塞内卡病毒A SYBR GreenⅠ荧光定量PCR检测方法的建立

Establishment of a SYBR GreenⅠreal-time PCR assay for detection of Senecavirus A

为深入研究塞内卡病毒A的病原学及流行病学,本研究分离获得了1株塞内卡病毒A的重庆地区毒株并将其命名为SVA-XN2021。针对VP1基因,设计了1对特异性引物,将SVA-XN2021株编码区克隆入载体pcDNA3.1(+),构建了以重组标准阳性质粒pcDNA3.1(+)-SVA为模板的SVA的SYBR GreenⅠ荧光定量PCR检测方法,并进行了特异性、敏感性和重复性试验。结果显示,本研究建立的荧光定量PCR检测方法的Ct值与标准品模板在1.0×10^(1)~1.0×10^(6)copies/μL的范围内线性关系良好,相关系数为0.9948。且用该方法对PEDV、TGEV、PDCoV等猪常见的腹泻病样品检测时无扩增,表明其特异性良好;该方法检测下限可达到1.0×10^(1)copies/μL,比常规RT-PCR敏感性高10倍;组内变异系数介于0.1%~0.29%之间,组间变异系数介于0.1%~0.81%之间,说明该检测方法重复性较好。上述结果表明,本研究建立的SYBR GreenⅠ荧光定量PCR方法可以作为SVA的重要诊断技术,有利于SVA的防治。

To further study the etiology and epidemiology of Senecavirus A,one Senecavirus A Chongqing strain was isolated and named SVA-XN2021.The coding region of SVA-XN2021 strain was synthesized.In this study,a pair of specific primers were designed based on VP1 gene,the coding region of SVA-XN2021 strain was cloned into the vector pc DNA3.1(+),a SYBR GreenⅠfluorescence quantitative PCR method for SVA was established based on the recombinant pc DNA3.1(+)-SVA standard positive plasmid,and the specificity,sensitivity and repeatability were tested.In result,the Ctvalue of the q RT-PCR established in this study has a good linear relationship with the standard template in the range from 1.0×10^(1)copies/μL to 1.0×10^(6)copies/μL,and the correlation coefficient is 0.9948.This method has good specificity for PEDV,TGEV,PDCo V and other porcine viruses.In addition,the detection limit of this method is 1.0×10^(1)copies/μL,which is significantly higher than that of the conventional RT-PCR.The intra-group coefficient of variation of this method is between 0.1%and 0.29%,and the inter-group coefficient of variation is from 0.1%to 0.81%,it proves that the detection method has ideal repeatability.Therefore,the SYBR GreenⅠfluorescence q RT-PCR method established in this study can be used as an important diagnostic technology reserve and is conducive to the prevention and treatment of SVA.