750 16.631 re f 580.125 468.693 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 474.719 Td /F1 9.8 Tf [(5.50)] TJ ET 0.267 0.267 0.267 rg 26.625 468.693 238.447 0.750 re f 26.625 452.811 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 458.838 Td /F1 9.8 Tf [(7.5E+00)] TJ ET 0.267 0.267 0.267 rg 264.322 468.693 79.396 0.750 re f 264.322 452.811 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 458.838 Td /F1 9.8 Tf [(36.43)] TJ ET 0.267 0.267 0.267 rg 342.968 468.693 81.560 0.750 re f 342.968 452.811 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 458.838 Td /F1 9.8 Tf [(40.91)] TJ ET 0.267 0.267 0.267 rg 423.778 468.693 79.396 0.750 re f 423.778 452.811 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 458.838 Td /F1 9.8 Tf [(36.29)] TJ ET 0.267 0.267 0.267 rg 502.424 468.693 78.451 0.750 re f 502.424 452.811 0.750 16.631 re f 580.125 452.811 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 458.838 Td /F1 9.8 Tf [(6.30)] TJ ET 0.267 0.267 0.267 rg 26.625 452.811 238.447 0.750 re f 26.625 436.930 238.447 0.750 re f 26.625 436.930 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 442.956 Td /F1 9.8 Tf [(1.14E+00)] TJ ET 0.267 0.267 0.267 rg 264.322 452.811 79.396 0.750 re f 264.322 436.930 79.396 0.750 re f 264.322 436.930 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 442.956 Td /F1 9.8 Tf [(39.76)] TJ ET 0.267 0.267 0.267 rg 342.968 452.811 81.560 0.750 re f 342.968 436.930 81.560 0.750 re f 342.968 436.930 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 442.956 Td /F1 9.8 Tf [(UD)] TJ ET 0.267 0.267 0.267 rg 423.778 452.811 79.396 0.750 re f 423.778 436.930 79.396 0.750 re f 423.778 436.930 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 442.956 Td /F1 9.8 Tf [(UD)] TJ ET 0.267 0.267 0.267 rg 502.424 452.811 78.451 0.750 re f 502.424 436.930 78.451 0.750 re f 502.424 436.930 0.750 16.631 re f 580.125 436.930 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 442.956 Td /F1 9.8 Tf [(UD)] TJ ET BT 26.250 364.834 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 344.879 Td /F1 9.8 Tf [(The MERS-CoV causing a severe respiratory disease erupted in the Near East last year. In outbreak situations, rapid diagnostic )] TJ ET BT 26.250 332.975 Td /F1 9.8 Tf [(tools can generally help to confirm infection in suspected patients and to test contacts in order to contain spread of the )] TJ ET BT 26.250 321.070 Td /F1 9.8 Tf [(infectious agent. For wide scale testing, robust field-deployable test systems provide an advantage and allow testing in make )] TJ ET BT 26.250 309.165 Td /F1 9.8 Tf [(shift laboratories or at an outbreak site.)] TJ ET BT 26.250 289.760 Td /F1 9.8 Tf [(The performance of the isothermal RT-RPA assay for the detection of MERS-CoV RNA described here equals that of a )] TJ ET BT 26.250 277.856 Td /F1 9.8 Tf [(published real-time RT-PCR assay )] TJ ET 0.267 0.267 0.267 rg BT 179.032 279.363 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 183.851 277.856 Td /F1 9.8 Tf [( in terms of sensitivity and specificity at a run time of only 10 minutes. RNA concentrations )] TJ ET BT 26.250 265.951 Td /F1 9.8 Tf [(in samples collected from MERS-CoV-infected patient between days 11-16 of infection were 1.2 x10)] TJ ET BT 457.600 269.839 Td /F1 8.7 Tf [(6)] TJ ET BT 462.418 265.951 Td /F1 9.8 Tf [(, 5370, 2691, and 1031, )] TJ ET BT 26.250 254.046 Td /F1 9.8 Tf [(for lower respiratory tract, oronasal swabs, urine, and stool, respectively )] TJ ET 0.267 0.267 0.267 rg BT 338.903 255.553 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 348.541 254.046 Td /F1 9.8 Tf [(. Therefore, the detection limit of our RT-RPA assay )] TJ ET BT 26.250 242.141 Td /F1 9.8 Tf [(\(10 RNA copies\) is ideal for identifying MERS-CoV. Unfortunately, no clinical samples were available in our hand to test.)] TJ ET BT 26.250 222.737 Td /F1 9.8 Tf [(The run time of the RT-RPA was 10 minutes \(figure 1 and 2B\), and including nucleic acid extraction from samples, results can )] TJ ET BT 26.250 210.832 Td /F1 9.8 Tf [(therefore be obtained in about 30 minutes. Reagents are available as dried pellets \(cost 4 Euro/test\) and the detection device is )] TJ ET BT 26.250 198.927 Td /F1 9.8 Tf [(comparably cheap \(around 4500 Euro\). Rapid point-of-care diagnosis and field investigations are feasible as recent use of this )] TJ ET BT 26.250 187.022 Td /F1 9.8 Tf [(technology during the FMDV outbreak in Egypt in 2012 has shown )] TJ ET 0.267 0.267 0.267 rg BT 315.103 188.530 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 319.922 187.022 Td /F1 9.8 Tf [(. We recently successfully operated a mobile RT-RPA unit )] TJ ET BT 26.250 175.118 Td /F1 9.8 Tf [(consisting of a nucleic acid extraction kit, a RT-RPA kit, a pipette set, a magnetic separator, a tubescanner, and a laptop in )] TJ ET BT 26.250 163.213 Td /F1 9.8 Tf [(Kedougou, Senegal using a solar panel \(manuscript in preparation\). The experiences made indicate that mobile RT-RPA is a )] TJ ET BT 26.250 151.308 Td /F1 9.8 Tf [(very good alternative for mobile point-of-care detection, since infrastructure and equipment needs are below that of laboratory )] TJ ET BT 26.250 139.403 Td /F1 9.8 Tf [(based or mobile RT-PCR )] TJ ET 0.267 0.267 0.267 rg BT 137.858 140.911 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 142.677 143.292 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 145.086 140.911 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 149.905 143.292 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 152.314 140.911 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 157.133 139.403 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 119.999 Td /F1 9.8 Tf [(During the Hajj, around three million pilgrims from allover the world visit Mecca, Saudi Arabia for an average of ten days . )] TJ ET BT 26.250 108.094 Td /F1 9.8 Tf [(Physical stress on the pilgrims may increase the susceptibility to diseases )] TJ ET 0.267 0.267 0.267 rg BT 347.025 109.601 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 356.662 108.094 Td /F1 9.8 Tf [( and the density of the susceptibles might therefore )] TJ ET BT 26.250 96.189 Td /F1 9.8 Tf [(be conducive to transmission of MERS-CoV. Therefore, implementation of mobile RT-RPA units during the Hajj might be an )] TJ ET BT 26.250 84.284 Td /F1 9.8 Tf [(easy tool to detect MERS-CoV infected cases on site to identify acute cases and prevent the spread of the virus. RT-RPA might )] TJ ET BT 26.250 72.380 Td /F1 9.8 Tf [(also help to improve affordable monitoring for MERS-CoV by public health laboratories worldwide.)] TJ ET Q q 15.000 25.347 577.500 751.653 re W n 0.965 0.965 0.965 rg 26.250 712.873 555.000 64.127 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 712.873 m 581.250 712.873 l 581.250 713.623 l 26.250 713.623 l f q 35.250 724.123 537.000 52.877 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: Probit regression analysis of MERS-CoV RT-RPA assay)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(The probit analysis was performed using Statistica software on data of the eight runs of 10)] TJ ET BT 424.353 753.826 Td /F1 8.7 Tf [(7)] TJ ET BT 429.172 748.106 Td /F1 9.8 Tf [(-10)] TJ ET BT 443.260 753.826 Td /F1 8.7 Tf [(1 )] TJ ET BT 450.488 748.106 Td /F1 9.8 Tf [(RNA molecular standard. )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(The limit of detection at 95% probability \(21 RNA