H10N8禽流感病毒Real time RT-PCR检测方法的建立  

The development of real time RT-PCR for avian influenza virus H10N8

作　　者：薄洪[1] 高荣保[1] 李晓丹[1] 张烨[1] 邹淑梅[1] 朱云[1] 刘琳玉 王大燕[1] 舒跃龙[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京102206

出　　处：《中华实验和临床病毒学杂志》2015年第1期74-76,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：2014年国家自然科学基金资助（81373107）

摘　　要：目的 建立针对H10N8禽流感病毒特异、灵敏的real time RT-PCR检测方法.方法 通过比对现有H10N8病毒血凝素（HA）和神经氨酸酶（NA）全长基因序列,分别针对HA和NA基因设计引物和探针6套（P1-P6）和3套（P7-P9）,建立鉴别H10N8禽流感病毒real time RT-PCR方法,分别对筛选的引物进行特异性、灵敏性和临床标本检测适应性等评价.结果 特异性检测显示6套针对HA的引物和探针中的3套能够检测H10N8病毒,其中2套（P5和P6）与其他非H10亚型流感病毒无交叉反应;3套针对NA的引物和探针均能检测H10N8病毒,其中2套（P7和P9）与其他非N8亚型流感病毒无交叉反应.灵敏性检测显示,P5和P6以及P7和P9均能检出最大稀释度为109的H10N8病毒RNA,重复性显示4组的Ct值CV均＜3％.使用P6和P9对3例H10N8病例呼吸道标本份进行real time RT-PCR检测均为阳性,其中2例病毒分离阳性,4份病毒分离阳性的活禽相关环境样本检测均为阳性.结论 建立了H10N8禽流感病毒荧光定量PCR检测方法,该方法具有良好的特异性和敏感性.Objective Establish real time RT-PCR methods for detecting H10N8 subtype of avian influenza virusesand evaluate the sensitivity and specificity of this methods.Methods Compared the existing hemagglutinin （HA） and neuraminidase （NA） gene sequences of H10N8 subtype respectively,primers and probes of HA and NA were designed.Six sets of HA primers and probes （P1-P6） and three sets of NA primers and probes （P7-P9） were evaluated for specificity,sensitivity and adaptability.Results All primers and probes were used to detect the H10N8 subtype and other subtypes influenza viruses.Two sets of primers and probes （P5 and P6） showed no cross reaction with other H10 subtype influenza viruses.Three sets of NA primers and probes （P7,P8 and P9） could detect the H10N8 virus.P7and P9 showed no reaction with non-N8 subtype influenza viruses.Sensitivity assay showed that P5,P6,P7 and P9 could detect 10-9dilution of H10N8 viral RNA.Reproducibility assay indicated that the Ct values of four repetitive groups less than 3% CV.P6 and P9 detected the clinical respiratory specimens of three H10N8 cases and four viruses isolated from environmental samples of live poultry markets,real time RT-PCR test were all positive results.Conclusions Quantitative PCR methods for detecting H10N8 subtype of avian influenza virus have been established and the method holds good specificity and sensitivity.

关 键 词：流感病毒A型 鸟 逆转录聚合酶链反应 

分 类 号：R440[医药卫生—诊断学]