猪圆环病毒2型实时定量PCR检测方法的建立

Development of real-time quantitative PCR assay of porcine circovirus type 2

用PCR技术从一临床病猪组织中扩增出猪圆环病毒2型(PCV2)417 bp片段,并克隆到pGEM-T Easy载体中,构建成含有PCV2基因片段的重组质粒。应用该重组质粒进行实时荧光定量PCR,建立了PCV2 DNA的实时PCR标准曲线及其直线回归方程。结果显示该方法重复性好,其检测灵敏度达到102拷贝/μL,检测样品时特异性强。此研究表明,所建立的PCV2 DNA实时定量PCR方法具有重复性好、敏感性高与特异性强等特点,能够用于PCV2的定量检测。

A segment of 417 base pair（bp） for porcine circovirus type 2（PCV2） gene was amplified by PCR assay from the tissues of a clinical diseased pig,and was cloned into the pGEM-T easy vector.Then,one recombinant plasmid with the 417 bp segment was constructed.The standard curve and the corresponding linear regression equation of PCV2 DNA level were obtained by real-time fluorescent quantitative PCR with the recombinant plasmid.The results showed that this method was easily reproducible and high specific while used to detect samples,its sensitivity was proved to be 102 copies/L.The present study indicated that the real-time PCR assay of PCV2 DNA was of high specificity,sensitivity and reproducibility,and provided a method to quantify PCV2.