DOI QR코드

DOI QR Code

Comparison of Molecular Assays for the Rapid Detection and Simultaneous Subtype Differentiation of the Pandemic Influenza A (H1N1) 2009 Virus

  • Lee, Mi Kyung (Department of Laboratory Medicine, Chung-Ang University College of Medicine) ;
  • Kim, Hye Ryoun (Department of Laboratory Medicine, Chung-Ang University College of Medicine)
  • Received : 2011.07.27
  • Accepted : 2012.03.22
  • Published : 2012.08.28

Abstract

In April 2009, the H1N1 pandemic influenza virus emerged as a novel influenza virus. The aim of this study was to compare the performances of several molecular assays, including conventional reverse transcription polymerase chain reaction (RT-PCR), two real-time reverse transcription (rRT)-PCRs, and two multiplex RTPCRs. A total of 381 clinical specimens were collected from patients (223 men and 158 women), and both the Seeplex RV7 assay and rRT-PCR were ordered on different specimens within one week after collection. The concordance rate for the two methods was 87% (332/381), and the discrepancy rate was 13% (49/381). The positive rates for the molecular assays studied included 93.1% for the multiplex Seeplex RV7 assay, 93.1% for conventional reverse transcription (cRT)-PCR, 89.7% for the multiplex Seeplex Flu ACE Subtyping assay, 82.8% for protocol B rRT-PCR, and 58.6% for protocol A rRT-PCR. Our results showed that the multiplex Seeplex assays and the cRT-PCR yielded higher detection rates than rRT-PCRs for detecting the influenza A (H1N1) virus. Although the multiplex Seeplex assays had the advantage of simultaneous detection of several viruses, they were time-consuming and troublesome. Our results show that, although rRT-PCR had the advantage, the detection rates of the molecular assays varied depending upon the source of the influenza A (H1N1)v virus. Our findings also suggest that rRT-PCR sometimes detected virus in extremely low abundance and thus required validation of analytical performance and clinical correlation.

