Skip to main content

Advertisement

SpringerLink
Log in

Rapid Subtyping and Pathotyping of Avian Influenza Virus using Chip-based RT-PCR

  • Original Article
  • Published:
BioChip Journal Aims and scope Submit manuscript

Abstract

Frequent outbreaks of avian influenza viruses (AIV), which are influenza A viruses, result in heavy economic and national damages. However, current diagnostic methods require about 5–7 days for AIV to be confirmed, which allows enough time for the widespread dissemination of the virus. Therefore, there is a need for rapid and accurate diagnosis of AIV. Here, we developed a diagnostic method using chip-based quantitative reverse transcription-polymerase chain reaction (RT-qPCR) for the rapid identification of the major subtypes H5, H7, and H9 of AIV that influence the pathogenicity of the virus. Specific primer pairs and probes were designed to target the highly conserved Matrix (M) gene regions in influenza A viruses. Specific primer pairs and probes for subtyping were developed by targeting the conserved sequences in the haemagglutinin (HA) gene specific to each subtype. For pathotyping, specific primer pairs and probes for the highly pathogenic avian influenza viruses (HPAIV) were generated by targeting the HA cleavage site, which is highly related to the pathogenicity of the virus. Using the primer pairs and probe sets, we synthesized the major sequence of each type and constructed a standard curve to confirm their detection limit. In addition, we used a chip-based RT-qPCR method to test the samples collected from the faeces of wild birds, cloacal and oropharyngeal swab samples of infected chicken, and allantoic fluids samples incubated with inoculated eggs. As a result, AIV was accurately detected in all samples. Our study shows that by using a single DNA chip in a portable chip-based RT-qPCR device and specific primer pairs and probes, rapid and accurate AIV detection and subtyping and pathotyping is possible, which will reduce the spread of the virus, thereby reducing the economic and national damages.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Table 1
Figure 1
Figure 2
Table 2

Similar content being viewed by others

References

  1. Hause, B.M., Ducatez, M., Collin, E.A., Ran, Z., Liu, R., Sheng, Z., Armien, A., Kaplan, B., Chakravarty, S., Hoppe, A.D., Webby, R.J., Simonson, R.R. & Li, F. Isolation of a novel swine influenza virus from Oklahoma in 2011 which is distantly related to Human Influenza C viruses. PLoS Pathog.9, e100 3176 (2013).

    Article  Google Scholar 

  2. Perdue, M.L. & Swayne, D.E. Public health risk from avian influenza viruses. Avian Dis.49, 317–327 (2005).

    Article  Google Scholar 

  3. Senne, D., Panigrahy, B., Kawaoka, Y., Pearson, J. E., Süss, J., Lipkind, M., Kida, H. & Webster, R.G. Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the HA cleavage site as a marker of pathogenicity potential. Avian Dis.40, 425–437 (1996).

    Article  CAS  Google Scholar 

  4. Koopmans, M., Wilbrink, B., Conyn, M., Natrop, G., van der Nat, H., Vennema, H., Meijer, A., van Steenbergen, J., Fouchier, R. & Osterhaus, A. Transmission of H7N7 avian influenza A virus to human beings during a large outbreak in commercial poultry farms in the Netherlands. Lancet363, 587–593 (2004).

    Article  Google Scholar 

  5. Swayne, D.E. 2016. Animal Influenza, Second Edition, in: Suarez, D.L. (Eds.), Influenza A virus. John Wiley & Sons, Inc., Hobokenk, pp. 1–30.

    Chapter  Google Scholar 

  6. Kim, H.-R., Lee, Y.-J., Park, C.-K., Oem, J.-K., Lee, O.-S., Kang, H.-M., Choi, J.-G. & Bae, Y.-C. Highly pathogenic avian influenza (H5N1) outbreaks in wild birds and poultry, South Korea. Emerging Infect. Dis.18, 480–483 (2012).

    Article  Google Scholar 

  7. Hoffmann, E., Stech, J., Guan, Y., Webster, R.G. & Perez, D.R. Universal primer set for the full-length amplification of all influenza A viruses. Arch. Virol.146, 2275–2289 (2001).

    Article  CAS  Google Scholar 

  8. Behlke, M.A., Huang, L., Bogh, L., Rose, S. & Devor, E. J. Fluorescence and fluorescence applications. Integr. DNA Technol. 1–13. (2005).

  9. Marras, S.A.E., Kramer, F.R. & Tyagi, S. Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res.30, e122 (2002).

    Article  Google Scholar 

  10. Atkinson, C., Emery, V. & Griffiths, P. Development of a novel single tube nested PCR for enhanced detection of cytomegalovirus DNA from dried blood spots. J. Virol. Methods196, 40–44 (2014).

