摘要
根据GenBank中H6亚型禽流感病毒(AIV)的HA基因、N1亚型AIV的NA基因和所有亚型AIV的M基因序列,分别设计并筛选出3对特异性引物,优化引物之间的浓度,建立了H6N1亚型AIV三重RT-PCR检测方法。对该法进行特异性、敏感性和临床样品检测。结果显示,所建立的方法对H6N1亚型AIV可特异性扩增出447(H6亚型AIV)、325(N1亚型AIV)和669bp(AIV)目的条带,对H6亚型AIV扩增出447、669bp目的条带,对N1亚型AIV扩增出325、669bp目的条带,对其他亚型AIV仅扩增出669bp目的条带,对常见禽病病原体均未扩增出任何条带;该法对H6N1亚型AIV检测下限为0.1pg;305份临床样品检测结果与病毒分离鉴定一致。本研究所建立的H6N1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H6、N1亚型AIV和所有亚型AIV,为H6N1亚型AIV的检测提供1种简便、快速和有效的方法。
According to the sequences of HA,NA and M genes of H6,N1 and all subtypes avian influenza（AIV）in GenBank,three pairs of specific primers were designed respectively,and the concentrations among primers and reaction contains were optimized,and specificity,sensitivity and clinical samples were tested to develop a triplex RT-PCR assay for detection H6N1.It showed that H6N1 subtype AIV could be amplified into three specific bands by this RT-PCR,the lengths of these bands were 447（H6-AIV）,325（N1-AIV）and 669bp（all AIV）,respectively.Samples containing H6 or N1subtype AIV could be amplified into two specific bands,which were 447and669 bp,325and 669 bp,respectively.Samples containing other subtypes AIV could be amplified into a 669 bp specific band.No specific band was amplified from other avian pathogenic virus.The limit of detection for H6N1 subtype AIV was 0.1pg.In the detection of 305 clinical samples,the results were coincident with the viral isolation completely.In conclusion,this triplex RT-PCR assay is a specific,sensitive method for the detection of H6,N1 and all subtypes AIV in the same tube.It can be applied in rapid differential diagnosis for clinical samples,and also provide a rapid,simple and effective tool to determine the infection of H6N1 and all subtypes AIV.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2015年第10期1626-1630,共5页
Chinese Journal of Veterinary Science
基金
广西特聘专家专项资助项目(2011B020)
广西科技攻关重大专项资助项目(14121003-4-2)
广西水产畜牧兽医局科研项目(桂渔牧科201452001)