molecules\) is depicted by a Rhomboid.)] TJ ET Q BT 26.250 695.849 Td /F1 9.8 Tf [(Using a ten-fold serial dilution of RNA extracted from MERS-CoV tissue culture supernatant, the UpE real-time RT-PCR assays )] TJ ET 0.267 0.267 0.267 rg BT 26.250 685.451 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 35.887 683.944 Td /F1 9.8 Tf [( detected down to a dilution of 10)] TJ ET BT 178.978 687.832 Td /F1 8.7 Tf [(-6)] TJ ET BT 186.683 683.944 Td /F1 9.8 Tf [(, whereas the Orf1A )] TJ ET 0.267 0.267 0.267 rg BT 276.100 685.451 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 280.919 683.944 Td /F1 9.8 Tf [( and NC real-time RT-PCR assays as well as the RT-RPA assay )] TJ ET BT 26.250 672.039 Td /F1 9.8 Tf [(detected RNA down to a dilution of 10)] TJ ET BT 189.923 675.928 Td /F1 8.7 Tf [(-5)] TJ ET BT 197.628 672.039 Td /F1 9.8 Tf [(, corresponding to 7.5 genome equivalents/reaction \(3000 genome equivalents/ml\) )] TJ ET BT 26.250 660.135 Td /F1 9.8 Tf [(\(Table 3\). The RT-RPA assay specificity was determined using several coronaviruses and respiratory viruses \(Table 1\) as well )] TJ ET BT 26.250 648.230 Td /F1 9.8 Tf [(as human genome. None of them were detected.)] TJ ET q 26.250 624.613 555.000 13.736 re W n 0.271 0.267 0.267 rg BT 26.250 627.360 Td /F1 9.8 Tf [(Table 3. Sensitivity of RT-RPA assay in comparison with three real-time RT-PCR assays)] TJ ET Q 0.965 0.965 0.965 rg 26.250 583.937 555.000 33.175 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 617.113 m 581.250 617.113 l 580.500 616.363 l 27.000 616.363 l f 581.250 617.113 m 581.250 583.937 l 580.500 583.937 l 580.500 616.363 l f 26.250 617.113 m 26.250 583.937 l 27.000 583.937 l 27.000 616.363 l f 0.271 0.267 0.267 rg BT 33.000 602.548 Td /F1 9.0 Tf [(Ten-fold serial dilution of RNA extracted from MERS-CoV culture supernatant was used. UD is undetected; C)] TJ ET BT 467.160 600.032 Td /F1 8.0 Tf [(T)] TJ ET BT 472.048 602.548 Td /F1 9.0 Tf [(, cycle threshold; TT, )] TJ ET BT 33.000 593.269 Td /F1 9.0 Tf [(threshold time in minute)] TJ ET 1.000 1.000 1.000 rg 26.250 436.555 555.000 147.382 re f 0.267 0.267 0.267 rg 26.625 582.812 238.447 0.750 re f 26.625 567.306 0.750 16.256 re f 26.625 548.474 0.750 18.832 re f 26.625 532.218 0.750 16.256 re f 0.271 0.267 0.267 rg BT 31.875 572.957 Td /F4 9.8 Tf [(Genome equivalence/reaction)] TJ ET 0.267 0.267 0.267 rg 264.322 582.812 79.021 0.750 re f 343.343 582.812 80.810 0.750 re f 424.153 582.812 79.021 0.750 re f 264.322 566.931 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 572.957 Td /F4 9.8 Tf [(Real-time RT-PCR)] TJ ET 0.267 0.267 0.267 rg 502.424 582.812 78.451 0.750 re f 502.424 567.306 0.750 16.256 re f 580.125 567.306 0.750 16.256 re f 502.424 548.099 0.750 19.207 re f 580.125 548.099 0.750 19.207 re f 0.271 0.267 0.267 rg BT 507.674 572.957 Td /F4 9.8 Tf [(RT-RPA)] TJ ET 0.267 0.267 0.267 rg 264.322 566.931 79.396 0.750 re f 264.322 548.099 0.750 19.582 re f 0.271 0.267 0.267 rg BT 269.572 554.715 Td /F4 9.8 Tf [(UpE )] TJ ET 0.267 0.267 0.267 rg BT 291.783 555.164 Td /F4 8.7 Tf [(13)] TJ ET 342.968 566.931 81.560 0.750 re f 342.968 548.099 0.750 19.582 re f 0.271 0.267 0.267 rg BT 348.218 554.715 Td /F4 9.8 Tf [(Orf1A )] TJ ET 0.267 0.267 0.267 rg BT 378.014 555.164 Td /F4 8.7 Tf [(3)] TJ ET 423.778 566.931 79.396 0.750 re f 423.778 548.099 0.750 19.582 re f 0.271 0.267 0.267 rg BT 429.028 557.076 Td /F4 9.8 Tf [(NC)] TJ ET 0.267 0.267 0.267 rg 264.322 548.099 79.396 0.750 re f 264.322 532.218 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 538.244 Td /F4 9.8 Tf [(C? Mean)] TJ ET 0.267 0.267 0.267 rg 342.968 548.099 81.560 0.750 re f 342.968 532.218 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 538.244 Td /F4 9.8 Tf [(C? Mean)] TJ ET 0.267 0.267 0.267 rg 423.778 548.099 79.396 0.750 re f 423.778 532.218 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 538.244 Td /F4 9.8 Tf [(C? Mean)] TJ ET 0.267 0.267 0.267 rg 502.424 548.099 78.451 0.750 re f 502.424 532.218 0.750 16.631 re f 580.125 532.218 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 538.244 Td /F4 9.8 Tf [(TT Mean)] TJ ET 0.267 0.267 0.267 rg 26.625 532.218 238.447 0.750 re f 26.625 516.336 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 522.363 Td /F1 9.8 Tf [(9.92E+04)] TJ ET 0.267 0.267 0.267 rg 264.322 532.218 79.396 0.750 re f 264.322 516.336 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 522.363 Td /F1 9.8 Tf [(21.97)] TJ ET 0.267 0.267 0.267 rg 342.968 532.218 81.560 0.750 re f 342.968 516.336 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 522.363 Td /F1 9.8 Tf [(23.93)] TJ ET 0.267 0.267 0.267 rg 423.778 532.218 79.396 0.750 re f 423.778 516.336 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 522.363 Td /F1 9.8 Tf [(22.02)] TJ ET 0.267 0.267 0.267 rg 502.424 532.218 78.451 0.750 re f 502.424 516.336 0.750 16.631 re f 580.125 516.336 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 522.363 Td /F1 9.8 Tf [(4.30)] TJ ET 0.267 0.267 0.267 rg 26.625 516.336 238.447 0.750 re f 26.625 500.455 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 506.481 Td /F1 9.8 Tf [(1.23E+04)] TJ ET 0.267 0.267 0.267 rg 264.322 516.336 79.396 0.750 re f 264.322 500.455 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 506.481 Td /F1 9.8 Tf [(25.10)] TJ ET 0.267 0.267 0.267 rg 342.968 516.336 81.560 0.750 re f 342.968 500.455 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 506.481 Td /F1 9.8 Tf [(27.14)] TJ ET 0.267 0.267 0.267 rg 423.778 516.336 79.396 0.750 re f 423.778 500.455 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 506.481 Td /F1 9.8 Tf [(24.84)] TJ ET 0.267 0.267 0.267 rg 502.424 516.336 78.451 0.750 re f 502.424 500.455 0.750 16.631 re f 580.125 500.455 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 506.481 Td /F1 9.8 Tf [(4.70)] TJ ET 0.267 0.267 0.267 rg 26.625 500.455 238.447 0.750 re f 26.625 484.574 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 490.600 Td /F1 9.8 Tf [(1.30E+03)] TJ ET 0.267 0.267 0.267 rg 264.322 500.455 79.396 0.750 re f 264.322 484.574 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 490.600 Td /F1 9.8 Tf [(28.58)] TJ ET 0.267 0.267 0.267 rg 342.968 500.455 81.560 0.750 re f 342.968 484.574 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 490.600 Td /F1 9.8 Tf [(31.32)] TJ ET 0.267 0.267 0.267 rg 423.778 500.455 79.396 0.750 re f 423.778 484.574 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 490.600 Td /F1 9.8 Tf [(28.37)] TJ ET 0.267 0.267 0.267 rg 502.424 500.455 78.451 0.750 re f 502.424 484.574 0.750 16.631 re f 580.125 484.574 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 490.600 Td /F1 9.8 Tf [(5.00)] TJ ET 0.267 0.267 0.267 rg 26.625 484.574 238.447 0.750 re f 26.625 468.693 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 474.719 Td /F1 9.8 Tf [(9.66E+01)] TJ ET 0.267 0.267 0.267 rg 264.322 484.574 79.396 0.750 re f 264.322 468.693 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 474.719 Td /F1 9.8 Tf [(32.50)] TJ ET 0.267 0.267 0.267 rg 342.968 484.574 81.560 0.750 re f 342.968 468.693 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 474.719 Td /F1 9.8 Tf [(36.49)] TJ ET 0.267 0.267 0.267 rg 423.778 484.574 79.396 0.750 re f 423.778 468.693 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 474.719 Td /F1 9.8 Tf [(31.63)] TJ ET 0.267 0.267 0.267 rg 502.424 484.574 78.451 0.750 re f 502.424 468.693 0.750 16.631 re