1887

Journal of Medical Microbiology logo

Volume 56, Issue 5

Real-time RT-PCR for H5N1 avian influenza A virus detection Free

Abstract

The recent recurrence of highly pathogenic avian influenza virus A H5N1 was firstly reported in mid-December 2003 and continued through 2005. This study describes a sensitive and specific real-time RT-PCR method for the detection of influenza A subtype H5 and for monitoring virus loads. Using serial dilutions of influenza A H5N1 cultures, this assay reproducibly determined the lowest detection limit to be approximately 5×10 50 % egg infective doses (EID). In contrast, the minimum detection limit was approximately 3 EID in conventional RT-PCR with WHO primers and 10 EID in antigen-capture ELISA. In tests of serial dilutions of -transcribed influenza A H5 gene RNA, there was linear amplification from 40 copies to 4×10 copies of target RNA per reaction and approximately six copies, and sometimes even as few as three copies, of target RNA tested positive in our assay. Thirty-five throat swabs from ill birds were tested: 33 samples tested positive using this assay. In comparison, 27, 13 and 19 samples tested positive using conventional RT-PCR, antigen-capture ELISA and virus isolation, respectively. To evaluate further the sensitivity of this real-time RT-PCR, a standard panel and 60 H5N1 isolates that contained different clades of influenza virus A/H5N1 were tested and all tested positive. To evaluate the specificity of the assay, 60 throat swabs from patients infected with influenza virus A H1 were tested; all were negative. Thirteen other viruses were also tested and all tested negative.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): EID50, 50 % egg infective dose , FAM, 6-carboxyfluorescein , HA, haemagglutinin , HPAIV, highly pathogenic avian influenza virus , RRT-PCR, real-time RT-PCR , TAMRA, 6-carboxytetramethylrhodamine and WHO, World Health Organization
SGM
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.47014-0
2007-05-01
2024-04-27
Loading full text...

Full text loading...

/deliver/fulltext/jmm/56/5/603.html?itemId=/content/journal/jmm/10.1099/jmm.0.47014-0&mimeType=html&fmt=ahah

