H10亚型和N8亚型禽流感病毒三重RT-PCR检测方法的建立

Development of a triplex RT-PCR assay for detection of H10N8 subtype avian influenza virus

关键词:  禽流感病毒  H10亚型  N8亚型  三重RT-PCR

Key words:  Avian influenza virus  H10N8 subtype  Triplex RT-PCR

摘要:

根据基因库中H10亚型禽流感病毒(AIV)HA基因、N8亚型AIV NA基因和所有亚型AIV M基因序列,分别设计筛选出3对特异性引物,优化引物之间的浓度,建立了H10亚型和N8亚型AIV三重RT-PCR检测方法。该法对H10N8亚型AIV可特异性扩增出267(H10亚型AIV)、464(N8亚型AIV)和693 bp(AIV)目的条带,对H10亚型AIV扩增出267、693 bp目的条带,对N8亚型AIV扩增出464、693 bp目的条带,对其他亚型AIV仅扩增出693 bp目的条带,对常见禽病病原体均未扩增出任何条带；该法对H10亚型和N8亚型AIV检测下限为10_³拷贝/μL。120份临床样品检测结果与病毒分离鉴定一致。本研究建立的H10亚型和N8亚型AIV 三重RT-PCR检测方法特异性强、灵敏度高,可同时快速鉴别检测AIV和H10亚型和N8亚型AIV,为H10N8亚型AIV的检测提供一种简便、快速和有效的方法。

Abstract:

According to the sequences of HA, NA and M genes of H10, N8 and all subtypes avian influenza (AIV) in GenBank, three pairs of specific primers were designed respectively, and the concentration of the primers were optimized to develop a triplex RT-PCR assay for detection H10 and N8 subtypes. It was shown that H10N8 subtype AIV could be amplified into three specific bands by this RT-PCR, the lengths of these bands were 267 (H10-AIV), 464 (N1-AIV) and 693 bp (all AIV), respectively. Samples containing H10 or N8 subtypeAIV could be amplified into two specific bands, which were 267 and 693 bp, 464 and 693 bp, respectively. Samples containing other subtypes AIV could be amplified into a 693 bp specific band. No specific band was amplified from other avian pathogenic virus. The limit of detection for H10 and N8 subtypes AIV was 103 copies/μL. In the detection of one hundred twenty clinical samples, the results of were triplex RT-PCR accorded with the viral isolation completely. In conclusion, this triplex RT-PCR assay is a specific, sensitive method for the detection of H10, N8 and all subtypes AIV in the same tube. It can be applied in rapid differential diagnosis for H10 subtype, N8 subtype and all subtype AIV, and also provide a rapid, simple and effective tool to determine the infection of H10N8 AIV.