f 580.125 468.693 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 474.719 Td /F1 9.8 Tf [(5.50)] TJ ET 0.267 0.267 0.267 rg 26.625 468.693 238.447 0.750 re f 26.625 452.811 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 458.838 Td /F1 9.8 Tf [(7.5E+00)] TJ ET 0.267 0.267 0.267 rg 264.322 468.693 79.396 0.750 re f 264.322 452.811 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 458.838 Td /F1 9.8 Tf [(36.43)] TJ ET 0.267 0.267 0.267 rg 342.968 468.693 81.560 0.750 re f 342.968 452.811 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 458.838 Td /F1 9.8 Tf [(40.91)] TJ ET 0.267 0.267 0.267 rg 423.778 468.693 79.396 0.750 re f 423.778 452.811 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 458.838 Td /F1 9.8 Tf [(36.29)] TJ ET 0.267 0.267 0.267 rg 502.424 468.693 78.451 0.750 re f 502.424 452.811 0.750 16.631 re f 580.125 452.811 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 458.838 Td /F1 9.8 Tf [(6.30)] TJ ET 0.267 0.267 0.267 rg 26.625 452.811 238.447 0.750 re f 26.625 436.930 238.447 0.750 re f 26.625 436.930 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 442.956 Td /F1 9.8 Tf [(1.14E+00)] TJ ET 0.267 0.267 0.267 rg 264.322 452.811 79.396 0.750 re f 264.322 436.930 79.396 0.750 re f 264.322 436.930 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 442.956 Td /F1 9.8 Tf [(39.76)] TJ ET 0.267 0.267 0.267 rg 342.968 452.811 81.560 0.750 re f 342.968 436.930 81.560 0.750 re f 342.968 436.930 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 442.956 Td /F1 9.8 Tf [(UD)] TJ ET 0.267 0.267 0.267 rg 423.778 452.811 79.396 0.750 re f 423.778 436.930 79.396 0.750 re f 423.778 436.930 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 442.956 Td /F1 9.8 Tf [(UD)] TJ ET 0.267 0.267 0.267 rg 502.424 452.811 78.451 0.750 re f 502.424 436.930 78.451 0.750 re f 502.424 436.930 0.750 16.631 re f 580.125 436.930 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 442.956 Td /F1 9.8 Tf [(UD)] TJ ET BT 26.250 364.834 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 344.879 Td /F1 9.8 Tf [(The MERS-CoV causing a severe respiratory disease erupted in the Near East last year. In outbreak situations, rapid diagnostic )] TJ ET BT 26.250 332.975 Td /F1 9.8 Tf [(tools can generally help to confirm infection in suspected patients and to test contacts in order to contain spread of the )] TJ ET BT 26.250 321.070 Td /F1 9.8 Tf [(infectious agent. For wide scale testing, robust field-deployable test systems provide an advantage and allow testing in make )] TJ ET BT 26.250 309.165 Td /F1 9.8 Tf [(shift laboratories or at an outbreak site.)] TJ ET BT 26.250 289.760 Td /F1 9.8 Tf [(The performance of the isothermal RT-RPA assay for the detection of MERS-CoV RNA described here equals that of a )] TJ ET BT 26.250 277.856 Td /F1 9.8 Tf [(published real-time RT-PCR assay )] TJ ET 0.267 0.267 0.267 rg BT 179.032 279.363 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 183.851 277.856 Td /F1 9.8 Tf [( in terms of sensitivity and specificity at a run time of only 10 minutes. RNA concentrations )] TJ ET BT 26.250 265.951 Td /F1 9.8 Tf [(in samples collected from MERS-CoV-infected patient between days 11-16 of infection were 1.2 x10)] TJ ET BT 457.600 269.839 Td /F1 8.7 Tf [(6)] TJ ET BT 462.418 265.951 Td /F1 9.8 Tf [(, 5370, 2691, and 1031, )] TJ ET BT 26.250 254.046 Td /F1 9.8 Tf [(for lower respiratory tract, oronasal swabs, urine, and stool, respectively )] TJ ET 0.267 0.267 0.267 rg BT 338.903 255.553 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 348.541 254.046 Td /F1 9.8 Tf [(. Therefore, the detection limit of our RT-RPA assay )] TJ ET BT 26.250 242.141 Td /F1 9.8 Tf [(\(10 RNA copies\) is ideal for identifying MERS-CoV. Unfortunately, no clinical samples were available in our hand to test.)] TJ ET BT 26.250 222.737 Td /F1 9.8 Tf [(The run time of the RT-RPA was 10 minutes \(figure 1 and 2B\), and including nucleic acid extraction from samples, results can )] TJ ET BT 26.250 210.832 Td /F1 9.8 Tf [(therefore be obtained in about 30 minutes. Reagents are available as dried pellets \(cost 4 Euro/test\) and the detection device is )] TJ ET BT 26.250 198.927 Td /F1 9.8 Tf [(comparably cheap \(around 4500 Euro\). Rapid point-of-care diagnosis and field investigations are feasible as recent use of this )] TJ ET BT 26.250 187.022 Td /F1 9.8 Tf [(technology during the FMDV outbreak in Egypt in 2012 has shown )] TJ ET 0.267 0.267 0.267 rg BT 315.103 188.530 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 319.922 187.022 Td /F1 9.8 Tf [(. We recently successfully operated a mobile RT-RPA unit )] TJ ET BT 26.250 175.118 Td /F1 9.8 Tf [(consisting of a nucleic acid extraction kit, a RT-RPA kit, a pipette set, a magnetic separator, a tubescanner, and a laptop in )] TJ ET BT 26.250 163.213 Td /F1 9.8 Tf [(Kedougou, Senegal using a solar panel \(manuscript in preparation\). The experiences made indicate that mobile RT-RPA is a )] TJ ET BT 26.250 151.308 Td /F1 9.8 Tf [(very good alternative for mobile point-of-care detection, since infrastructure and equipment needs are below that of laboratory )] TJ ET BT 26.250 139.403 Td /F1 9.8 Tf [(based or mobile RT-PCR )] TJ ET 0.267 0.267 0.267 rg BT 137.858 140.911 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 142.677 143.292 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 145.086 140.911 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 149.905 143.292 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 152.314 140.911 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 157.133 139.403 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 119.999 Td /F1 9.8 Tf [(During the Hajj, around three million pilgrims from allover the world visit Mecca, Saudi Arabia for an average of ten days . )] TJ ET BT 26.250 108.094 Td /F1 9.8 Tf [(Physical stress on the pilgrims may increase the susceptibility to diseases )] TJ ET 0.267 0.267 0.267 rg BT 347.025 109.601 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 356.662 108.094 Td /F1 9.8 Tf [( and the density of the susceptibles might therefore )] TJ ET BT 26.250 96.189 Td /F1 9.8 Tf [(be conducive to transmission of MERS-CoV. Therefore, implementation of mobile RT-RPA units during the Hajj might be an )] TJ ET BT 26.250 84.284 Td /F1 9.8 Tf [(easy tool to detect MERS-CoV infected cases on site to identify acute cases and prevent the spread of the virus. RT-RPA might )] TJ ET BT 26.250 72.380 Td /F1 9.8 Tf [(also help to improve affordable monitoring for MERS-CoV by public health laboratories worldwide.)] TJ ET Q q 15.000 25.347 577.500 751.653 re W n 0.965 0.965 0.965 rg 26.250 712.873 555.000 64.127 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 712.873 m 581.250 712.873 l 581.250 713.623 l 26.250 713.623 l f q 35.250 724.123 537.000 52.877 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: Probit regression analysis of MERS-CoV RT-RPA assay)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(The probit analysis was performed using Statistica software on data of the eight runs of 10)] TJ ET BT 424.353 753.826 Td /F1 8.7 Tf [(7)] TJ ET BT 429.172 748.106 Td /F1 9.8 Tf [(-10)] TJ ET BT 443.260 753.826 Td /F1 8.7 Tf [(1 )] TJ ET BT 450.488 748.106 Td /F1 9.8 Tf [(RNA molecular standard. )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(The limit of detection at 95% probability \(21 RNA molecules\) is