Development of a Multiplex RT-qPCR for the Detection of Different Clades of Avian Influenza in Poultry.

Tran Bac Le,Woonsung Na,Hye Kwon Kim,Dae Gwin Jeong,Daesub Song,Sun-Woo Yoon,Van Phan Le,Min-Suk Song

doi:10.3390/v12010100

Abstract

Since the initial detection of H5N1, a highly pathogenic avian influenza (HPAI) virus, in 1996 in China, numerous HPAI H5 lineages have been classified, and they continue to pose a threat to animal and human health. In this study, we developed a novel primer/probe set that can be employed to simultaneously detect pan-H5 HPAI and two clades, 2.3.2.1 and 2.3.4.4, of H5Nx viruses using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The sensitivity and specificity of these primer sets and probes were confirmed with a number of different subtypes of influenza virus and the H5-HA gene plasmid DNA. In particular, the multiplex RT-qPCR assay was successfully applied to the simultaneous detection of H5 HPAI and different virus clades in clinical field samples from a poultry farm. Therefore, this multiplex assay and a novel detection primer set and probes will be useful for the laboratory diagnosis and epidemiological field studies of different circulating H5 HPAI virus clades in poultry and migratory wild birds.

Highlights

Influenza A virus (IAV), a member of the genus Orthomyxovirus, causes a highly contagious respiratory disease in avian species and mammals [1]
For the screening of influenza positive field samples, all filed samples were analyzed using M-gene-specific reverse transcription quantitative polymerase chain reaction (RT-qPCR) according to the WHO manual on animal influenza diagnosis and surveillance [21], and only M-gene positive field samples were used in the present study
In order to enhance the simultaneous specificity of a target gene, the H5 highly pathogenic avian influenza (HPAI) HA gene sequences from different years and various clades detection of downloaded a target gene,from the H5

Summary

Introduction

Influenza A virus (IAV), a member of the genus Orthomyxovirus, causes a highly contagious respiratory disease in avian species and mammals [1]. Avian influenza (AI) viruses are classified into two pathotypes—low pathogenic avian influenza (LPAI) and HPAI—based on the pathogenicity in chickens and molecular determinants of the multibasic cleavage site motif in the hemagglutinin (HA) protein [3,4]. During the circulation of HPAI virus subtypes H5Nx and H7Nx in poultry, they proved to be highly pathogenic with mortality of up to 100%. According to the WHO report [5], different subtypes of H5 HPAI viruses pose a substantial threat to global health with high mortality rate and huge economic burden to poultry farms

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