Keywords

References

  1. Bellau-Pujol, S., A. Vabret, L. Legrand, J. Dina, S. Gouarin, J. Etitjean-Lecherbonner, et al. 2005. Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses. J. Virol. Methods 126: 53-63. https://doi.org/10.1016/j.jviromet.2005.01.020
  2. Carr, M. J., R. Gunson, A. Maclean, S. Coughlan, M. Fitzgerald, M. Scully, et al. 2009. Development of a real-time RT-PCR for the detection of swine-lineage influenza A (H1N1) virus infections. J. Clin. Virol. 45: 196-199. https://doi.org/10.1016/j.jcv.2009.06.001
  3. Choi, Y. J., H. S. Nam, J. S. Park, H. J. Kim, K. B. Park, M. H. Jeon, et al. 2010. Comparative analysis of the multiple test methods for the detection of Pandemic Influenza A/H1N1 2009 virus. J. Microbiol. Biotechnol. 20: 1450-1456. https://doi.org/10.4014/jmb.1004.04039
  4. Hall, R. J., M. Peacey, Q. S. Huang, and P. E. Carter. 2009. Rapid method to support diagnosis of swine origin influenza virus infection by sequencing of real-time PCR amplicons from diagnostic assays. J. Clin. Microbiol. 47: 3053-3054. https://doi.org/10.1128/JCM.01000-09
  5. Hindiyeh, M., V. Levy, R. Azarr, N. Varsano, L. Regev, Y. Shalev, et al. 2005. Evaluation of a multiplex real-time reverse transcriptase PCR assay for detection and differentiation of influenza viruses A and B during the 2001-2002 influenza season in Israel. J. Clin. Microbiol. 43: 589-595. https://doi.org/10.1128/JCM.43.2.589-595.2005
  6. Hymas, W. C., A. Mills, S. Ferguson, J. Langer, R. C. She, W. Mahoney, and D. R. Hillyyard. 2010. Development of a multiplex real-time RT-PCR assay for detection of influenza A, influenza B, RSV and typing of the 2009-H1N1 influenza virus. J. Virol. Methods 167: 113-118. https://doi.org/10.1016/j.jviromet.2010.03.020
  7. Jiang, T., X. Kang, Y. Deng, H. Zhao, X. Li, X. Yu, et al. 2010. Development of a real-time RT-PCR assay for a novel influenza A (H1N1) virus. J. Virol. Methods 163: 470-473. https://doi.org/10.1016/j.jviromet.2009.09.021
  8. Karre, T., H. F. Maguire, D. Butcher, A. Graepler, D. Weed, and M. L. Wilson. 2010. Comparison of Becton Dickinson Directigen EZ Flu A+B test against the CDC real-time PCR assay for detection of 2009 pandemic influenza A/H1N1 virus. J. Clin. Microbiol. 48: 343-344. https://doi.org/10.1128/JCM.02063-09
  9. Kim, S. R., C. S. Ki, and N. Y. Lee. 2009. Rapid detection and identification of 12 respiratory viruses using a dual priming oligonucleotide system-based multiplex PCR assay. J. Virol. Methods 156: 111-116. https://doi.org/10.1016/j.jviromet.2008.11.007
  10. Kok, T., S. Wati, B. Bayly, D. Devonshire-Gill, and G. Higgins. 2000. Comparison of six nucleic acid extraction methods for detection of viral DNA or RNA sequences in four different non-serum specimen types. J. Clin. Virol. 16: 59-63. https://doi.org/10.1016/S1386-6532(99)00066-9
  11. Lam, W. Y., T. F. Leung, N. Lee, J. L. Cheung, A. C. Yeung, Y. I. Ho, et al. 2010. Development and comparison of molecular assays for the rapid detection of the pandemic influenza A (H1N1) 2009 virus. J. Med. Virol. 82: 675-683. https://doi.org/10.1002/jmv.21725
  12. Liao, R. S., L. L. Tomalty, A. Majury, and D. E. Zoutman. 2009. Comparison of viral isolation and multiplex real-time reverse transcription-PCR for confirmation of respiratory syncytial virus and influenza virus detection by antigen immunoassays. J. Clin. Microbiol. 47: 527-532. https://doi.org/10.1128/JCM.01213-08
  13. Lu, Q., X. Q. Zhang, S. L. Pond, S. Reed, R. T. Schooley, and Y. T. Liu. 2009. Detection in 2009 of the swine origin influenza A (H1N1) virus by a subtyping microarray. J. Clin. Microbiol. 47: 3060-3061. https://doi.org/10.1128/JCM.01243-09
  14. Neumann, G., T. Noda, and Y. Kawaoka. 2009. Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature 459: 931-939. https://doi.org/10.1038/nature08157
  15. Pabbaraju, K., S. Wong, A. A. Wong, G. D. Appleyard, L. Chui, X. L. Pang, et al. 2009. Design and validation of real-time reverse transcription-PCR assays for detection of pandemic (H1N1) 2009 virus. J. Clin. Microbiol. 47: 3454-3460. https://doi.org/10.1128/JCM.01103-09
  16. Panning, M., M. Eickmann, O. Landt, M. Monazahian, S. Olschlager, S. Baumgarte, et al. 2009. Detection of influenza A(H1N1)v virus by real-time RT-PCR. Euro Surveill. 14: 19329.
  17. Poon, L. L., K. H. Chan, G. J. Smith, C. S. Leung, Y. Guan, K. Y. Yuen, et al. 2009. Molecular detection of a novel human influenza (H1N1) of pandemic potential by conventional and real-time quantitative RT-PCR assays. Clin. Chem. 55: 1555-1558. https://doi.org/10.1373/clinchem.2009.130229
  18. Vasoo, S., J. Stevens, and K. Singh. 2009. Rapid antigen tests for diagnosis of pandemic (swine) influenza A/H1N1. Clin. Infect. Dis. 49: 1090-1093. https://doi.org/10.1086/644743
  19. Wenzel, J. J., H. Walch, M. Bollwein, H. H. Niller, W. Ankenbauer, R. Mauritz, et al. 2009. Library of prefabricated locked nucleic acid hydrolysis probes facilitates rapid development of reverse-transcription quantitative real-time PCR assays for detection of novel influenza A/H1N1/09 virus. Clin. Chem. 55: 2218-2222. https://doi.org/10.1373/clinchem.2009.136192
  20. Whiley, D. M., S. Bialasiewicz, C. Bletchly, C. E. Faux, B. Harrower, A. R. Gould, et al. 2009. Detection of novel influenza A(H1N1) virus by real-time RT-PCR. J. Clin. Virol. 45: 203-204. https://doi.org/10.1016/j.jcv.2009.05.032