depicted by a Rhomboid.)] TJ ET Q BT 26.250 695.849 Td /F1 9.8 Tf [(Using a ten-fold serial dilution of RNA extracted from MERS-CoV tissue culture supernatant, the UpE real-time RT-PCR assays )] TJ ET 0.267 0.267 0.267 rg BT 26.250 685.451 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 35.887 683.944 Td /F1 9.8 Tf [( detected down to a dilution of 10)] TJ ET BT 178.978 687.832 Td /F1 8.7 Tf [(-6)] TJ ET BT 186.683 683.944 Td /F1 9.8 Tf [(, whereas the Orf1A )] TJ ET 0.267 0.267 0.267 rg BT 276.100 685.451 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 280.919 683.944 Td /F1 9.8 Tf [( and NC real-time RT-PCR assays as well as the RT-RPA assay )] TJ ET BT 26.250 672.039 Td /F1 9.8 Tf [(detected RNA down to a dilution of 10)] TJ ET BT 189.923 675.928 Td /F1 8.7 Tf [(-5)] TJ ET BT 197.628 672.039 Td /F1 9.8 Tf [(, corresponding to 7.5 genome equivalents/reaction \(3000 genome equivalents/ml\) )] TJ ET BT 26.250 660.135 Td /F1 9.8 Tf [(\(Table 3\). The RT-RPA assay specificity was determined using several coronaviruses and respiratory viruses \(Table 1\) as well )] TJ ET BT 26.250 648.230 Td /F1 9.8 Tf [(as human genome. None of them were detected.)] TJ ET q 26.250 624.613 555.000 13.736 re W n 0.271 0.267 0.267 rg BT 26.250 627.360 Td /F1 9.8 Tf [(Table 3. Sensitivity of RT-RPA assay in comparison with three real-time RT-PCR assays)] TJ ET Q 0.965 0.965 0.965 rg 26.250 583.937 555.000 33.175 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 617.113 m 581.250 617.113 l 580.500 616.363 l 27.000 616.363 l f 581.250 617.113 m 581.250 583.937 l 580.500 583.937 l 580.500 616.363 l f 26.250 617.113 m 26.250 583.937 l 27.000 583.937 l 27.000 616.363 l f 0.271 0.267 0.267 rg BT 33.000 602.548 Td /F1 9.0 Tf [(Ten-fold serial dilution of RNA extracted from MERS-CoV culture supernatant was used. UD is undetected; C)] TJ ET BT 467.160 600.032 Td /F1 8.0 Tf [(T)] TJ ET BT 472.048 602.548 Td /F1 9.0 Tf [(, cycle threshold; TT, )] TJ ET BT 33.000 593.269 Td /F1 9.0 Tf [(threshold time in minute)] TJ ET 1.000 1.000 1.000 rg 26.250 436.555 555.000 147.382 re f 0.267 0.267 0.267 rg 26.625 582.812 238.447 0.750 re f 26.625 567.306 0.750 16.256 re f 26.625 548.474 0.750 18.832 re f 26.625 532.218 0.750 16.256 re f 0.271 0.267 0.267 rg BT 31.875 572.957 Td /F4 9.8 Tf [(Genome equivalence/reaction)] TJ ET 0.267 0.267 0.267 rg 264.322 582.812 79.021 0.750 re f 343.343 582.812 80.810 0.750 re f 424.153 582.812 79.021 0.750 re f 264.322 566.931 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 572.957 Td /F4 9.8 Tf [(Real-time RT-PCR)] TJ ET 0.267 0.267 0.267 rg 502.424 582.812 78.451 0.750 re f 502.424 567.306 0.750 16.256 re f 580.125 567.306 0.750 16.256 re f 502.424 548.099 0.750 19.207 re f 580.125 548.099 0.750 19.207 re f 0.271 0.267 0.267 rg BT 507.674 572.957 Td /F4 9.8 Tf [(RT-RPA)] TJ ET 0.267 0.267 0.267 rg 264.322 566.931 79.396 0.750 re f 264.322 548.099 0.750 19.582 re f 0.271 0.267 0.267 rg BT 269.572 554.715 Td /F4 9.8 Tf [(UpE )] TJ ET 0.267 0.267 0.267 rg BT 291.783 555.164 Td /F4 8.7 Tf [(13)] TJ ET 342.968 566.931 81.560 0.750 re f 342.968 548.099 0.750 19.582 re f 0.271 0.267 0.267 rg BT 348.218 554.715 Td /F4 9.8 Tf [(Orf1A )] TJ ET 0.267 0.267 0.267 rg BT 378.014 555.164 Td /F4 8.7 Tf [(3)] TJ ET 423.778 566.931 79.396 0.750 re f 423.778 548.099 0.750 19.582 re f 0.271 0.267 0.267 rg BT 429.028 557.076 Td /F4 9.8 Tf [(NC)] TJ ET 0.267 0.267 0.267 rg 264.322 548.099 79.396 0.750 re f 264.322 532.218 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 538.244 Td /F4 9.8 Tf [(C? Mean)] TJ ET 0.267 0.267 0.267 rg 342.968 548.099 81.560 0.750 re f 342.968 532.218 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 538.244 Td /F4 9.8 Tf [(C? Mean)] TJ ET 0.267 0.267 0.267 rg 423.778 548.099 79.396 0.750 re f 423.778 532.218 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 538.244 Td /F4 9.8 Tf [(C? Mean)] TJ ET 0.267 0.267 0.267 rg 502.424 548.099 78.451 0.750 re f 502.424 532.218 0.750 16.631 re f 580.125 532.218 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 538.244 Td /F4 9.8 Tf [(TT Mean)] TJ ET 0.267 0.267 0.267 rg 26.625 532.218 238.447 0.750 re f 26.625 516.336 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 522.363 Td /F1 9.8 Tf [(9.92E+04)] TJ ET 0.267 0.267 0.267 rg 264.322 532.218 79.396 0.750 re f 264.322 516.336 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 522.363 Td /F1 9.8 Tf [(21.97)] TJ ET 0.267 0.267 0.267 rg 342.968 532.218 81.560 0.750 re f 342.968 516.336 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 522.363 Td /F1 9.8 Tf [(23.93)] TJ ET 0.267 0.267 0.267 rg 423.778 532.218 79.396 0.750 re f 423.778 516.336 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 522.363 Td /F1 9.8 Tf [(22.02)] TJ ET 0.267 0.267 0.267 rg 502.424 532.218 78.451 0.750 re f 502.424 516.336 0.750 16.631 re f 580.125 516.336 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 522.363 Td /F1 9.8 Tf [(4.30)] TJ ET 0.267 0.267 0.267 rg 26.625 516.336 238.447 0.750 re f 26.625 500.455 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 506.481 Td /F1 9.8 Tf [(1.23E+04)] TJ ET 0.267 0.267 0.267 rg 264.322 516.336 79.396 0.750 re f 264.322 500.455 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 506.481 Td /F1 9.8 Tf [(25.10)] TJ ET 0.267 0.267 0.267 rg 342.968 516.336 81.560 0.750 re f 342.968 500.455 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 506.481 Td /F1 9.8 Tf [(27.14)] TJ ET 0.267 0.267 0.267 rg 423.778 516.336 79.396 0.750 re f 423.778 500.455 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 506.481 Td /F1 9.8 Tf [(24.84)] TJ ET 0.267 0.267 0.267 rg 502.424 516.336 78.451 0.750 re f 502.424 500.455 0.750 16.631 re f 580.125 500.455 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 506.481 Td /F1 9.8 Tf [(4.70)] TJ ET 0.267 0.267 0.267 rg 26.625 500.455 238.447 0.750 re f 26.625 484.574 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 490.600 Td /F1 9.8 Tf [(1.30E+03)] TJ ET 0.267 0.267 0.267 rg 264.322 500.455 79.396 0.750 re f 264.322 484.574 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 490.600 Td /F1 9.8 Tf [(28.58)] TJ ET 0.267 0.267 0.267 rg 342.968 500.455 81.560 0.750 re f 342.968 484.574 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 490.600 Td /F1 9.8 Tf [(31.32)] TJ ET 0.267 0.267 0.267 rg 423.778 500.455 79.396 0.750 re f 423.778 484.574 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 490.600 Td /F1 9.8 Tf [(28.37)] TJ ET 0.267 0.267 0.267 rg 502.424 500.455 78.451 0.750 re f 502.424 484.574 0.750 16.631 re f 580.125 484.574 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 490.600 Td /F1 9.8 Tf [(5.00)] TJ ET 0.267 0.267 0.267 rg 26.625 484.574 238.447 0.750 re f 26.625 468.693 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 474.719 Td /F1 9.8 Tf [(9.66E+01)] TJ ET 0.267 0.267 0.267 rg 264.322 484.574 79.396 0.750 re f 264.322 468.693 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 474.719 Td /F1 9.8 Tf [(32.50)] TJ ET 0.267 0.267 0.267 rg 342.968 484.574 81.560 0.750 re f 342.968 468.693 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 474.719 Td /F1 9.8 Tf [(36.49)] TJ ET 0.267 0.267 0.267 rg 423.778 484.574 79.396 0.750 re f 423.778 468.693 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 474.719 Td /F1 9.8 Tf [(31.63)] TJ ET 0.267 0.267 0.267 rg 502.424 484.574 78.451 0.750 re f 502.424 468.693 0.750 16.631 re f 580.125 