Vet Med - Czech, 2017, 62(4):211-220 | DOI: 10.17221/179/2016-VETMED

Multiplex real-time reverse transcription polymerase chain reaction for differential detection of H5, N1, and N8 genes of highly pathogenic avian influenza virusesOriginal Paper

Y.R. Park1, E.M. Kim1, Y.J. Lee2, S.G. Yeo1, C.K. Park*,1
1 College of Veterinary Medicine and Animal Disease Intervention Center, Kyungpook National University, Daegu, Republic of Korea
2 Avian Disease Division, Animal and Plant Quarantine Agency, Gimcheon, Republic of Korea

Rapid and differential diagnosis of highly pathogenic avian influenza virus (HPAIV) subtype H5 is essential for the effective prevention and control of outbreaks caused by this pathogen. In this study, we describe a one-step multiplex real-time reverse transcription polymerase chain reaction (mRRT-PCR), using H5-, N1-, and N8-specific primers and probes, for differential detection of two HPAIVs (H5N1 and H5N8) and other H5-subtype AIVs. Using the mRRT-PCR assay, we were able to detect H5N1, H5N8, and other H5-subtype AIVs in a one-tube reaction, with high specificity; furthermore, using an in silico PCR program, we confirmed that this assay can detect nearly all H5, N1, and N8 genes of AIVs currently available in the Influenza Sequence Database. The limit of detection of the assay was determined to be as low as 100 copies/reaction for each target gene, and was comparable to limits of detection of previously reported mRRT-PCR assays. Thus, the mRRT-PCR assay described here can serve as a rapid and reliable differential diagnostic tool for the monitoring and surveillance of H5N1, H5N8, and other H5-subtype AIVs in countries where these pathogens are problematic.

Keywords: highly pathogenic avian influenza virus; real-time RT-PCR; H5N1; H5N8

Published: April 30, 2017  Show citation

ACS AIP APA ASA Harvard Chicago Chicago Notes IEEE ISO690 MLA NLM Turabian Vancouver
Park YR, Kim EM, Lee YJ, Yeo SG, Park CK. Multiplex real-time reverse transcription polymerase chain reaction for differential detection of H5, N1, and N8 genes of highly pathogenic avian influenza viruses. Vet Med - Czech. 2017;62(4):211-220. doi: 10.17221/179/2016-VETMED.
Share...
Download citation
  • BibTeX (.bib)
  • Bookends (.ris)
  • EasyBib (.ris)
  • EndNote (.enw)
  • EndNote 8 (.xml)
  • ISI WoS (.isi)
  • Medlars (.medlars)
  • Mendeley (.ris)
  • MODS (.xml)
  • Papers (.ris)
  • RefWorks (.txt)
  • RefManager (.ris)
  • RIS (.ris)
  • MS Word (.xml)
  • Zotero (.ris)
    • Open full article

