猪传染性胃肠炎病毒猪流行性腹泻病毒猪轮状病毒多重荧光RT-PCR检测方法的建立

Establishment and application of multiplex RT-PCR procedure for detection of three virus from swine

根据TGEV的M基因、PEDV的M基因和PoRV的VP2基因序列,通过优化引物、探针浓度和退火温度,确定了最佳的反应体系和扩增程序,成功建立了这3种病毒的TaqMan探针单重荧光定量PCR方法。在此基础上,经过进一步的条件优化,建立了检测TGEV、PEDV、PoRV的三重实时荧光定量PCR方法,该方法对TGEV、PEDV、PoRV的检测灵敏度分别为2.49 copies/μL、4.36 copies/μL、4.96 copies/μL,TGEV、PEDV、PoRV的组内重复试验的CV最大值分别为2.5%、3.8%、4.3%,组间重复性试验的CV最大值分别为3.7%、3.4%、3.2%,均不超过5%,表明建立的方法重复性好;用此方法对PRV、PCV1、PRRSV病毒样本进行检测,均没有交叉反应,表明该方法特异性好;用建立的方法对临床40份病料样品进行了检测,结果显示,TGEV、PEDV、PoRV的阳性检出率分别为5%、30%、12.5%;其中TGEV与PEDV混合感染率为2.5%;PEDV与PoRV混合感染率为5%;多重荧光定量PCR方法与单一荧光RT-PCR方法的检测结果均一致,表明建立的方法具有很好的临床应用价值。

Based on the M gene sequence of TGEV and PEDV and VP2 gene sequence of Po RV,the optimal reaction system and amplification procedure were established by optimizing primer,probe concentration and annealing temperature,and the Quantitative PCR method of Taq Man probes for three viruses is successfully established.On this basis,after further optimization of conditions,a triple real-time fluorescent quantitative PCR method for detecting TGEV,PEDV,and Po RV was established.The detection sensitivity of this method for TGEV,PEDV,and Po RV were 2.49 copies/μL,4.36 copies/μL,and 4.96 copies/μL respectively.The maximum value of CV in repeated trials detected by TGEV,PEDV and Po RV were2.5%,3.8%,4.3%,and the maximum value of CV in repeated trials between groups were 3.7%,3.4%,3.2%,which are no more than 5%.indicating that the established method has good reproducibility.Using this method to detect PRV,PCV1,and PRRSV virus samples,there is no cross-reaction,indicating that the me thod is specific.Using the established method to detect 40 clinical diseases,the samples were tested,and the positive rates of TGEV,PEDV,and Po RV were 5%,30%,and 12.5%respectively.The mixed infection rate of TGEV and PEDV was 2.5%,the mixed infection rate of PEDV and Po RV was 5%.The results of the multiple fluorescence quantitative PCR method are consistent with those of the detection of a single fluorescent RT-PCR method,indicating that the established method has good clinical application value.