猪德尔塔冠状病毒荧光定量PCR检测方法的建立

Development of Fluorescence Quantitative PCR Assay for Detection of Porcine Deltacoronavirus

根据GenBank登录的猪德尔塔冠状病毒(PDCoV)毒株序列设计特异性引物,建立了检测PDCoV的实时荧光PCR方法。该方法在标准品浓度2.49×10~8~2.49×10~2 copies/μL范围内线性关系良好;特异性试验显示,其他几种常见猪病病毒未见典型扩增曲线;敏感性试验显示,最低检测下限为24.9 copies/μL;重复性试验显示,批内和批间变异系数小于3%。用该方法检测62份猪腹泻粪便样品,相比常规PCR方法,前者的检出率更高;两种方法检测1 168份进境活猪粪便拭子样品,结果均为阴性。结果表明,本研究所建立的方法适用于国内猪场的PDCoV的检测和进境活猪的监控检测,也可作为PDCoV的诊断及分子流行病学调查的技术手段。

Based on the sequence analysis results of different porcine deltacoronavirus（PDCoV）strains published in GenBank,specific primers were designed,and then a real-time fiuorescence quantitative PCR method was established. Results showed that the linear determination range of standards was 2.49×108 to 2.49×102 copies/μL; according to the specificity analysis,the amplifications of other porcine viruses were all negative results; the sensitivity test showed the minimum detection limit was 24.9 copies/μL; the repeatability tests showed the intra-assay and inter-assay variable coefficients were less than 3%. Otherwise,62 fecal samples collected from domestic pig farms were detected by the established and conventional PCR method,and the results indicated the real-time PCR had a higher positive detection rate. Using the two methods to detect 1 168 fecal samples collected from imported pigs,all test results were negative. As a conclusion,the method established in this study is not only suitable for PDCoV detection of the clinical samples in domestic pig farms,but also suitable for monitoring and detecting of live pigs imported from foreign countries. Also,the method would provide an effective method for diagnosis and molecular epidemiological investigation of PDCoV.