This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Drosten, C. Articles by Koay, E. S. C. Search for Related Content PubMed PubMed Citation Articles by Drosten, C. Articles by Koay, E. S. C.

Evaluation of Advanced Reverse Transcription-PCR Assays and an Alternative PCR Target Region for Detection of Severe Acute Respiratory Syndrome-Associated Coronavirus

Department of Virology, Bernhard-Nocht Institute for Tropical Medicine, Hamburg,¹ Institute of Medical Virology, Johann-Wolfgang-Goethe University, Frankfurt, Germany,⁴ Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital,² Department of Infectious Diseases, Tan Tock Seng Hospital,³ Department of Pathology, National University of Singapore, Singapore,⁶ Department of Microbiology, Faculty of Medicine, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, Special Administrative Region, People’s Republic of China⁵

Received 10 October 2003/ Returned for modification 14 December 2003/ Accepted 14 February 2004

First-generation reverse transcription-PCR (RT-PCR) assays for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) gave false-negative results in a considerable fraction of patients. In the present study, we evaluated two second-generation, replicase (R) gene-based, real-time RT-PCR test kits—the RealArt HPA coronavirus LC kit (Artus, Hamburg, Germany) and the LightCycler SARS-CoV quantification kit (Roche, Penzberg, Germany)—and a real-time RT-PCR assay for the nucleocapsid (N) gene. Detecting the N-gene RNA might be advantageous due to its high abundance in cells. The kits achieved sensitivities of 70.8% (Artus) and 67.1% (Roche) in 66 specimens from patients with confirmed SARS (samples primarily from the upper and lower respiratory tract and stool). The sensitivity of the N-gene assay was 74.2%. The differences in all of the sensitivities were not statistically significant (P = 0.680 [analysis of variance]). Culture cells initially contained five times more N- than R-gene RNA, but the respective levels converged during 4 days of virus replication. In clinical samples the median concentrations of R- and N-gene RNA, respectively, were 1.2 x 10⁶ and 2.8 x 10⁶ copies/ml (sputum and endotracheal aspirates), 4.3 x 10⁴ and 5.5 x 10⁴ copies/ml (stool), and 5.5 x 10² and 5.2 x 10² copies/sample (throat swabs and saliva). Differences between the samples types were significant but not between the types of target RNA. All (n = 12) samples from the lower respiratory tract tested positive in all tests. In conclusion, the novel assays are more sensitive than the first-generation tests, but they still do not allow a comprehensive ruling out of SARS. Methods for the routine sampling of sputum without infection risk are needed to improve SARS RT-PCR.

This article has been cited by other articles:

• Dong, J., Olano, J. P., McBride, J. W., Walker, D. H. (2008). Emerging Pathogens: Challenges and Successes of Molecular Diagnostics. J. Mol. Diagn. 10: 185-197 [Abstract] [Full Text]  
• Cheng, V. C. C., Lau, S. K. P., Woo, P. C. Y., Yuen, K. Y. (2007). Severe Acute Respiratory Syndrome Coronavirus as an Agent of Emerging and Reemerging Infection. Clin. Microbiol. Rev. 20: 660-694 [Abstract] [Full Text]  
• de Souza Luna, L. K., Heiser, V., Regamey, N., Panning, M., Drexler, J. F., Mulangu, S., Poon, L., Baumgarte, S., Haijema, B. J., Kaiser, L., Drosten, C. (2007). Generic Detection of Coronaviruses and Differentiation at the Prototype Strain Level by Reverse Transcription-PCR and Nonfluorescent Low-Density Microarray. J. Clin. Microbiol. 45: 1049-1052 [Abstract] [Full Text]  
• Louie, L., Simor, A. E., Chong, S., Luinstra, K., Petrich, A., Mahony, J., Smieja, M., Johnson, G., Gharabaghi, F., Tellier, R., Willey, B. M., Poutanen, S., Mazzulli, T., Broukhanski, G., Jamieson, F., Louie, M., Richardson, S., Ontario Laboratory Working Group for the Rapid Dia, (2006). Detection of Severe Acute Respiratory Syndrome Coronavirus in Stool Specimens by Commercially Available Real-Time Reverse Transcriptase PCR Assays. J. Clin. Microbiol. 44: 4193-4196 [Abstract] [Full Text]  
• Drosten, C., Panning, M., Drexler, J. F., Hansel, F., Pedroso, C., Yeats, J., de Souza Luna, L. K., Samuel, M., Liedigk, B., Lippert, U., Sturmer, M., Doerr, H. W., Brites, C., Preiser, W. (2006). Ultrasensitive Monitoring of HIV-1 Viral Load by a Low-Cost Real-Time Reverse Transcription-PCR Assay with Internal Control for the 5' Long Terminal Repeat Domain. Clin. Chem. 52: 1258-1266 [Abstract] [Full Text]  
• Espy, M. J., Uhl, J. R., Sloan, L. M., Buckwalter, S. P., Jones, M. F., Vetter, E. A., Yao, J. D. C., Wengenack, N. L., Rosenblatt, J. E., Cockerill, F. R. III, Smith, T. F. (2006). Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing. Clin. Microbiol. Rev. 19: 165-256 [Abstract] [Full Text]  
• Mahony, J. B., Richardson, S. (2005). Molecular Diagnosis of Severe Acute Respiratory Syndrome: The State of the Art. J. Mol. Diagn. 7: 551-559 [Abstract] [Full Text]  
• Yip, S. P., To, S. S. T., Leung, P. H.M., Cheung, T. S., Cheng, P. K.C., Lim, W. W.L. (2005). Use of Dual TaqMan Probes to Increase the Sensitivity of 1-Step Quantitative Reverse Transcription-PCR: Application to the Detection of SARS Coronavirus. Clin. Chem. 51: 1885-1888 [Full Text]  
• Inoue, M., Barkham, T., Keong, L. K., Gee, L. S., Wanjin, H. (2005). Performance of Single-Step Gel-Based Reverse Transcription-PCR (RT-PCR) Assays Equivalent to That of Real-Time RT-PCR Assays for Detection of the Severe Acute Respiratory Syndrome-Associated Coronavirus. J. Clin. Microbiol. 43: 4262-4265 [Abstract] [Full Text]  
• Poon, L. L. M., Wong, B. W. Y., Chan, K. H., Ng, S. S. F., Yuen, K. Y., Guan, Y., Peiris, J. S. M. (2005). Evaluation of Real-Time Reverse Transcriptase PCR and Real-Time Loop-Mediated Amplification Assays for Severe Acute Respiratory Syndrome Coronavirus Detection. J. Clin. Microbiol. 43: 3457-3459 [Abstract] [Full Text]  
• Tong, T, Lai, T S.-t. (2005). The severe acute respiratory syndrome coronavirus in tears. Br. J. Ophthalmol. 89: 392-392 [Full Text]