高致病性A(H5N8)亚型禽流感病毒NA基因核酸检测方法的建立  

Establishment of a Real-time PCR Method for Detection of the NA Gene of the Highly Pathogenic Avian Influenza A (H5N8) Virus

作　　者：李晓丹[1] 张烨[1] 薄洪[1] 杨磊[1] 舒跃龙[1] 王大燕[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,卫生和计划生育委员会医学病毒和病毒病重点实验室,北京102206

出　　处：《病毒学报》2017年第3期457-461,共5页Chinese Journal of Virology

基　　金：国家重点研发计划(项目号:2016YFD0500208),题目:人感染动物流感病毒

摘　　要：建立以Real-time PCR为基础的新型高致病性A(H5N8)亚型禽流感病毒NA基因检测方法。针对2016年6月起频繁暴发的H5N8禽流感疫情,从GenBank和Global Initiative on Sharing All Influenza Data(GISAID)下载2014年以来的H5N8亚型禽流感病毒的NA序列,通过序列比对,在相对保守区域设计适用于实时荧光逆转录聚合酶链式反应(rRT-PCR)的引物和探针。选用28株不同NA亚型的流感病毒进行特异性验证,结果显示本文设计的引物探针组合能够特异性检测高致病性H5N8亚型禽流感病毒的NA基因。灵敏度检测结果显示,本文设计的引物探针组合能检出最低23个拷贝的RNA。本文建立了高致病性H5N8亚型禽流感病毒NA基因特异性荧光定量检测方法,与世界卫生组织(WHO)推荐的A型流感病毒M基因、H5基因检测引物探针的最低检测限一致,可以组合用于H5N8亚型禽流感病毒的检测。We wished to develop an assay for identification of the neuraminidase （NA） gene of thehighly pathogenic avian influenza A（H5N8） virus NA gene by real-time reverse transcription-polymerase chain reaction method. NA gene sequences of theavian influenza HSN8 virus deposited in GenBank and GISAID since 2014 were downloaded. After alignment, primers and a probe targeted to the conserved region were designed. Cross-reactivity was tested using 28 influenza viruses with different NA subtypes. Results showed that only the NA gene originating from the avian influenza A（HSN8） virus could be detected. The results of sensitivity assay revealed that the detection limit of detection of the primers and probe designed in this study was 23 RNA copies per reaction. Overall,we od foridentification of NA gene identification of the highly method can be applied to the eharacterizationof this virus tion-recommended protocol for detection of the matrixand offered an efficient and reliable diagnostic methpathogenic avian influenza A（HSN8） virus. Our in combinationwith the World Health Organiza- hemagglutiningenes of this virus.

关 键 词：流感 A(H5N8) 实时荧光逆转录聚合酶链式反应(Real-time RT-PCR) 

分 类 号：R373.1[医药卫生—病原生物学]