猪环曲病毒TaqMan荧光定量RT-PCR检测方法的建立和应用

Development and application of the TaqMan real-time reverse transcription polymerase chain reaction for detection of porcine torovirus

根据猪环曲病毒N基因保守序列设计特异性引物与TaqMan探针,建立猪环曲病毒荧光定量RT-PCR检测方法,并评价其特异性、敏感性和重复性。结果显示,该方法能检测猪环曲病毒,而对猪流行性腹泻病毒、传染性胃肠炎病毒、猪星状病毒、A群猪轮状病毒、猪博卡病毒、猪嵴病毒和伪犬病病毒等核酸的检测均无交叉反应,具有良好的特异性;建立的定量标准曲线Ct值和模板终浓度在109~102copies/L范围内有良好的线性关系,该方法检测灵敏度可达102copies/L;重复性试验的组内、组间的变异系数均小于2.5%。对88份猪粪便样品进行检测,荧光定量RT-PCR检出30份猪环曲病毒阳性,常规RT-PCR检出28份猪环曲病毒阳性,两种方法的符合率为93.34%。本研究建立的荧光定量RT-PCR可用于猪环曲病毒的快速检测和流行病学调查。

The real-timeRT-PCR assay was developed using the specific primers and a TaqMan probe according to the N gene conserved sequence of porcine torovirus. Specificity,sensitivity and repeatability of the method were evaluated. The result showed this method had no cross reactions with other diarrhea virus（porcine epidemic diarrhea virus,transmissible gastroenteritis virus,porcine astrovirus, group A porcine rotavirus,porcine bocavirus,porcine kobuvirus,porcine pseudorabies virus）.The Ct value of standard curve had a linear relationship to the log of the final concentration of template in the range of 10^9-10^2 copies/μ L. The detection limit was I00 copies/N L. The coefficient of variation was less than 2.5% both for intra and inter assay. Among the 88 fecal samples from porcine,30 and 28 were positive based on the TaqMan real-time PCR and conventional RT-PCR methods,respectively,and the total concordance of the two methods was approximately 93.34%.In conclusion,the real-time RT-PCR could be used for the detection and the epidemiological investigation of porcine torovirus.