468.693 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 474.719 Td /F1 9.8 Tf [(5.50)] TJ ET 0.267 0.267 0.267 rg 26.625 468.693 238.447 0.750 re f 26.625 452.811 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 458.838 Td /F1 9.8 Tf [(7.5E+00)] TJ ET 0.267 0.267 0.267 rg 264.322 468.693 79.396 0.750 re f 264.322 452.811 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 458.838 Td /F1 9.8 Tf [(36.43)] TJ ET 0.267 0.267 0.267 rg 342.968 468.693 81.560 0.750 re f 342.968 452.811 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 458.838 Td /F1 9.8 Tf [(40.91)] TJ ET 0.267 0.267 0.267 rg 423.778 468.693 79.396 0.750 re f 423.778 452.811 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 458.838 Td /F1 9.8 Tf [(36.29)] TJ ET 0.267 0.267 0.267 rg 502.424 468.693 78.451 0.750 re f 502.424 452.811 0.750 16.631 re f 580.125 452.811 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 458.838 Td /F1 9.8 Tf [(6.30)] TJ ET 0.267 0.267 0.267 rg 26.625 452.811 238.447 0.750 re f 26.625 436.930 238.447 0.750 re f 26.625 436.930 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 442.956 Td /F1 9.8 Tf [(1.14E+00)] TJ ET 0.267 0.267 0.267 rg 264.322 452.811 79.396 0.750 re f 264.322 436.930 79.396 0.750 re f 264.322 436.930 0.750 16.631 re f 0.271 0.267 0.267 rg BT 269.572 442.956 Td /F1 9.8 Tf [(39.76)] TJ ET 0.267 0.267 0.267 rg 342.968 452.811 81.560 0.750 re f 342.968 436.930 81.560 0.750 re f 342.968 436.930 0.750 16.631 re f 0.271 0.267 0.267 rg BT 348.218 442.956 Td /F1 9.8 Tf [(UD)] TJ ET 0.267 0.267 0.267 rg 423.778 452.811 79.396 0.750 re f 423.778 436.930 79.396 0.750 re f 423.778 436.930 0.750 16.631 re f 0.271 0.267 0.267 rg BT 429.028 442.956 Td /F1 9.8 Tf [(UD)] TJ ET 0.267 0.267 0.267 rg 502.424 452.811 78.451 0.750 re f 502.424 436.930 78.451 0.750 re f 502.424 436.930 0.750 16.631 re f 580.125 436.930 0.750 16.631 re f 0.271 0.267 0.267 rg BT 507.674 442.956 Td /F1 9.8 Tf [(UD)] TJ ET BT 26.250 364.834 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 344.879 Td /F1 9.8 Tf [(The MERS-CoV causing a severe respiratory disease erupted in the Near East last year. In outbreak situations, rapid diagnostic )] TJ ET BT 26.250 332.975 Td /F1 9.8 Tf [(tools can generally help to confirm infection in suspected patients and to test contacts in order to contain spread of the )] TJ ET BT 26.250 321.070 Td /F1 9.8 Tf [(infectious agent. For wide scale testing, robust field-deployable test systems provide an advantage and allow testing in make )] TJ ET BT 26.250 309.165 Td /F1 9.8 Tf [(shift laboratories or at an outbreak site.)] TJ ET BT 26.250 289.760 Td /F1 9.8 Tf [(The performance of the isothermal RT-RPA assay for the detection of MERS-CoV RNA described here equals that of a )] TJ ET BT 26.250 277.856 Td /F1 9.8 Tf [(published real-time RT-PCR assay )] TJ ET 0.267 0.267 0.267 rg BT 179.032 279.363 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 183.851 277.856 Td /F1 9.8 Tf [( in terms of sensitivity and specificity at a run time of only 10 minutes. RNA concentrations )] TJ ET BT 26.250 265.951 Td /F1 9.8 Tf [(in samples collected from MERS-CoV-infected patient between days 11-16 of infection were 1.2 x10)] TJ ET BT 457.600 269.839 Td /F1 8.7 Tf [(6)] TJ ET BT 462.418 265.951 Td /F1 9.8 Tf [(, 5370, 2691, and 1031, )] TJ ET BT 26.250 254.046 Td /F1 9.8 Tf [(for lower respiratory tract, oronasal swabs, urine, and stool, respectively )] TJ ET 0.267 0.267 0.267 rg BT 338.903 255.553 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 348.541 254.046 Td /F1 9.8 Tf [(. Therefore, the detection limit of our RT-RPA assay )] TJ ET BT 26.250 242.141 Td /F1 9.8 Tf [(\(10 RNA copies\) is ideal for identifying MERS-CoV. Unfortunately, no clinical samples were available in our hand to test.)] TJ ET BT 26.250 222.737 Td /F1 9.8 Tf [(The run time of the RT-RPA was 10 minutes \(figure 1 and 2B\), and including nucleic acid extraction from samples, results can )] TJ ET BT 26.250 210.832 Td /F1 9.8 Tf [(therefore be obtained in about 30 minutes. Reagents are available as dried pellets \(cost 4 Euro/test\) and the detection device is )] TJ ET BT 26.250 198.927 Td /F1 9.8 Tf [(comparably cheap \(around 4500 Euro\). Rapid point-of-care diagnosis and field investigations are feasible as recent use of this )] TJ ET BT 26.250 187.022 Td /F1 9.8 Tf [(technology during the FMDV outbreak in Egypt in 2012 has shown )] TJ ET 0.267 0.267 0.267 rg BT 315.103 188.530 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 319.922 187.022 Td /F1 9.8 Tf [(. We recently successfully operated a mobile RT-RPA unit )] TJ ET BT 26.250 175.118 Td /F1 9.8 Tf [(consisting of a nucleic acid extraction kit, a RT-RPA kit, a pipette set, a magnetic separator, a tubescanner, and a laptop in )] TJ ET BT 26.250 163.213 Td /F1 9.8 Tf [(Kedougou, Senegal using a solar panel \(manuscript in preparation\). The experiences made indicate that mobile RT-RPA is a )] TJ ET BT 26.250 151.308 Td /F1 9.8 Tf [(very good alternative for mobile point-of-care detection, since infrastructure and equipment needs are below that of laboratory )] TJ ET BT 26.250 139.403 Td /F1 9.8 Tf [(based or mobile RT-PCR )] TJ ET 0.267 0.267 0.267 rg BT 137.858 140.911 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 142.677 143.292 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 145.086 140.911 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 149.905 143.292 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 152.314 140.911 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 157.133 139.403 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 119.999 Td /F1 9.8 Tf [(During the Hajj, around three million pilgrims from allover the world visit Mecca, Saudi Arabia for an average of ten days . )] TJ ET BT 26.250 108.094 Td /F1 9.8 Tf [(Physical stress on the pilgrims may increase the susceptibility to diseases )] TJ ET 0.267 0.267 0.267 rg BT 347.025 109.601 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 356.662 108.094 Td /F1 9.8 Tf [( and the density of the susceptibles might therefore )] TJ ET BT 26.250 96.189 Td /F1 9.8 Tf [(be conducive to transmission of MERS-CoV. Therefore, implementation of mobile RT-RPA units during the Hajj might be an )] TJ ET BT 26.250 84.284 Td /F1 9.8 Tf [(easy tool to detect MERS-CoV infected cases on site to identify acute cases and prevent the spread of the virus. RT-RPA might )] TJ ET BT 26.250 72.380 Td /F1 9.8 Tf [(also help to improve affordable monitoring for MERS-CoV by public health laboratories worldwide.)