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Peer-reviewedCC BY-NCJournal of Virus Eradication

Establishment and performance evaluation of quadruplex RT-qPCR method for universal avian influenza virus detection and H5/H7/H9 subtyping

  • Peng Liu1
  • Xiuwen Zhang2
  • Yi Wang3
  • Yan Liang4
  • Yangyang Liu4
  • Chenyang Wang1
  • Fei Yu5
  • Qing'an Han6
  • Yuzhong Ma7
7 affiliations
  1. 1College of Veterinary Medicine, Hebei Agricultural University, Baoding, 071001, China; Yanyun Laboratory, Baoding, 071001, China; Hebei Key Laboratory of Analysis and Control of Zoonotic Pathogenic Microorganisms, Baoding, 071001, China
  2. 2Baoding Key Laboratory for Biotic Resources and Diagnostics, CasMed Biotech Co., Ltd., Baoding, 071000, China; College of Life Sciences, Hebei Agricultural University, Baoding, 071001, China; Hebei Key Laboratory of Analysis and Control of Zoonotic Pathogenic Microorganisms, Baoding, 071001, China
  3. 3Yanyun Laboratory, Baoding, 071001, China; Baoding Key Laboratory for Biotic Resources and Diagnostics, CasMed Biotech Co., Ltd., Baoding, 071000, China; Hebei Key Laboratory of Analysis and Control of Zoonotic Pathogenic Microorganisms, Baoding, 071001, China
  4. 4Baoding Key Laboratory for Biotic Resources and Diagnostics, CasMed Biotech Co., Ltd., Baoding, 071000, China; Hebei Key Laboratory of Analysis and Control of Zoonotic Pathogenic Microorganisms, Baoding, 071001, China
  5. 5Yanyun Laboratory, Baoding, 071001, China; College of Life Sciences, Hebei Agricultural University, Baoding, 071001, China; Hebei Key Laboratory of Analysis and Control of Zoonotic Pathogenic Microorganisms, Baoding, 071001, China; Corresponding author. Yanyun Laboratory, Baoding, 071001, China.
  6. 6Hebei Animal Disease Control Center, Shijiazhuang, 050000, China; Hebei Key Laboratory of Analysis and Control of Zoonotic Pathogenic Microorganisms, Baoding, 071001, China; Corresponding author. Hebei Animal Disease Control Center, Shijiazhuang, 050000, China.
  7. 7College of Veterinary Medicine, Hebei Agricultural University, Baoding, 071001, China; Corresponding author.
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Abstract

Avian influenza viruses (AIVs) are zoonotic pathogens that exhibit periodic pandemics globally, posing significant public health risks. In this study, we developed a quadruplex RT-qPCR method that targets the M gene for universal AIV detection and the HA gene for simultaneous differentiation of the H5, H7, and H9 subtypes. The assay achieved a detection limit of 1 copy/μL for the H5, H7, and H9 reference materials based on HA gene quantification, equivalent to 5 copies per reaction. No cross-reactivity was observed with a panel of common avian pathogens. The reproducibility evaluation demonstrated both intra-batch and inter-batch coefficients of variation (CVs) to be less than 5%. Furthermore, the assay results were consistent with the expected outcomes for the reference materials tested, and the subtyping results were in agreement with whole-genome sequencing for the environmental samples. These findings indicate that the quadruplex RT-qPCR assay is a reliable tool for broad AIV detection and H5/H7/H9 subtyping, supporting its application in AIV surveillance.

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Cite

BibTeXRIS
BibTeX
@article{liu2026establishment,
  title = {{Establishment and performance evaluation of quadruplex RT-qPCR method for universal avian influenza virus detection and H5/H7/H9 subtyping}},
  author = {Peng Liu and Xiuwen Zhang and Yi Wang and Yan Liang and Yangyang Liu and Chenyang Wang and Fei Yu and Qing'an Han and Yuzhong Ma},
  journal = {Journal of Virus Eradication},
  year = {2026},
  month = oct,
  doi = {10.1016/j.jve.2026.100638},
  url = {https://doi.org/10.1016/j.jve.2026.100638},
  note = {CC BY-NC},
}
RIS
TY  - JOUR
TI  - Establishment and performance evaluation of quadruplex RT-qPCR method for universal avian influenza virus detection and H5/H7/H9 subtyping
AU  - Peng Liu
AU  - Xiuwen Zhang
AU  - Yi Wang
AU  - Yan Liang
AU  - Yangyang Liu
AU  - Chenyang Wang
AU  - Fei Yu
AU  - Qing'an Han
AU  - Yuzhong Ma
PY  - 2026
DA  - 2026/10/16
JO  - Journal of Virus Eradication
DO  - 10.1016/j.jve.2026.100638
UR  - https://doi.org/10.1016/j.jve.2026.100638
AB  - Avian influenza viruses (AIVs) are zoonotic pathogens that exhibit periodic pandemics globally, posing significant public health risks. In this study, we developed a quadruplex RT-qPCR method that targets the M gene for universal AIV detection and the HA gene for simultaneous differentiation of the H5, H7, and H9 subtypes. The assay achieved a detection limit of 1 copy/μL for the H5, H7, and H9 reference materials based on HA gene quantification, equivalent to 5 copies per reaction. No cross-reactivity was observed with a panel of common avian pathogens. The reproducibility evaluation demonstrated both intra-batch and inter-batch coefficients of variation (CVs) to be less than 5%. Furthermore, the assay results were consistent with the expected outcomes for the reference materials tested, and the subtyping results were in agreement with whole-genome sequencing for the environmental samples. These findings indicate that the quadruplex RT-qPCR assay is a reliable tool for broad AIV detection and H5/H7/H9 subtyping, supporting its application in AIV surveillance.
SN  - 2055-6640
SN  - 2055-6659
ER  -