H5N6禽流感病毒双重实时荧光RT-PCR的建立与应用  

Development and application of dual real time RT-PCR for avian influenza H5N6 virus

作　　者：谭翰清[1] 程洁萍 朱颖梅[1] 谭海芳[1] 黄强[1] 苏乐斌 林凤[1] 邓廷国 黎碧坚[1] 

机构地区：[1]广东省肇庆市疾病预防控制中心,肇庆526060

出　　处：《中华实验和临床病毒学杂志》2017年第1期62-65,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：广东省医学科学技术研究基金（A2015573）;肇庆市科技创新计划项目（2015E1810）

摘　　要：目的 建立H5N6禽流感病毒TaqMan-MGB探针双重实时荧光RT-PCR,用于疑似病例快速诊断及活禽交易市场外环境监测.方法 根据GenBank中H5N6禽流感病毒HA和NA高保守序列设计特异引物和TaqMan-MGB探针,建立和优化双重实时荧光RT-PCR体系,进行特异性、灵敏度、重复性试验,并在临床应用中与商品化试剂盒比较.结果 H5N6双重实时荧光RT-PCR体系可在80 min内完成检测,与其它亚型流感病毒及常见呼吸道病原体无交叉反应,最低检测限达到10拷贝/反应,H5和N6靶基因标准曲线的相关系数R2分别为0.999和0.993,Ct值的变异系数分别为0.151％-0.549％和0.213％-0.575％,验证试验的阳性和阴性符合率均为100％.结论 H5N6禽流感病毒TaqMan-MGB探针双重实时荧光RT-PCR特异、灵敏、稳定,可在疑似病例的早期快速应急检测及活禽交易市场外环境持续性监测方面具有较好的应用价值.Objective To establish a TaqMan-MGB probe-based real-time fluorescence RT-PCR assay for avian influenza H5N6 virus used in rapid diagnosis for suspected cases and surveillance for outer environment of live poultry markets.Methods Based on the conservative sequences of avian influenza H5N6 virus for HA and NA gene published on GenBank,specific primers and TaqMan-MGB probes were designed to develop and optimize for the dual real-time RT-PCR assay.Specificity,sensitivity,repeatability and comparison tests were carried out.Results This dual real-time RT-PCR detection can be completed within 80 minutes.There was no cross-reaction with other subtypes of influenza virus and common respiratory pathogens.The minimum detection limit could be up to 10 copies/reaction.The correlation coefficient of standard curve for the gene of H5 and N6 were 0.999 and 0.993,and the coefficients of variation for cycle threshold were range from 0.151%-0.549% and 0.213%-0.575 %,respectively.The positive and negative coincidence rates of the validation test were 100%.Conclusions This TaqMan-MGB probe-based dual real-time RT-PCR for avian influenza H5N6 virus was rapid,specific and sensitive.It will have a good use in early emergency detection of suspected cases and continuous monitoring of external environment in live poultry trade market.

关 键 词：H5N6禽流感病毒 实时荧光RT—PCR TaqMan.MGB探针 血凝素基因 神经氨酸酶 基因 

分 类 号：R440[医药卫生—诊断学] R511.7[医药卫生—临床医学]