Rapid differential detection of H1 and H3 subtype swine influenza viruses by a TaqMan-MGB based duplex one-step real time RT-PCR assay
Kaibiao Chen1, Ming Kong1, Jiao Liu1, Jun Jiao1, Zixiong Zeng1, Xinxin Bu1, Yayao Yan1, Yu Chen1, Ruyi Gao1, Xiaowen Liu1, 2, 3, Xiaoquan Wang1, 2, 3, Jiao Hu1, 2, 3, Shunlin Hu1, 2, 3, Xinan Jiao1,2,3, Min Gu1, 2, 3*, Xiufan Liu1, 2, 3
1Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu225009, China
2Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, China
3Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, China
Corresponding author:
Min Gu, Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu 225009, China
E-mail: gumin@yzu.edu.cn ; Telephone: +86 514 87972247; Fax: +86 514 87972591Abstract:
Background: Swine influenza is not only an economically important respiratory disease in swine, but also constantly poses a threat to human health. Hence, developing a rapid, sensitive and efficient detection method of swine influenza virus (SIV) is highly essential.
Method: By aligning the HA gene sequences of SIV circulating in China in recent 10 years, a H1 primer-probe set targeting both Eurasian avian-like H1N1 (EA H1N1) and Pandemic 2009 H1N1 (Pdm09 H1N1) lineages plus a H3 prime-probe set targeting the prevalent human-like H3N2 (HL H3N2) subtype were designed, respectively. Further, a TaqMan-MGB based duplex one-step real time RT-PCR (RRT-PCR) assay was established, and the sensitivity, specificity and repeatability were evaluated.
Results: The duplex RRT-PCR exhibited good sensitivity with the detection limit of 5 copies/μL HA plasmid for each of the EA H1N1, Pdm09 H1N1 and HL H3N2 subtype SIVs, and matched a detection accuracy of 94.4% (17/18) with traditional virus isolation through chicken embryo inoculation using experimentally infected mice lung samples. Besides, the method showed high repeatability both within-run and between-runs, and no cross-reactivity against some commonly circulated porcine viruses in China. Furthermore, the duplex RRT-PCR method revealed a relatively higher prevalent rate of H1 than H3 subtype SIV in 166 nasal swabs from pigs in some slaughterhouse during October~December, 2019.
Conclusions: This developed assay could be very helpful for rapid differential detection and routine surveillance of EA H1N1, Pdm H1N1 and HL H3N2 subtype SIVs in China.
Key words: swine influenza virus, H1 and H3, Taqman-MGB, real-time RT-PCR, virus detection