猪传染性胃肠炎病毒RT-CPA检测方法构建及初步应用

Construction and Preliminary Application of Porcine Transmissible Gastroenteritis Virus RT-CPA Detection Method

为开发一种猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)RT-CPA快速且准确的检测方法。本试验根据TGEV S基因的D序列,设计了3对引物,使用构建的重组质粒作为标准阳性对照,通过优化筛选条件并应用可视化核酸试纸条,成功建立了一种一步法TGEV RT-CPA检测方法。本实验筛选最优条件为:Mg^(2+)浓度1.0 mmol/L,dNTP浓度0.8 mmol/L,Betaine浓度1.0 mol/L,BstDNA聚合酶浓度8.0 U,反应温度63℃,反应时间60 min,AMV逆转录酶5 U。本研究的扩增产物通过凝胶电泳显示出梯形条带,证明了其对TGEV的特异性检测和重复性良好,灵敏度高达10拷贝/μL。初步应用显示,该方法与TGEV抗原快速检测试剂盒的符合率超过90%,显示了高灵敏度。TGEV RT-CPA方法免去了RNA提取和cDNA合成的步骤,能够完成一步检测,且该方法不需要特殊仪器,适用于现场快速检测TGEV,为快速诊断和防控猪传染性胃肠炎提供了新的技术选择。

To develop a rapid and accurate method for the detection of porcine Transmissible gastroenteritis virus(TGEV)RT-CPA.In this experiment,3 pairs of primers were designed according to the D sequence of TGEV S gene,and the constructed recombinant plasmid was used as the standard positive control.A one-step TGEV RT-CPA detection method was successfully established by optimizing the screening conditions and applying visual nucleic acid strips.The experimental conditions included Mg^(2+)concentration of 1.0 mmol/L,dNTP concentration of 0.8 mmol/L,Betaine concentration of 1.0 mmol/L,Bst DNA polymerase concentration of 8.0 U,reaction temperature of 63℃,reaction time of 60 minutes,and AMV reverse transcriptase of 5 U.The amplifi cation product in this study showed trapezoidal bands by gel electrophoresis,which proved its specifi city and good repeatability for TGEV detection with a sensitivity of up to 10 copies/μL.Preliminary application shows that the coincidence rate of this method with TGEV antigen rapid detection kit is more than 90%,showing high sensitivity.TGEV RT-CPA method can complete one step detection without the steps of RNA extraction and cDNA synthesis.In addition,the method does not require special instruments and is suitable for the rapid detection of TGEV in the fi eld,providing a new technology for the rapid diagnosis and control of transmissible gastroenteritis of pigs.