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 Mahony, J. B. Articles by Richardson, S. Search for Related Content PubMed PubMed Citation Articles by Mahony, J. B. Articles by Richardson, S.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, April 2004, p. 1471-1476, Vol. 42, No. 4
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.4.1471-1476.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Performance and Cost Evaluation of One Commercial and Six In-House Conventional and Real-Time Reverse Transcription-PCR Assays for Detection of Severe Acute Respiratory Syndrome Coronavirus

James B. Mahony,^1,2* Astrid Petrich,^1,2 Lisa Louie,³ Xinyu Song,² Sylvia Chong,² Marek Smieja,^1,2 Max Chernesky,^1,2 Mark Loeb,¹ Susan Richardson,⁴ and the Ontario Laboratory Working Group for the Rapid Diagnosis of Emerging Infections

McMaster University,¹ Father Sean O'Sullivan Research Centre, St. Joseph's Healthcare, Hamilton, and,² Sunnybrook and Women's College Health Sciences Centre, University of Toronto and Heathcare Network,³ The Hospital for Sick Children Toronto, Ontario L8N 4A6, Canada⁴

Received 10 October 2003/ Returned for modification 26 November 2003/ Accepted 14 December 2003

We evaluated seven reverse transcription-PCR (RT-PCR) assays, including six in-house assays and one commercial assay for the detection of severe acute respiratory syndrome coronavirus (SARS-CoV) RNA in clinical specimens. RT-PCR assays targeted different genomic regions and included three conventional assays (one nested and two non-nested) run on a conventional heat block and four real-time assays performed in a LightCycler (LC; Roche Diagnostics). All in-house assays were optimized for assay parameters, including MgCl₂, primer, and probe concentrations. The commercial assay was the RealArt HPA CoV RT-PCR assay (Artus), which was run in the LC. Testing serial dilutions of cultured SARS-CoV showed that the analytical sensitivity of the assays ranged from 10^–8 to 10^–6, corresponding to 1 and 100 copies of viral RNA, respectively. Significant differences in analytical sensitivities were observed between assays (P < 0.01, probit regression analysis for 50% sensitivity levels for the top two assays versus the others). Testing 68 clinical specimens (including 17 respiratory tract specimens, 29 urine samples, and 22 stools or rectal swabs) demonstrated that six of the seven assays detected at least 17 of 18 positives (defined as positive in at least two assays), and two of the assays had a sensitivity of 100%. There were no significant differences in sensitivity between the assays (P = 0.5 [Cochrance Q test, least sensitive 15 of 18 versus 18 of 18]). The specificities of the assays ranged from 94.0 to 100% without significant differences (P = 0.25 to 0.5 [McNemar test]). The reagent and technologist cost of performing the in-house PCR assays ranged from $5.46 to $9.81 Canadian dollars (CDN) per test. The commercial assay cost was considerably higher at $40.37 per test. The results demonstrated good performance for all assays, providing laboratories that need to do SARS RNA testing with a choice of assay formats.

Contributing members of the Working Group are listed in Acknowledgments.

This article has been cited by other articles:

• Marshall, D. J., Reisdorf, E., Harms, G., Beaty, E., Moser, M. J., Lee, W.-M., Gern, J. E., Nolte, F. S., Shult, P., Prudent, J. R. (2007). Evaluation of a Multiplexed PCR Assay for Detection of Respiratory Viral Pathogens in a Public Health Laboratory Setting. J. Clin. Microbiol. 45: 3875-3882 [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]  
• Petrich, A., Mahony, J., Chong, S., Broukhanski, G., Gharabaghi, F., Johnson, G., Louie, L., Luinstra, K., Willey, B., Akhaven, P., Chui, L., Jamieson, F., Louie, M., Mazzulli, T., Tellier, R., Smieja, M., Cai, W., Chernesky, M., Richardson, S. E., for the Ontario Laboratory Working Group for the R, (2006). Multicenter comparison of nucleic Acid extraction methods for detection of severe acute respiratory syndrome coronavirus RNA in stool specimens.. J. Clin. Microbiol. 44: 2681-2688 [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]  
• 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]  
• Liu, I-J., Chen, P.-J., Yeh, S.-H., Chiang, Y.-P., Huang, L.-M., Chang, M.-F., Chen, S.-Y., Yang, P.-C., Chang, S.-C., Wang, W.-K., the SARS Research Group of the National Taiwan Uni, (2005). Immunofluorescence Assay for Detection of the Nucleocapsid Antigen of the Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus in Cells Derived from Throat Wash Samples of Patients with SARS. J. Clin. Microbiol. 43: 2444-2448 [Abstract] [Full Text]