实时荧光定量PCR检测H1N1亚型猪流感病毒

Development of Rea1-Time Fluorescent Quantitative PCR Assay to Detect H1N1 Subtype Swine Influenza Virus

根据GenBank中H1N1亚型猪流感病毒NP基因的序列(DQ889686),利用Premier express设计并合成1对引物和相应的TaqMan探针,从猪流感病毒HN407株接种的鸡胚尿囊液中提取总RNA,经RT-PCR扩增NP基因。将鉴定正确的NP基因片段克隆入pGEM-T Easy载体中,转化大肠杆菌JM109,经PCR及测序鉴定后得到阳性重组质粒。以该阳性重组质粒为荧光定量PCR标准品模板建立标准曲线。对探针浓度、引物浓度、镁离子浓度和退火温度进行优化,建立了最佳的荧光定量PCR反应体系和扩增程序。经临床应用表明荧光定量PCR的建立为早期诊断猪流感病毒、定量分析猪流感病毒感染程度奠定了基础。

A pair of primers and a TaqMan probe were designed according to NP gene nucleotide sequence （DQ889686） of H1N1 subtype swine influenza virus （SIV） in GenBank. NP gene was amplified by RT-PCR from RNA extracted from allantoic fluid infected by HN407 isolate of SIV. The PCR product was cloned into pGEM-T Easy vector and sequenced. The positive recombined plasmid was used as a positive quantitative template to establish a standard curve. By optimizing the probe＇ s concentration, Mg^2＋ concentration, primers concentration and the annealing temperature, real-time fluorescent quantitative PCR was established. Clinical detection of H1N1 subtype SIV by real-time fluorescent quantitative PCR showed that the constructed method paved the way for the early and rapid detection of SIV and quantitative analysis for the infect degree of SIV.