Rapid Differential Detection of H1 and H3 Subtype Swine Influenza Viruses by a TaqMan-MGB Based Duplex One-Step Real Time RT-PCR Assay

Abstract

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. 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 evaluated. The duplex RRT-PCR possessed the detection limit of 5 copies/μL HA plasmid for each of the EA H1N1, Pdm09 H1N1 and HL H3N2 subtype SIVs, and matched an overall detection sensitivity of 100% and specificity of 91.67% 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. This developed assay could be very helpful for rapid differential detection and routine surveillance of EA H1N1, Pdm09 H1N1 and HL H3N2 subtype SIVs in China.

Clinical impact of the PAI-1 4G/5G polymorphism in Chinese patients with venous thromboembolism

“…It is worth noting that the major advantages of the TaqMan-MGB probe are that it forms a stable binding to the target gene and suppresses the production of non-specific amplification products [29,30]. Recently, TaqMan-MGB RT-PCR has been used to detect drug resistance in Helicobacter pylori [31], subtype analysis of swine influenza virus [32], mutation analysis of hereditary optic neuropathy [33] and SNPs detection in diabetes susceptibility genes [34]. Given that there are currently no standardized detection procedures for FVL and PAI-1 genotypes in China, our study may lay the foundation for the practical application of the TaqMan-MGB RT-PCR method in the future.…”