    Article  CAS  Google Scholar 

  11. Koshkin, A.A., Singh, S.K., Nielsen, P., Rajwanshi, V.K., Kumar, R., Meldgaard, M., Olsen, C.E. & Wengel, J. LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron54, 3607–3630 (1998).

    Article  CAS  Google Scholar 

  12. Astakhova, K. Toward non-enzymatic ultrasensitive identification of single nucleotide polymorphisms by optical methods. Chemosensors2, 193–206. (2014).

    Article  Google Scholar 

  13. Ugozzoli, L.A., Latorra, D., Pucket, R. Arar, K. & Hamby, K. Real-time genotyping with oligonucleotide probes containing locked nucleic acids. Anal. Biochem.324, 143–152 (2004).

    Article  CAS  Google Scholar 

  14. Ahn, J.J., Kim, Y., Hong, J.Y., Kim, G.W., Kim, S. Y. & Hwang, S.Y. Probe-based fluorescence melting curve analysis for differentiating Larimichthys polyactis and Larimichthys crocea. Food Anal. Methods9, 2036–2041 (2016).

    Article  Google Scholar 

  15. Braasch, D.A. & Corey, D.R. Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. Chem. Biol.8, 1–7 (2001).

    Article  CAS  Google Scholar 

  16. Schmittgen, T.D. & Livak, K.J. Analyzing real-time PCR data by the comparative C T method. Nat. Protoc.3, 1101–1108 (2008).

    Article  CAS  Google Scholar 

  17. Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W. & Shipley, G.L. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem.55, 611–622 (2009).

    Article  CAS  Google Scholar 

  18. Schefe, J.H., Lehmann, K.E., Buschmann, I.R., Unger, T. & Funke-Kaiser, H. Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s C T difference” formula. J. Mol. Med.84, 901–910 (2006).

    Article  CAS  Google Scholar 

  19. Holland, P.M., Abramson, R.D., Watson R. & Gelfand, D.H. Detection of specific polymerase chain reaction product by utilizing the 5′—3′exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. U. S. A.88, 7276–7280 (1991).

    Article  CAS  Google Scholar 

  20. Arya, M., Shergill, I. S., Williamson, M., Gommersall, L., Arya, N. & Patel, H. R. Basic principles of real-time quantitative PCR. Expert Rev. Mol. Diagn.5, 209–219 (2005).

    Article  CAS  Google Scholar 

  21. Hall, T.A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser.41, 95–98 (1999).

    CAS  Google Scholar 

  22. Kwon, S.H., Lee, S., Jang, J., Seo, Y. & Lim, H.Y. A point-of-care diagnostic system to influenza viruses using chip-based ultra-fast PCR. J. Med. Virol.90, 1019–1102. (2018).

    Article  CAS  Google Scholar 

  23. Schrader, C., Schielke, A., Ellerbroek, L. & Johne, R. PCR inhibitors-occurrence, properties and removal. J. Appl. Microbiol.113, 1014–1026 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through Animal Disease Management Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (116103032HD020).

Author information

Authors and Affiliations

  1. Department of Bio-Nanotechnology, Hanyang University, 55, Hanyangdeahak-ro, Sangnok-gu, Ansan, Gyeonggi-do, 15588, Korea

    Na young Kwon, Seol young Kim & Seung Yong Hwang

  2. BioCore Co. Ltd, 33, Digital-ro 9-gil, Geumcheon-gu, Seoul, 08511, Korea

    Jeong Jin Ahn, Ji-Hoon Kim & Seung Yong Hwang

  3. College of Veterinary Medicine, Konkuk University, 120, Neungdong-ro, Gwangjin-gu, Seoul, 05029, Korea

    Ji Ho Lee & Chang-Seon Song

  4. US Agricultural Department, 355 E Hancock Ave # 320, Athens, GA, 30601, USA

    Jung-Hoon Kwon

Authors
  1. Na young Kwon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeong Jin Ahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ji-Hoon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Seol young Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ji Ho Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jung-Hoon Kwon
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chang-Seon Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Seung Yong Hwang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Seung Yong Hwang.

Additional information

Conflict of Interests

The authors declare no competing financial interests.

These authors contrilbuted equally.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kwon, N.y., Ahn, J.J., Kim, JH. et al. Rapid Subtyping and Pathotyping of Avian Influenza Virus using Chip-based RT-PCR. BioChip J 13, 333–340 (2019). https://doi.org/10.1007/s13206-019-3405-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13206-019-3405-2

Keywords

Search

Navigation