猪捷申病毒TaqMan实时荧光定量RT-PCR检测方法的建立和初步应用

Development and Preliminary Application of a Real-time TaqMan Fluorescent Quantitative RT-PCR Assay for Detection of Porcine Teschovirus

为建立猪捷申病毒(PTV)的TaqMan实时荧光定量RT-PCR(RT-qPCR)检测方法,在PTV基因组序列保守区设计引物和TaqMan探针,构建重组质粒作为阳性标准品,通过优化反应条件,建立了检测PTV的RT-qPCR方法,并进行特异性、敏感性和重复性试验。结果表明,该方法仅对PTV出现特异性扩增反应,与猪流行性腹泻病毒、猪丁型冠状病毒、猪传染性胃肠炎病毒、A群猪轮状病毒等常见猪病毒均无交叉反应。该方法的标准曲线Ct值与4.6×10^(8)~4.6×10_(2)拷贝/μL的质粒浓度范围具有良好线性关系,标准曲线方程为Ct=-3.201×log X+40.527,线性相关系数(R^(2))为0.996,检测下限为4.6×10^(1)拷贝/μL。对不同浓度的pMD18-PTV进行组内与组间重复检测,每个浓度重复试验的Ct值的变异系数均小于4.0%,具有良好的重现性。应用所建立的方法对235份临床猪腹泻样品进行检测,PTV阳性样品32份,样品阳性率为13.62%。本试验建立的RT-qPCR方法为PTV实验室检测及病毒研究分析提供了可靠的技术手段。

To establish a real-time TaqMan fluorescent quantitative RT-PCR(RT-qPCR)assay for the detection of porcine teschovirus(PTV),primers and TaqMan fluorescent probe were designed according to the conserved region of PTV genome sequence.A recombinant plasmid was constructed as the positive standard sample,and RT-qPCR was developed by optimization of reacting conditions.Furthermore,the specificity,sensitivity and repeatability of the established RT-qPCR were tested.Results showed that there was no cross-reaction with conventional porcine viruses,such as porcine epidemic diarrhea virus,porcine transmissible gastroenteritis,porcine deltacoronavirus,porcine rotavirus group A,and et al.The assay was linear for the template plasmid pMD18-PTV in the range from 4.6×10^(8) copies/μL to 4.6×10^(2) copies/μL,the standard equation was Ct=-3.201×log X+40.527,correlation coefficient was R^(2)=0.996,and detection limit was low to 4.6×10^(1) copies/μL.Also,the assay showed the good reproducibility with the coefficient of variation(CV)less than 4%both in intra-assay and inter-assay.Thirty-two of the 235 clinical fecal samples collected from different areas in Guangxi were positive for PTV tested by the developed assay.In conclusion,the development of this assay provides a convenient,specific and sensitive diagnostic method for the PTV detection and epidemiological investigation.