Development and evaluation of a TaqMan MGB RT-PCR assay for detection of H5 and N8 subtype influenza virus

Fan Yang,Fumin Liu,Lihua Xu,Haibo Wu,Nanping Wu,Hangping Yao

doi:10.1186/s12879-020-05277-z

Abstract

BackgroundHighly pathogenic influenza A (H5N8) viruses have caused several worldwide outbreaks in birds and are of potential risk to humans. Thus, a specific, rapid and sensitive method for detection is urgently needed.MethodsIn the present study, TaqMan minor groove binder probes and multiplex real-time RT-PCR primers were designed to target the H5 hemagglutinin and N8 neuraminidase genes. A total of 38 strains of avian influenza viruses and other viruses were selected to test the performance of the assay.ResultsThe results showed that only H5 and N8 avian influenza viruses yielded a positive signal, while all other subtypes avian influenza viruses and other viruses were negative. High specificity, repeatability, and sensitivity were achieved, with a detection limit of 10 copies per reaction.ConclusionsThe developed assay could be a powerful tool for rapid detection of H5N8 influenza viruses in the future.

Highlights

Pathogenic influenza A (H5N8) viruses have caused several worldwide outbreaks in birds and are of potential risk to humans
HPAIV H5N8 virus spread from Asia to North America and caused an outbreak leading to heavy losses of poultry in commercial farms in 2014–15 [8, 9]
The reassortant HPAIV H5N2 was composed of Eurasian HPAIV H5N8 and North American lineage avian influenza viruses (AIVs), causing several outbreaks in Canada and North America [10], and affecting 232 farms in 15 states and more than 50 million birds in 2015 in the US [11]

Summary

Introduction

Pathogenic influenza A (H5N8) viruses have caused several worldwide outbreaks in birds and are of potential risk to humans. A specific, rapid and sensitive method for detection is urgently needed. Pathogenic avian influenza (HPAI) viruses are a threat to humans and animals, and cause considerable economic damage. The first H5N1 HPAI virus was detected in 1996 in a domestic goose in Guangdong, China (Gs/GD lineage), and caused deaths in wild birds, poultry and humans, and has since spread to over 80 countries in Asia, Europe, Africa and North America [1]. In early 2014, reassortant clade 2.3.4.4 H5N8 HPAI virus caused outbreaks in poultry in South Korea [4], and by late 2014, it had spread to Japan, Russia and Europe, with multiple cases reported from wild birds, including apparently healthy birds [5,6,7]. In 2016 and 2019, the HPAI H5N8 virus caused successive epidemics in Nigeria, Cameroon, Egypt, Saudi Arabia and Namibia [12–

Methods

Results

Conclusion