猪环曲病毒TaqMan荧光定量RT-PCR检测方法的建立和应用

发布时间：2019-01-06 05:50

【摘要】：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。
[Abstract]:According to the conserved sequence of porcine cyclo virus N gene, specific primers and TaqMan probes were designed to establish a fluorescent quantitative RT-PCR assay for detection of porcine cyclopus virus, and to evaluate its specificity, sensitivity and reproducibility. The results showed that the method could be used to detect porcine cyclopyclovir, but to porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine stellate virus, group A rotavirus, porcine Boca virus. The detection of porcine cristae virus and pseudocanine disease virus had no cross reaction and had good specificity. The Ct value of the established quantitative standard curve has a good linear relationship with the final concentration of the template in the range of 109~102copies/L. The sensitivity of the method can reach 102 copias / L, and the coefficient of variation between groups is less than 2.5 in the repeatability test group. Among 88 fecal samples, 30 were positive by fluorescent quantitative RT-PCR and 28 by routine RT-PCR. The coincidence rate of the two methods was 93.34. The fluorescent quantitative RT-PCR developed in this study can be used for rapid detection and epidemiological investigation of porcine Cyclovir.
【作者单位】：浙江省动物疫病预防控制中心;
【基金】：浙江省公益技术研究项目(2016C32053)
【分类号】：S852.651

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