双重RT-qPCR鉴别CSFV和BVDV方法的建立及初步应用

Establishment and Preliminary Application of Dual Fluorescence Quantitative RT-PCR in Identifying CSFV and BVDV Methods

为建立猪瘟病毒(CSFV)和牛病毒性腹泻病毒(BVDV)的鉴别检测方法,本研究根据CSFV Npro基因、BVDV 5'-UTR基因保守区域设计特异性引物和Taq Man探针,构建阳性重组质粒,优化反应条件,建立了双重RT-q PCR检测方法。该方法 CSFV和BVDV的检测灵敏度均为100拷贝/μL,具有良好的特异性和重复性;对206份猪病料进行检测,CSFV阳性59份、BVDV皆为阴性,与本实验室常用单项RT-q PCR试剂盒检测结果一致。本研究建立的双重RT-q PCR整个检测过程不到3 h,采取闭管反应,检测完毕直接处理,避免开盖造成气溶胶污染,具有简单、快捷、灵敏度高、生物安全性好等优点,可用于CSFV和BVDV的临床鉴别检测,为2种病毒的交叉污染的检测提供一种方便、快捷的方法。

In order to establish a method for simultaneous detection of CSFV and BVDV,the double RT-q PCR was established by designing specific primers and Taq Man probes based on conserved regions of CSFV Nprogene and BVDV 5 ＇-UTR gene. The standard curve was made and the reaction conditions were optimized. The sensitivity of CSFV and BVDV were 100 copies/μl,with good specificity and respeatability. 206 pig samples were tested,of which 59 was CSFV positive and BVDV were negative,The CSFV test results were consistent with the single RT-q PCR kit used in our laboratory. In this study,the whole detection process of RT-q PCR was less than 3 h,and the aerosol contamination was avoided by the direct treatment of closed tube reaction. The advantages of the double fluorescent quantitative RT-PCR established in this study is simple,rapid,high sensitivity and good biosafety,which can be used for the differential detection of CSFV and BVDV in swine infection.