中国人兽共患病学报2013，Vol.29Issue(1)：59-63,5.DOI:10.3969/cjz.j.issn.1002-2694.2013.01.013
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 GreenⅠreal-time PCR for detection of novel influenza A H1N1 and seasonal influenza viruses
摘要
Abstract
The purpose of this study is to develop a SYBR Green Ⅰ real-time fluorescence quantitative PCR assay for rapid 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 Green Ⅰ 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 amplification 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 sensitive, low-cost, high-throughput and suitable for subtyping of human influenza virus without fluorescence probe.关键词
甲型H1N1/实时荧光定量PCR/SYBR GreenⅠKey words
novel influenza A H1N1/ real-time fluorescence quantitative PCR/ SYBR Green Ⅰ分类
医药卫生引用本文复制引用
李文悌,孙菲,王晓春..SYBR GreenⅠ实时PCR检测甲型H1N1与季节性流感病毒方法的建立[J].中国人兽共患病学报,2013,29(1):59-63,5.基金项目
湖南省科技厅项目(2011sk3260) （2011sk3260）
湖南省出入境检验检疫局科技项目(2009hnk001) （2009hnk001）
长沙市科技局科技项目(K0904150-11) （K0904150-11）
中南大学研究生学位论文创新基金(2011ssxt253) （2011ssxt253）