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(5)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Outbreaks)] TJ ET Q endstream endobj 261 0 obj << /Type /Annot /Subtype /Link /A 262 0 R /Border [0 0 0] /H /I /Rect [ 26.2500 684.6497 35.8873 693.4680 ] >> endobj 262 0 obj << /Type /Action >> endobj 263 0 obj << /Type /Annot /Subtype /Link /A 264 0 R /Border [0 0 0] /H /I /Rect [ 276.1003 684.6497 280.9189 693.4680 ] >> endobj 264 0 obj << /Type /Action >> endobj 265 0 obj << /Type /Annot /Subtype /Link /A 266 0 R /Border [0 0 0] /H /I /Rect [ 291.7826 554.3627 301.4199 563.1810 ] >> endobj 266 0 obj << /Type /Action >> endobj 267 0 obj << /Type /Annot /Subtype /Link /A 268 0 R /Border [0 0 0] /H /I /Rect [ 378.0138 554.3627 382.8325 563.1810 ] >> endobj 268 0 obj << /Type /Action >> endobj 269 0 obj << /Type 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0.267 RG q 15.000 34.946 577.500 742.054 re W n 0.271 0.267 0.267 rg BT 26.250 750.278 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 730.324 Td /F1 9.8 Tf [(Authors thank Ron Fouchier, Erasmus University, Rotterdam, Netherlands for providing the MERS-CoV isolate and Christian )] TJ ET BT 26.250 718.419 Td /F1 9.8 Tf [(Drosten for genome analysis. The MERS-CoV sample preparation was performed at the Robert Koch-Institut in Berlin, Germany )] TJ ET BT 26.250 706.515 Td /F1 9.8 Tf [(under the ECDC Framework Service Contract Ref. No. ECDC/2008/011. The respiratory virus panel for cross reactivity was )] TJ ET BT 26.250 694.610 Td /F1 9.8 Tf [(provided by Landesgesundheitsamt Niedersachsen, Germany and Brunhilde Schweiger, National Reference Center for )] TJ ET BT 26.250 682.705 Td /F1 9.8 Tf [(influenza viruses, Robert Koch Institute, Germany.)] TJ ET BT 26.250 653.603 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 626.148 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 626.148 Td /F1 9.8 Tf [(Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. Isolation of a novel coronavirus from a man with )] TJ ET BT 26.250 614.244 Td /F1 9.8 Tf [(pneumonia in Saudi Arabia. N Engl J Med. 2012 Nov 8;367\(19\):1814-20. PubMed PMID:23075143.)] TJ ET BT 26.250 594.839 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 594.839 Td /F1 9.8 Tf [(WHO Middle East Respiratory Syndrome Coronavirus update)] TJ ET 0.267 0.267 0.267 rg BT 26.250 586.353 Td /F1 7.5 Tf [(REFERENCE LINK)] TJ ET 0.271 0.267 0.267 rg BT 26.250 567.803 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 567.803 Td /F1 9.8 Tf [(Corman VM, Mller MA, Costabel U, Timm J, Binger T, Meyer B, Kreher P, Lattwein E, Eschbach-Bludau M, Nitsche A, )] TJ ET BT 26.250 555.898 Td /F1 9.8 Tf [(Bleicker T, Landt O, Schweiger B, Drexler JF, Osterhaus AD, Haagmans BL, Dittmer U, Bonin F, Wolff T, Drosten C. Assays for )] TJ ET BT 26.250 543.993 Td /F1 9.8 Tf [(laboratory confirmation of novel human coronavirus \(hCoV-EMC\) infections. Euro Surveill. 2012 Dec 6;17\(49\). PubMed )] TJ ET BT 26.250 532.089 Td /F1 9.8 Tf [(PMID:23231891.)] TJ ET BT 26.250 512.684 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 512.684 Td /F1 9.8 Tf [(Piepenburg O, Williams CH, Stemple DL, Armes NA. DNA detection using recombination proteins. PLoS Biol. 2006 )] TJ ET BT 26.250 500.779 Td /F1 9.8 Tf [(Jul;4\(7\):e204. PubMed PMID:16756388.)] TJ ET BT 26.250 481.374 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 481.374 Td /F1 9.8 Tf [(Abd El Wahed A, El-Deeb A, El-Tholoth M, Abd El Kader H, Ahmed A, Hassan S, Hoffmann B, Haas B, Shalaby MA, Hufert )] TJ ET BT 26.250 469.470 Td /F1 9.8 Tf [(FT, Weidmann M. A Portable Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Foot-)] TJ ET BT 26.250 457.565 Td /F1 9.8 Tf [(and-Mouth Disease Virus. PLoS One. 2013 Aug 20;8\(8\):e71642. PubMed PMID:23977101.)] TJ ET BT 26.250 438.160 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 438.160 Td /F1 9.8 Tf [(Euler M, Wang Y, Heidenreich D, Patel P, Strohmeier O, Hakenberg S, Niedrig M, Hufert FT, Weidmann M. Development of )] TJ ET BT 26.250 426.255 Td /F1 9.8 Tf [(a panel of recombinase polymerase amplification assays for detection of biothreat agents. J Clin Microbiol. 2013 Apr;51\(4\):1110-)] TJ ET BT 26.250 414.351 Td /F1 9.8 Tf [(7. PubMed PMID:23345286.)] TJ ET BT 26.250 394.946 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 394.946 Td /F1 9.8 Tf [(Euler M, Wang Y, Nentwich O, Piepenburg O, Hufert FT, Weidmann M. Recombinase polymerase amplification assay for )] TJ ET BT 26.250 383.041 Td /F1 9.8 Tf [(rapid detection of Rift Valley fever virus. J Clin Virol. 2012 Aug;54\(4\):308-12. PubMed PMID:22683006.)] TJ ET BT 26.250 363.636 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 363.636 Td /F1 9.8 Tf [(Euler M, Wang Y, Otto P, Tomaso H, Escudero R, Anda P, Hufert FT, Weidmann M. Recombinase polymerase amplification )] TJ ET BT 26.250 351.732 Td /F1 9.8 Tf [(assay for rapid detection of Francisella tularensis. J Clin Microbiol. 2012 Jul;50\(7\):2234-8. PubMed PMID:22518861.)] TJ ET BT 26.250 332.327 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 332.327 Td /F1 9.8 Tf [(Weidmann M, Sanchez-Seco MP, Sall AA, Ly PO, Thiongane Y, L MM, Schley H, Hufert FT. Rapid detection of important )] TJ ET BT 26.250 320.422 Td /F1 9.8 Tf [(human pathogenic Phleboviruses. J Clin Virol. 2008 Feb;41\(2\):138-42. PubMed PMID:18006376.)] TJ ET BT 26.250 301.017 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 301.017 Td /F1 9.8 Tf [(Drosten C, Seilmaier M, Corman VM, Hartmann W, Scheible G, Sack S, Guggemos W, Kallies R, Muth D, Junglen S, Mller )] TJ ET BT 26.250 289.113 Td /F1 9.8 Tf [(MA, Haas W, Guberina H, Rhnisch T, Schmid-Wendtner M, Aldabbagh S, Dittmer U, Gold H, Graf P, Bonin F, Rambaut A, )] TJ ET BT 26.250 277.208 Td /F1 9.8 Tf [(Wendtner CM. Clinical features and virological analysis of a case of Middle East respiratory syndrome coronavirus infection. )] TJ ET BT 26.250 265.303 Td /F1 9.8 Tf [(Lancet Infect Dis. 2013 Sep;13\(9\):745-51. PubMed PMID:23782859.)] TJ ET BT 26.250 245.898 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 245.898 Td /F1 9.8 Tf [(Ahmed QA, Arabi YM, Memish ZA. Health risks at the Hajj. Lancet. 2006 Mar 25;367\(9515\):1008-15. PubMed )] TJ ET BT 26.250 233.994 Td /F1 9.8 Tf [(PMID:16564364.)] TJ ET BT 26.250 214.589 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 214.589 Td /F1 9.8 Tf [(Corman VM, Eckerle I, Bleicker T, Zaki A, Landt O, Eschbach-Bludau M, van Boheemen S, Gopal R, Ballhause M, )] TJ ET BT 26.250 202.684 Td /F1 9.8 Tf [(Bestebroer TM, Muth D, Mller MA, Drexler JF, Zambon M, Osterhaus AD, Fouchier RM, Drosten C. Detection of a novel )] TJ ET BT 26.250 190.779 Td /F1 9.8 Tf [(human coronavirus by real-time reverse-transcription polymerase chain reaction. Euro Surveill. 2012 Sep 27;17\(39\). PubMed )] TJ ET BT 26.250 178.875 Td /F1 9.8 Tf [(PMID:23041020.)] TJ ET BT 26.250 159.470 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 159.470 Td /F1 9.8 Tf [(Razanajatovo NH, Richard V, Hoffmann J, Reynes JM, Razafitrimo GM, Randremanana RV, Heraud JM. Viral etiology of )] TJ ET BT 26.250 147.565 Td /F1 9.8 Tf [(influenza-like illnesses in Antananarivo, Madagascar, July 2008 to June 2009. PLoS One. 2011 Mar 3;6\(3\):e17579. PubMed )] TJ ET BT 26.250 135.660 Td /F1 9.8 Tf [(PMID:21390235.)] TJ ET BT 26.250 116.256 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 116.256 Td /F1 9.8 Tf [(Pyrc K, Bosch BJ, Berkhout B, Jebbink MF, Dijkman R, Rottier P, van der Hoek L. Inhibition of human coronavirus NL63 )] TJ ET BT 26.250 104.351 Td /F1 9.8 Tf [(infection at early stages of the replication cycle. Antimicrob Agents Chemother. 