References

  1. CDC 1997; Isolation of avian influenza A (H5N1) viruses from humans – Hong Kong, May–December 1997. MMWR Morb Mortal Wkly Rep 46:1204–1207
     [Google Scholar]
  2. CDC 2004; Cases of influenza A (H5N1) – Thailand, 2004. MMWR Morb Mortal Wkly Rep 53:100–103
     [Google Scholar]
  3. Chen W., Xu Z., Mu J., He B., Yang L., Lin L., Meng S., Mu F., Gan H. other authors 2004; Real-time quantitative fluorescent reverse transcriptase-PCR for detection of severe acute respiratory syndrome-associated coronavirus RNA. Mol Diagn 8:231–235 [CrossRef]
     [Google Scholar]
  4. FAO 2005; Economic and social impacts of avian influenza. http://www.fao.org/AG/AGAINFO/subjects/en/health/diseases-cards/cd/documents/Economic-and-social-impacts-of-avian-influenza-Geneva.pdf
  5. Holland P. M., Abramson R. D., Watson R., Gelfand D. H. 1991; Detection of specific polymerase chain reaction product by utilizing the 5′ to 3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 88:7276–7280 [CrossRef]
     [Google Scholar]
  6. Kubar A., Saygun I., Ozdemir A., Yapar M., Slots J. 2005; Real-time polymerase chain reaction quantification of human cytomegalovirus and Epstein–Barr virus in periodontal pockets and the adjacent gingiva of periodontitis lesions. J Periodontal Res 40:97–104 [CrossRef]
     [Google Scholar]
  7. Kuypers J., Wright N., Ferrenberg J., Huang M. L., Cent A., Corey L., Morrow R. 2006; Comparison of real-time PCR assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children. J Clin Microbiol 44:2382–2388 [CrossRef]
     [Google Scholar]
  8. Lee M. S., Chang P. C., Shien J. H., Cheng M. C., Shieh H. K. 2001; Identification and subtyping of avian influenza viruses by reverse transcription-PCR. J Virol Methods 97:13–22 [CrossRef]
     [Google Scholar]
  9. Liu J., Xiao H., Lei F., Zhu Q., Qin K., Zhang X. W., Zhang X. L., Zhao D., Wang G. other authors 2005; Highly pathogenic H5N1 influenza virus infection in migratory birds. Science 309:1206 [CrossRef]
     [Google Scholar]
  10. Livak K. J., Flood S. J., Marmaro J., Giusti W., Deetz K. 1995; Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 4:357–362 [CrossRef]
     [Google Scholar]
  11. Munch M., Nielsen L. P., Handberg K. J., Jorgensen P. H. 2001; Detection and subtyping (H5 and H7) of avian type A influenza virus by reverse transcription-PCR and PCR-ELISA. Arch Virol 146:87–97 [CrossRef]
     [Google Scholar]
  12. Ng E. K. O., Cheng P. K. C., Ng A. Y. Y., Hoang T. L., Lim W. W. L. 2005; Influenza A H5N1 detection. Emerg Infect Dis 11:1303–1305 [CrossRef]
     [Google Scholar]
  13. Palmer S., Wiegand A. P., Maldarelli F., Bazmi H., Mican J. M., Polis M., Dewar R. L., Planta A., Liu S. other authors 2003; New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol 41:4531–4536 [CrossRef]
     [Google Scholar]
  14. Perkins S. M., Webb D. L., Torrance S. A., El Saleeby C., Harrison L. M., Aitken J. A., Patel A., DeVincenzo J. P. 2005; Comparison of a real-time reverse transcriptase PCR assay and a culture technique for quantitative assessment of viral load in children naturally infected with respiratory syncytial virus. J Clin Microbiol 43:2356–2362 [CrossRef]
     [Google Scholar]
  15. Starick E., Romer-Oberdorfer A., Werner O. 2000; Type- and subtype-specific RT-PCR assays for avian influenza A viruses (AIV). J Vet Med B Infect Dis Vet Public Health 47:295–301 [CrossRef]
     [Google Scholar]
  16. Tran T. H., Nguyen T. L., Nguyen T. D., Luong T. S., Pham P. M., Nguyen V. C., Pham T. S., Vo C. D., Le T. Q. other authors 2004; Avian influenza A (H5N1) in 10 patients in Vietnam. N Engl J Med 350:1179–1188 [CrossRef]
     [Google Scholar]
  17. Viseshakul N., Thanawongnuwech R., Amonsin A., Suradhat S., Payungporn S., Keawchareon J., Oraveerakul K., Wongyanin P., Plitkul S. other authors 2004; The genome sequence analysis of H5N1 avian influenza A virus isolated from the outbreak among poultry populations in Thailand. Virology 328:169–176 [CrossRef]
     [Google Scholar]
  18. Wang C., Tian X., Cao Z., Yu X., Sun M., Chen X., Wang H., Tian K. 2004; Establishment of H5 avian influenza virus capture ELISA employing biotin labeled monoclonal antibody. Acta Lab Anim Sci Sin 12:204–207
     [Google Scholar]
  19. WHO 2005; Recommended laboratory tests to identify avian influenza A virus in specimens from humans. http://www.who.int/csr/disease/avian_influenza/guidelines/avian_labtests2.pdf
  20. WHO 2006; Cumulative number of confirmed human cases of avian influenza A/(H5N1) reported to WHO. http://www.who.int/csr/disease/avian_influenza/country/cases_table_2006_05_23/en/index.html
  21. Wright K. E., Wilson G. A. R., Novosad D., Dimock C., Tan D., Weber J. M. 1995; Typing and subtyping of influenza viruses in clinical samples by PCR. J Clin Microbiol 33:1180–1184
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.47014-0
Loading
Real-time RT-PCR for H5N1 avian influenza A virus detection
J. Med. Microbiol. 56, 603 (2007); https://doi.org/10.1099/jmm.0.47014-0
/content/journal/jmm/10.1099/jmm.0.47014-0
/content/journal/jmm/10.1099/jmm.0.47014-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error