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A multiplex real-time RT-PCR assay for the detection of H1, H2 and H3 subtype avian influenza viruses

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Abstract

Avian influenza viruses (AIVs) are influenza A viruses, of which subtypes H1, H2 and H3 are highly transmissible in poultry and have the risk of transmission to human as well. It is important to establish an accurate, sensitive and convenient means of virus detection. In this study, we developed a multiplex real-time RT-PCR assay based on conserved sequences of the virus hemagglutinin and matrix, and designed primers and probes for the simultaneous and rapid detection of AIV subtypes H1, H2 and H3. We used different subtypes of AIVs and other avian respiratory viruses for evaluation of the specificity of this method. The results showed good sensitivity, specificity and reproducibility. The detection limit was 10–100 copies per reaction. The method also achieved good concordance with the virus isolation method when compared to 81 poultry samples evaluated. It provides a new method for detecting mixed infections of AIVs.

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Fig. 1

Data Availability

The datasets supporting the conclusions of this article are contained within the article and its supporting files.

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Funding

This study was funded by Grants from the National Science Foundation of the People’s Republic of China (Grant Number.32273092), Zhejiang Provincial Natural Science Foundation of China (Grant Number LY19H260006), the Fundamental Research Funds for the Central Universities (Grant Number 2022ZFJH003), and the Independent Task of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (Grant Number 2022ZZ02).

Author information

Authors and Affiliations

  1. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, National Clinical Research Center for Infectious Diseases, The First Affiliated Hospital, Zhejiang University, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China

    Sijing Yan, Fan Yang, Hangping Yao, Nanping Wu & Haibo Wu

  2. Jinan Microecological Biomedicine Shandong Laboratory, Jinan, 250117, China

    Hangping Yao, Nanping Wu & Haibo Wu

  3. Hangzhou Biotest Biotech Co.,Ltd, 27 Tuyi Road, Cangqian Street, Yuhang District, Hangzhou, 311121, Zhejiang, China

    Dalu Dong, Danna Wu & Chunsheng Ye

Authors
  1. Sijing Yan
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  2. Fan Yang
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  3. Hangping Yao
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  4. Dalu Dong
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  5. Danna Wu
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  6. Nanping Wu
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  7. Chunsheng Ye
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  8. Haibo Wu
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Contributions

SY, CY and HW conceived and designed the experiments; FY, DD, DW and HY performed the experiments; SY, NW and HW analyzed data; SY and HW wrote the paper. All authors contributed to the final review of the submitted version of the manuscript.

Corresponding authors

Correspondence to Chunsheng Ye or Haibo Wu.

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The animal experiment was approved by the First Affiliated Hospital, School of Medicine, Zhejiang University (Grant Number 2019-39).

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Edited by Juergen Richt.

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Yan, S., Yang, F., Yao, H. et al. A multiplex real-time RT-PCR assay for the detection of H1, H2 and H3 subtype avian influenza viruses. Virus Genes 59, 333–337 (2023). https://doi.org/10.1007/s11262-022-01963-z

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  • DOI: https://doi.org/10.1007/s11262-022-01963-z

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