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