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Rapid differential detection of subtype H1 and H3 swine influenza viruses using a TaqMan-MGB-based duplex one-step real-time RT-PCR assay

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Abstract

Swine influenza is an economically important respiratory disease in swine, but it also constantly poses a threat to human health. Therefore, developing rapid, sensitive, and efficient detection methods for swine influenza virus (SIV) is important. By aligning the haemagglutinin (HA) gene sequences of SIVs circulating in China over a 10-year period, an H1 primer-probe set targeting both Eurasian avian-like H1N1 (EA H1N1) and pandemic 2009 H1N1 ((H1N1)pdm09) lineages plus a H3 primer-probe set targeting the prevalent human-like H3N2 (HL H3N2) subtype were designed. Subsequently, a TaqMan-MGB-based duplex one-step real-time RT-PCR (RT-qPCR) assay was established and evaluated. The duplex RT-qPCR has a detection limit of 5 copies/μL of HA plasmid for EA H1N1, (H1N1)pdm09, and HL H3N2 subtype SIVs, and its overall detection sensitivity of 100% and specificity of 91.67% matches that of traditional virus isolation through chicken embryo inoculation using experimentally infected mouse lung samples. The method showed high repeatability both within run and between runs, and there was no cross-reactivity against several other porcine viruses that are commonly circulating in China. Furthermore, the duplex RT-qPCR method revealed a higher prevalence of subtype H1 than subtype H3 in 166 nasal swabs from pigs collected from one slaughterhouse between October and December 2019. This assay could be very helpful in the rapid differential detection and routine surveillance of EA H1N1, (H1N1)pdm09, and HL H3N2 SIVs in China.

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Acknowledgements

This work was supported by the National Key Research and Development Project of China (2016YFD0500202-1), the Jiangsu Provincial Natural Science Fund for Excellent Young Scholars (BK20170068), the Special Financial Grant from the China Postdoctoral Science Foundation (2017T100410), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Jiangsu Qinglan Project, and the High-End Talent Support Program of Yangzhou University.

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Authors and Affiliations

  1. Animal Infectious Diseases Laboratory, College of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, 225009, Jiangsu, China

    Kaibiao Chen, Ming Kong, Jiao Liu, Jun Jiao, Zixiong Zeng, Liwei Shi, Xinxin Bu, Yayao Yan, Yu Chen, Ruyi Gao, Xiaowen Liu, Xiaoquan Wang, Jiao Hu, Shunlin Hu, Xinan Jiao, Xiufan Liu & Min Gu

  2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China

    Xiaowen Liu, Xiaoquan Wang, Jiao Hu, Shunlin Hu, Xinan Jiao, Xiufan Liu & Min Gu

  3. Jiangsu Key Laboratory of Zoonoses, Yangzhou University, Yangzhou, China

    Xiaowen Liu, Xiaoquan Wang, Jiao Hu, Shunlin Hu, Xinan Jiao, Xiufan Liu & Min Gu

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  1. Kaibiao Chen
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Corresponding author

Correspondence to Min Gu.

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The authors declare that they have no conflict of interest.

Ethical approval

This study was performed in strict concordance with the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. The animal experiment protocol was approved by the Jiangsu Province Administrative Committee for Laboratory Animals (approval no. SYXK-SU-2017-0044) and complied with the Guidelines of Jiangsu Laboratory Animal Welfare and Ethics of Jiangsu Administrative Committee of Laboratory Animals.

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Chen, K., Kong, M., Liu, J. et al. Rapid differential detection of subtype H1 and H3 swine influenza viruses using a TaqMan-MGB-based duplex one-step real-time RT-PCR assay. Arch Virol 166, 2217–2224 (2021). https://doi.org/10.1007/s00705-021-05127-6

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