2006 Jun;50\(6\):2000-8. PubMed )] TJ ET BT 26.250 92.446 Td /F1 9.8 Tf [(PMID:16723558.)] TJ ET BT 26.250 73.041 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 73.041 Td /F1 9.8 Tf [(Drosten C, Gnther S, Preiser W, van der Werf S, Brodt HR, Becker S, Rabenau H, Panning M, Kolesnikova L, Fouchier )] TJ ET BT 26.250 61.137 Td /F1 9.8 Tf [(RA, Berger A, Burguire AM, Cinatl J, Eickmann M, Escriou N, Grywna K, Kramme S, Manuguerra JC, Mller S, Rickerts V, )] TJ ET BT 26.250 49.232 Td /F1 9.8 Tf [(Strmer M, Vieth S, Klenk HD, Osterhaus AD, Schmitz H, Doerr HW. Identification of a novel coronavirus in patients with severe )] TJ ET Q q 15.000 34.946 577.500 742.054 re W n 0.271 0.267 0.267 rg BT 26.250 750.278 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 730.324 Td /F1 9.8 Tf [(Authors thank Ron Fouchier, Erasmus University, Rotterdam, Netherlands for providing the MERS-CoV isolate and Christian )] TJ ET BT 26.250 718.419 Td /F1 9.8 Tf [(Drosten for genome analysis. The MERS-CoV sample preparation was performed at the Robert Koch-Institut in Berlin, Germany )] TJ ET BT 26.250 706.515 Td /F1 9.8 Tf [(under the ECDC Framework Service Contract Ref. No. ECDC/2008/011. The respiratory virus panel for cross reactivity was )] TJ ET BT 26.250 694.610 Td /F1 9.8 Tf [(provided by Landesgesundheitsamt Niedersachsen, Germany and Brunhilde Schweiger, National Reference Center for )] TJ ET BT 26.250 682.705 Td /F1 9.8 Tf [(influenza viruses, Robert Koch Institute, Germany.)] TJ ET BT 26.250 653.603 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 626.148 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 626.148 Td /F1 9.8 Tf [(Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. Isolation of a novel coronavirus from a man with )] TJ ET BT 26.250 614.244 Td /F1 9.8 Tf [(pneumonia in Saudi Arabia. N Engl J Med. 2012 Nov 8;367\(19\):1814-20. PubMed PMID:23075143.)] TJ ET BT 26.250 594.839 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 594.839 Td /F1 9.8 Tf [(WHO Middle East Respiratory Syndrome Coronavirus update)] TJ ET 0.267 0.267 0.267 rg BT 26.250 586.353 Td /F1 7.5 Tf [(REFERENCE LINK)] TJ ET 0.271 0.267 0.267 rg BT 26.250 567.803 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 567.803 Td /F1 9.8 Tf [(Corman VM, Mller MA, Costabel U, Timm J, Binger T, Meyer B, Kreher P, Lattwein E, Eschbach-Bludau M, Nitsche A, )] TJ ET BT 26.250 555.898 Td /F1 9.8 Tf [(Bleicker T, Landt O, Schweiger B, Drexler JF, Osterhaus AD, Haagmans BL, Dittmer U, Bonin F, Wolff T, Drosten C. Assays for )] TJ ET BT 26.250 543.993 Td /F1 9.8 Tf [(laboratory confirmation of novel human coronavirus \(hCoV-EMC\) infections. Euro Surveill. 2012 Dec 6;17\(49\). PubMed )] TJ ET BT 26.250 532.089 Td /F1 9.8 Tf [(PMID:23231891.)] TJ ET BT 26.250 512.684 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 512.684 Td /F1 9.8 Tf [(Piepenburg O, Williams CH, Stemple DL, Armes NA. DNA detection using recombination proteins. PLoS Biol. 2006 )] TJ ET BT 26.250 500.779 Td /F1 9.8 Tf [(Jul;4\(7\):e204. PubMed PMID:16756388.)] TJ ET BT 26.250 481.374 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 481.374 Td /F1 9.8 Tf [(Abd El Wahed A, El-Deeb A, El-Tholoth M, Abd El Kader H, Ahmed A, Hassan S, Hoffmann B, Haas B, Shalaby MA, Hufert )] TJ ET BT 26.250 469.470 Td /F1 9.8 Tf [(FT, Weidmann M. A Portable Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Foot-)] TJ ET BT 26.250 457.565 Td /F1 9.8 Tf [(and-Mouth Disease Virus. PLoS One. 2013 Aug 20;8\(8\):e71642. PubMed PMID:23977101.)] TJ ET BT 26.250 438.160 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 438.160 Td /F1 9.8 Tf [(Euler M, Wang Y, Heidenreich D, Patel P, Strohmeier O, Hakenberg S, Niedrig M, Hufert FT, Weidmann M. Development of )] TJ ET BT 26.250 426.255 Td /F1 9.8 Tf [(a panel of recombinase polymerase amplification assays for detection of biothreat agents. J Clin Microbiol. 2013 Apr;51\(4\):1110-)] TJ ET BT 26.250 414.351 Td /F1 9.8 Tf [(7. PubMed PMID:23345286.)] TJ ET BT 26.250 394.946 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 394.946 Td /F1 9.8 Tf [(Euler M, Wang Y, Nentwich O, Piepenburg O, Hufert FT, Weidmann M. Recombinase polymerase amplification assay for )] TJ ET BT 26.250 383.041 Td /F1 9.8 Tf [(rapid detection of Rift Valley fever virus. J Clin Virol. 2012 Aug;54\(4\):308-12. PubMed PMID:22683006.)] TJ ET BT 26.250 363.636 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 363.636 Td /F1 9.8 Tf [(Euler M, Wang Y, Otto P, Tomaso H, Escudero R, Anda P, Hufert FT, Weidmann M. Recombinase polymerase amplification )] TJ ET BT 26.250 351.732 Td /F1 9.8 Tf [(assay for rapid detection of Francisella tularensis. J Clin Microbiol. 2012 Jul;50\(7\):2234-8. PubMed PMID:22518861.)] TJ ET BT 26.250 332.327 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 332.327 Td /F1 9.8 Tf [(Weidmann M, Sanchez-Seco MP, Sall AA, Ly PO, Thiongane Y, L MM, Schley H, Hufert FT. Rapid detection of important )] TJ ET BT 26.250 320.422 Td /F1 9.8 Tf [(human pathogenic Phleboviruses. J Clin Virol. 2008 Feb;41\(2\):138-42. PubMed PMID:18006376.)] TJ ET BT 26.250 301.017 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 301.017 Td /F1 9.8 Tf [(Drosten C, Seilmaier M, Corman VM, Hartmann W, Scheible G, Sack S, Guggemos W, Kallies R, Muth D, Junglen S, Mller )] TJ ET BT 26.250 289.113 Td /F1 9.8 Tf [(MA, Haas W, Guberina H, Rhnisch T, Schmid-Wendtner M, Aldabbagh S, Dittmer U, Gold H, Graf P, Bonin F, Rambaut A, )] TJ ET BT 26.250 277.208 Td /F1 9.8 Tf [(Wendtner CM. Clinical features and virological analysis of a case of Middle East respiratory syndrome coronavirus infection. )] TJ ET BT 26.250 265.303 Td /F1 9.8 Tf [(Lancet Infect Dis. 2013 Sep;13\(9\):745-51. PubMed PMID:23782859.)] TJ ET BT 26.250 245.898 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 245.898 Td /F1 9.8 Tf [(Ahmed QA, Arabi YM, Memish ZA. Health risks at the Hajj. Lancet. 2006 Mar 25;367\(9515\):1008-15. PubMed )] TJ ET BT 26.250 233.994 Td /F1 9.8 Tf [(PMID:16564364.)] TJ ET BT 26.250 214.589 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 214.589 Td /F1 9.8 Tf [(Corman VM, Eckerle I, Bleicker T, Zaki A, Landt O, Eschbach-Bludau M, van Boheemen S, Gopal R, Ballhause M, )] TJ ET BT 26.250 202.684 Td /F1 9.8 Tf [(Bestebroer TM, Muth D, Mller MA, Drexler JF, Zambon M, Osterhaus AD, Fouchier RM, Drosten C. Detection of a novel )] TJ ET BT 26.250 190.779 Td /F1 9.8 Tf [(human coronavirus by real-time reverse-transcription polymerase chain reaction. Euro Surveill. 2012 Sep 27;17\(39\). PubMed )] TJ ET BT 26.250 178.875 Td /F1 9.8 Tf [(PMID:23041020.)] TJ ET BT 26.250 159.470 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 159.470 Td /F1 9.8 Tf [(Razanajatovo NH, Richard V, Hoffmann J, Reynes JM, Razafitrimo GM, Randremanana RV, Heraud JM. Viral etiology of )] TJ ET BT 26.250 147.565 Td /F1 9.8 Tf [(influenza-like illnesses in Antananarivo, Madagascar, July 2008 to June 2009. PLoS One. 2011 Mar 3;6\(3\):e17579. PubMed )] TJ ET BT 26.250 135.660 Td /F1 9.8 Tf [(PMID:21390235.)] TJ ET BT 26.250 116.256 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 116.256 Td /F1 9.8 Tf [(Pyrc K, Bosch BJ, Berkhout B, Jebbink MF, Dijkman R, Rottier P, van der Hoek L. Inhibition of human coronavirus NL63 )] TJ ET BT 26.250 104.351 Td /F1 9.8 Tf [(infection at early stages of the replication cycle. Antimicrob Agents Chemother. 