    References

    1. Choi JH, Kim MS, Lee JY, Lee NJ, Kwon D, Kang MG, Kang C (2013): Development and evaluation of multiplex realtime RT-PCR assays for seasonal, pandemic A/H1pdm09 and avian A/H5 influenza viruses detection. Journal of Microbiology 51, 252-257. Go to original source... Go to PubMed...
    2. Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH (1997): Multiplex PCR: Critical Parameters and Stepby-Step Protocol. BioTechniques 23, 504-511. Go to original source... Go to PubMed...
    3. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR (2001): Universal primer set for the full-length amplification of all influenza A viruses. Archives of Virology 146, 2275- 2289. Go to original source... Go to PubMed...
    4. Jeong J, Kang HM, Lee EK, Song BM, Kwon YK, Kim HR, Lee KJ, Hong MS, Jang I, Choi KS, Kim JY, Lee HJ, Moon OK, Jeong W, Choi J, Baek JH, Joo YS, Park YH, Lee HS, Lee YJ (2014): Highly pathogenic avian influenza virus (H5N8) in domestic poultry and its relationship with migratory birds in South Korea during 2014. Veterinary Microbiology 173, 249-257. Go to original source... Go to PubMed...
    5. Kalendar R, Lee D, Schulman AH (2011): Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Genomics 98, 137-144. Go to original source... Go to PubMed...
    6. Kim EM, Park CK (2015): Single-step real-time reverse transcription-polymerase chain reaction for simultaneous detection of H5N1 and H5N8 highly pathogenic avian influenza viruses. Journal of Animal and Veterinary Advances 14, 161-166.
    7. Kim HR, Park CK, Lee YJ, Woo GH, Lee KK, Oem JK, Kim SH, Jean YH, Bae YC, Yoon SS, Roh IS, Jeong OM, Kim HY, Choi JS, Byun JW, Song YK, Kwon JH, Joo YS (2010): An outbreak of highly pathogenic H5N1 avian influenza in Korea, 2008. Veterinary Microbiology 141, 362-366.
    8. Kim HR, Lee YJ, Park CK, Oem JK, Lee OS, Kang HM, Choi JG, Bae YC (2012): Highly pathogenic avian influenza (H5N1) outbreaks in wild birds and poultry, South Korea. Emerging Infectious Diseases 18, 480-483. Go to original source...
    9. Kim HR, Oem JK, Bae YC, Kang MS, Lee HS, Kwon YK (2013): Application of real-time reverse transcription polymerase chain reaction to the detection the matrix, H5 and H7 genes of avian influenza viruses in field samples from South Korea. Virology Journal 10, 85. Go to original source... Go to PubMed...
    10. Lee CW, Suarez DL (2004): Application of real-time RTPCR for the quantitation and competitive replication study of H5 and H7 subtype avian influenza virus. Journal of Virological Methods 119, 151-158. Go to original source... Go to PubMed...
    11. Lee YJ, Kang HM, Lee EK, Song BM, Jeong J, Kwon YK, Kim HR, Lee KJ, Hong MS, Jang I, Choi KS, Kim JY, Lee HJ, Kang MS, Jeong OM, Baek JH, Joo YS, Park YH, Lee HS (2014): Novel reassortant influenza A (H5N8) viruses, South Korea. Emerging Infectious Diseases 20, 1087- 1089. Go to original source... Go to PubMed...
    12. Li P, Zhang J, Muller CP, Chen J, Yang Z, Zhang R, Li J, He Y (2008): Development of a multiplex real-time polymerase chain reaction for the detection of influenza virus type A including H5 and H9 subtypes. Diagnostic Microbiology and Infectious Disease 61, 192-197. Go to original source... Go to PubMed...
    13. Markoulatos P, Siafakas N, Moncany M (2002): Multiplex polymerase chain reaction: a practical approach. Journal of Clinical Laboratory Analysis 16, 47-51. Go to original source... Go to PubMed...
    14. Monne I, Ormelli S, Salviato A, De Battisti C, Bettini F, Salomoni A, Drago A, Zecchin B, Capua I, Cattoli G (2008): Development and validation of a one-step realtime PCR assay for simultaneous detection of subtype H5, H7, and H9 avian influenza viruses. Journal of Clinical Microbiology 46, 1769-1773. Go to original source... Go to PubMed...
    15. Navarro E, Serrano-Heras G, Castanoa MJ, Solera J (2015): Real-time PCR detection chemistry. Clinica Chimica Acta 439, 231-250. Go to original source... Go to PubMed...
    16. Parida M, Shukla J, Sharma S, Santhosh SR, Ravi V, Mani R, Thomas M, Khare S, Rai A, Ratho RK, Pujari S, Mishra B, Rao PVL, Vijayaraghavan R (2011): Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of the swine-origin influenza A H1N1 virus. Journal of Molecular Diagnostics 13, 100-107. Go to original source... Go to PubMed...
    17. Payungporn S, Chutinimitkul S, Chaisingh A, Damrongwantanapokin S, Buranathai C, Amonsin A, Theamboonlers A, Poovorawan Y (2006): Single step multiplex real-time RT-PCR for H5N1 influenza A virus detection. Journal of Virological Methods 131, 143-147. Go to original source... Go to PubMed...
    18. Sidoti F, Rizzo F, Costa C, Astegiano S, Curtoni A, Mandola ML, Cavallo R, Bergallo M (2010): Development of real time RT-PCR assays for detection of type A influenza virus and for subtyping of avian H5 and H7 hemagglutinin subtypes. Molecular Biotechnology 44, 41-50. Go to original source... Go to PubMed...
    19. Smith GJD, Donis RO, World Health Organization/World Organisation for Animal Health/Food and Agriculture Organization (WHO/OIE/FAO) H5 Evolution Working Group (2015): Nomenclature updates resulting from the evolution of avian influenza A(H5) virus clades 2.1.3.2a, 2.2.1, and 2.3.4 during 2013-2014. Influenza and Other Respiratory Viruses 9, 271-276. Go to original source... Go to PubMed...
    20. Spackman E, Senne DA, Bulaga LL, Trock S, Suarez DL (2003): Development of multiplex real-time RT-PCR as a diagnostic tool for avian influenza. Avian Diseases 47, 1087-1090. Go to original source... Go to PubMed...
    21. Suwannakarn K, Payungporn S, Chieochansin T, Samransamruajkit R, Amonsin A, Songserm T, Chaisingh A, Chamnanpood P, Chutinimitkul S, Theamboonlers A, Poovorawan Y (2008): Typing (A/B) and subtyping (H1/ H3/H5) of influenza A viruses by multiplex real-time RTPCR assays. Journal of Virological Methods 152, 25-31. Go to original source... Go to PubMed...
    22. Tsukamoto K, Noguchi D, Suzuki K, Shishido M, Ashizawa T, Kim MC, Lee YJ, Tada T (2010): Broad detection of diverse H5 and H7 hemagglutinin genes of avian influenza viruses by real-time reverse transcription-PCR using primer and probe sets containing mixed bases. Journal of Clinical Microbiology 48, 4275-4278. Go to original source... Go to PubMed...
    23. WHO - World Health Organization (2014): WHO information for molecular diagnosis of influenza virus - update. World Health Organization, Geneva.
    24. WHO - World Health Organization (2015): Evolution of the influenza A (H5) haemagglutinin; WHO/OIE/FAO H5 working group reports a new clade designated 2.3.4.4. World Health Organization, Geneva.
    25. Wu C, Cheng X, He J, Lv X, Wang J, Deng R, Long Q, Wang X (2008): A multiplex real-time RT-PCR for detection and identification of influenza virus types A and B and subtypes H5 and N1. Journal of Virolological Methods 148, 81-88. Go to original source... Go to PubMed...

    This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY NC 4.0), which permits non-comercial use, distribution, and reproduction in any medium, provided the original publication is properly cited. No use, distribution or reproduction is permitted which does not comply with these terms.


    Return to the content