SYBR GreenⅠ实时PCR检测甲型H1N1与季节性流感病毒方法的建立  

Development of SYBR GreenⅠ real-time PCR for detection of novel influenza A H1N1 and seasonal influenza viruses

作　　者：李文悌[1] 孙菲 王晓春[1] 

机构地区：[1]中南大学湘雅医学院医学检验系,长沙410013 [2]湖南国际旅行卫生保健中心,长沙410016

出　　处：《中国人兽共患病学报》2013年第1期59-63,共5页Chinese Journal of Zoonoses

基　　金：湖南省科技厅项目(2011sk3260);湖南省出入境检验检疫局科技项目(2009hnk001);长沙市科技局科技项目(K0904150-11);中南大学研究生学位论文创新基金(2011ssxt253)~~

摘　　要：目的建立一种检测甲型H1N1、季节性H1N1、H3N2与乙型流感病毒的SYBR GreenⅠ实时荧光PCR方法。方法设计针对甲型H1N1、季节性H1N1、H3N2与乙型流感病毒HA基因的引物,分别进行SYBR GreenⅠ实时荧光定量PCR反应,同时进行熔解曲线和Tm值分析,并作灵敏度和重复性试验。结果该方法与H5、H7、H9亚型流感病毒及副流感病毒1、2、3型均无交叉反应。构建的荧光定量标准曲线循环阈值与模板浓度呈良好的线性关系,扩增效率为:95.588%,检测灵敏度为101copies/反应体系,结果重现性好。成功地验证性检验了32份盲样标本。结论本研究建立的SYBR GreenⅠ实时荧光PCR方法敏感、不需荧光标记探针、成本低及高通量,能用于人类流感病毒的分型检测。The purpose of this study is to develop a SYBR Green Ⅰ real-time fluorescence quantitative PCR assay for rap- id detection of novel influenza A H1N1 virus, seasonal H1N1, H3N2 and influenza B viruses. Specific primers were designed according to the conserved sequence of hemagglutinin （HA） gene of the four influenza viruses. The viruses were detected by SYBR GreenⅠ real-time fluorescence quantitative PCR with the analysis of dissociation curve （DC） and melting temperature （Tm）, respectively. Sensitivity assay and reproducibility assay was determined. The results showed that the assay had no cross-reaction with H5, H7, H9 subtype influenza virus and parainfluenza virus type 1, 2, 3. The standard curves established by standard plasmid showed fine linear relationships between threshold cycle （Ct） and template concentration. The amplifica- tion efficiency was 95. 588% and detection sensitivity was 101 copies/reaction system. The results had fine repetition, and 32 blind samples were detected successfully. It＇s suggested that this SYBR Green Ⅰ real-time fluorescence quantitative PCR is sen- Sitive, low-cost, high-throughput and suitable for suhtyping of human influenza virus without fluorescence probe.

关 键 词：甲型H1N1 实时荧光定量PCR SYBR GreenⅠ 

分 类 号：R373.1[医药卫生—病原生物学]