2006 Jun;50\(6\):2000-8. PubMed )] TJ ET BT 26.250 92.446 Td /F1 9.8 Tf [(PMID:16723558.)] TJ ET BT 26.250 73.041 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 73.041 Td /F1 9.8 Tf [(Drosten C, Gnther S, Preiser W, van der Werf S, Brodt HR, Becker S, Rabenau H, Panning M, Kolesnikova L, Fouchier )] TJ ET BT 26.250 61.137 Td /F1 9.8 Tf [(RA, Berger A, Burguire AM, Cinatl J, Eickmann M, Escriou N, Grywna K, Kramme S, Manuguerra JC, Mller S, Rickerts V, )] TJ ET BT 26.250 49.232 Td /F1 9.8 Tf [(Strmer M, Vieth S, Klenk HD, Osterhaus AD, Schmitz H, Doerr HW. Identification of a novel coronavirus in patients with severe )] TJ ET Q q 15.000 34.946 577.500 742.054 re W n 0.271 0.267 0.267 rg BT 26.250 750.278 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 730.324 Td /F1 9.8 Tf [(Authors thank Ron Fouchier, Erasmus University, Rotterdam, Netherlands for providing the MERS-CoV isolate and Christian )] TJ ET BT 26.250 718.419 Td /F1 9.8 Tf [(Drosten for genome analysis. The MERS-CoV sample preparation was performed at the Robert Koch-Institut in Berlin, Germany )] TJ ET BT 26.250 706.515 Td /F1 9.8 Tf [(under the ECDC Framework Service Contract Ref. No. ECDC/2008/011. The respiratory virus panel for cross reactivity was )] TJ ET BT 26.250 694.610 Td /F1 9.8 Tf [(provided by Landesgesundheitsamt Niedersachsen, Germany and Brunhilde Schweiger, National Reference Center for )] TJ ET BT 26.250 682.705 Td /F1 9.8 Tf [(influenza viruses, Robert Koch Institute, Germany.)] TJ ET BT 26.250 653.603 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 626.148 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 626.148 Td /F1 9.8 Tf [(Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. Isolation of a novel coronavirus from a man with )] TJ ET BT 26.250 614.244 Td /F1 9.8 Tf [(pneumonia in Saudi Arabia. N Engl J Med. 2012 Nov 8;367\(19\):1814-20. PubMed PMID:23075143.)] TJ ET BT 26.250 594.839 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 594.839 Td /F1 9.8 Tf [(WHO Middle East Respiratory Syndrome Coronavirus update)] TJ ET 0.267 0.267 0.267 rg BT 26.250 586.353 Td /F1 7.5 Tf [(REFERENCE LINK)] TJ ET 0.271 0.267 0.267 rg BT 26.250 567.803 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 567.803 Td /F1 9.8 Tf [(Corman VM, Mller MA, Costabel U, Timm J, Binger T, Meyer B, Kreher P, Lattwein E, Eschbach-Bludau M, Nitsche A, )] TJ ET BT 26.250 555.898 Td /F1 9.8 Tf [(Bleicker T, Landt O, Schweiger B, Drexler JF, Osterhaus AD, Haagmans BL, Dittmer U, Bonin F, Wolff T, Drosten C. Assays for )] TJ ET BT 26.250 543.993 Td /F1 9.8 Tf [(laboratory confirmation of novel human coronavirus \(hCoV-EMC\) infections. Euro Surveill. 2012 Dec 6;17\(49\). PubMed )] TJ ET BT 26.250 532.089 Td /F1 9.8 Tf [(PMID:23231891.)] TJ ET BT 26.250 512.684 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 512.684 Td /F1 9.8 Tf [(Piepenburg O, Williams CH, Stemple DL, Armes NA. 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PubMed )] TJ ET BT 26.250 233.994 Td /F1 9.8 Tf [(PMID:16564364.)] TJ ET BT 26.250 214.589 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 214.589 Td /F1 9.8 Tf [(Corman VM, Eckerle I, Bleicker T, Zaki A, Landt O, Eschbach-Bludau M, van Boheemen S, Gopal R, Ballhause M, )] TJ ET BT 26.250 202.684 Td /F1 9.8 Tf [(Bestebroer TM, Muth D, Mller MA, Drexler JF, Zambon M, Osterhaus AD, Fouchier RM, Drosten C. Detection of a novel )] TJ ET BT 26.250 190.779 Td /F1 9.8 Tf [(human coronavirus by real-time reverse-transcription polymerase chain reaction. Euro Surveill. 2012 Sep 27;17\(39\). PubMed )] TJ ET BT 26.250 178.875 Td /F1 9.8 Tf [(PMID:23041020.)] TJ ET BT 26.250 159.470 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 159.470 Td /F1 9.8 Tf [(Razanajatovo NH, Richard V, Hoffmann J, Reynes JM, Razafitrimo GM, Randremanana RV, Heraud JM. Viral etiology of )] TJ ET BT 26.250 147.565 Td /F1 9.8 Tf [(influenza-like illnesses in Antananarivo, Madagascar, July 2008 to June 2009. PLoS One. 2011 Mar 3;6\(3\):e17579. PubMed )] TJ ET BT 26.250 135.660 Td /F1 9.8 Tf [(PMID:21390235.)] TJ ET BT 26.250 116.256 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 116.256 Td /F1 9.8 Tf [(Pyrc K, Bosch BJ, Berkhout B, Jebbink MF, Dijkman R, Rottier P, van der Hoek L. Inhibition of human coronavirus NL63 )] TJ ET BT 26.250 104.351 Td /F1 9.8 Tf [(infection at early stages of the replication cycle. Antimicrob Agents Chemother. 2006 Jun;50\(6\):2000-8. PubMed )] TJ ET BT 26.250 92.446 Td /F1 9.8 Tf [(PMID:16723558.)] TJ ET BT 26.250 73.041 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 73.041 Td /F1 9.8 Tf [(Drosten C, Gnther S, Preiser W, van der Werf S, Brodt HR, Becker S, Rabenau H, Panning M, Kolesnikova L, Fouchier )] TJ ET BT 26.250 61.137 Td /F1 9.8 Tf [(RA, Berger A, Burguire AM, Cinatl J, Eickmann M, Escriou N, Grywna K, Kramme S, Manuguerra JC, Mller S, Rickerts V, )] TJ ET BT 26.250 49.232 Td /F1 9.8 Tf [(Strmer M, Vieth S, Klenk HD, Osterhaus AD, Schmitz H, Doerr HW. Identification of a novel coronavirus in patients with severe )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(6)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Outbreaks)] TJ ET Q endstream endobj 329 0 obj << /Type /Annot /Subtype /Link /A 330 0 R /Border [0 0 0] /H /I /Rect [ 26.2500 585.6592 91.2600 593.2905 ] >> endobj 330 0 obj << /Type /Action /S /URI /URI (http://wwwwhoint/csr/don/archive/disease/coronavirus_infections/en/indexhtml) >> endobj 331 0 obj << /Type /Annot /Subtype /Link /A 332 0 R /Border [0 0 0] /H /I /Rect [ 26.2500 585.6592 91.2600 593.2905 ] >> endobj 332 0 obj << /Type /Action /S /URI /URI (http://wwwwhoint/csr/don/archive/disease/coronavirus_infections/en/indexhtml) >> endobj 333 0 obj << /Type /Annot /Subtype /Link /A 334 0 R /Border [0 0 0] /H /I /Rect [ 26.2500 585.6592 91.2600 593.2905 ] >> endobj 334 0 obj << /Type /Action /S /URI /URI (http://wwwwhoint/csr/don/archive/disease/coronavirus_infections/en/indexhtml) >> endobj 335 0 obj << /Type /Page /Parent 3 0 R /Contents 336 0 R >> endobj 336 0 obj << /Length 3229 >> stream 0.271 0.267 0.267 rg q 15.000 675.572 577.500 101.429 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(acute respiratory syndrome. N Engl J Med. 2003 May 15;348\(20\):1967-76. PubMed PMID:12690091.)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Terlizzi ME, Massimiliano B, Francesca S, Sinesi F, Rosangela V, Stefano G, Costa C, Rossana C. Quantitative RT real )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(time PCR and indirect immunofluorescence for the detection of human parainfluenza virus 1, 2, 3. J Virol Methods. 2009 )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(Sep;160\(1-2\):172-7. PubMed PMID:19445964.)] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 704.857 Td /F1 9.8 Tf [(Patel P, Graser E, Robst S, Hillert R, Meye A, Hillebrand T, Niedrig M. rapidSTRIPE H1N1 test for detection of the )] TJ ET BT 26.250 692.952 Td /F1 9.8 Tf [(pandemic swine origin influenza A \(H1N1\) virus. J Clin Microbiol. 2011 Apr;49\(4\):1591-3. PubMed PMID:21248098.)] TJ ET Q q 15.000 675.572 577.500 101.429 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(acute respiratory syndrome. N Engl J Med. 2003 May 15;348\(20\):1967-76. PubMed PMID:12690091.)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Terlizzi ME, Massimiliano B, Francesca S, Sinesi F, Rosangela V, Stefano G, Costa C, Rossana C. Quantitative RT real )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(time PCR and indirect immunofluorescence for the detection of human parainfluenza virus 1, 2, 3. J Virol Methods. 2009 )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(Sep;160\(1-2\):172-7. PubMed PMID:19445964.)] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 704.857 Td /F1 9.8 Tf [(Patel P, Graser E, Robst S, Hillert R, Meye A, Hillebrand T, Niedrig M. rapidSTRIPE H1N1 test for detection of the )] TJ ET BT 26.250 692.952 Td /F1 9.8 Tf [(pandemic swine origin influenza A \(H1N1\) virus. 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