基于恒温隔绝式荧光PCR技术的猪流行性腹泻病毒检测方法的建立及应用

Establishment and application of a method for detection of porcine epidemic diarrhea virus based on insulated isothermal PCR

为建立猪流行性腹泻病毒（porcine epidemic diarrhea virus,PEDV）快速检测方法,参照Gen Bank中PEDV的N基因序列,设计引物及TaqMan探针,优化反应条件,建立了快速检测PEDV的荧光RT-PCR方法,并验证该法的敏感度、特异性和重复性。结果显示：该方法最佳反应体系为上游引物各1.5μL,下游引物各1.8μL,探针0.7μL,酶1μL,缓冲液12.5μL,模板2μL,补足DEPC水至25μL,获得的标准曲线方程为Ct=-3.15×lg拷贝数＋31.72,检测PEDV的敏感度可达0.32 TCID50/100μL,特异性好,对其他猪常见病毒检测结果均为阴性。结果表明：该方法适合猪流行性腹泻快速检测,为该病的诊断和防控奠定了基础。

This study was to establish a rapid detection of porcine epidemic diarrhea virus（ PEDV）,and by reference to the PEDV gene sequence in Gen Bank,to design primers and Taq Man probes,and to optimize the reaction conditions. A method for rapid detection of porcine epidemic diarrhea virus by fluorescent RT-PCR method was developed,and verified for sensitivity,specificity and reproducibility. According to the results of the fluorescent RT-PCR,The optimal reaction system was described as follows： the forward primer was 1. 5 μL,the reverse primer was 1. 8 μL,the probe was 0. 7 μL,the enzyme was 1μL,the Buffer was 12. 5 μL,the template was 2 μL,and the DEPC water was added up to 25 μL,the standard curve equation obtained was Ct =-3. 15×lgcopies＋31. 72. The sensitivity of the method to lively detect porcine epidemic diarrhea virus was 0. 32 TCID50/100 μL,with a favorable specificity. The results of detecting the common viruses in other pigs were negative. The method developed here was applicable to detection of porcine epidemic diarrhea,provide reference for diagnosis and prevention of the disease.