猪肠病毒G型实时定量PCR检测方法的建立及初步应用

为建立一种能够准确、快速地鉴别猪肠病毒G型的诊断方法,本研究参考猪肠病毒G型的3D基因序列设计1对特异性引物,扩增片段预计大小约为104 bp,将3D基因片段克隆到pMD18-T载体上的阳性重组质粒作为标准品,建立猪肠病毒G型的实时定量PCR检测方法,并应用到临床样品的检测。研究结果显示,该方法设计的引物具有高度的特异性;标准曲线的Cq值与质粒标准品模板在2.92×10^8copies/μL^2.92×10^2copies/μL范围内,呈良好的线性关系,相关系数为0.998,斜率为-4.023;组内与组间重复性试验显示变异系数很小,在0.26%~1.57%之间。该方法的最低检测限量可达2.92×10 copies/μL。使用本方法对2017-2019年广西部分地区的猪场临床腹泻粪便样品进行检测,猪肠病毒G型阳性检出率为18.42%,说明广西部分地区的猪场存在猪肠病毒G型的感染。本研究成功建立了猪肠病毒G型实时定量PCR检测方法,可用于猪肠病毒G型感染的临床检测。

Establishment and preliminary application of real-time quantitative PCR assay for the detection of porcine enterovirus G

In order to establish an accurate and rapid diagnostic method to identify porcine enterovirus G,a pair of specific primers with reference to the 3D gene sequence of porcine enterovirus G were designed in this study,the size of the amplified fragment was expected to be about 104 bp,and the positive recombinant plasmid composed of the 3D gene fragment and p MD18-T vector as the standard,to establish a real-time quantitative PCR assay for the detection of porcine enterovirus G,and applied to the detection of clinical samples. The results showed that the primers designed in this method were specific,the Cq value of the standard curve showed a good linear relationship with the plasmid standard template in the range of 2.92×10^8 copies/μL to 2.92×10^2 copies/μL,and the correlation coefficient was 0.998,the slope was-4.023. The variation coefficient of intra-and inter-group repeatability test was small,ranging from 0.26% to 1.57%. The minimum detection limit of this method is reach to 2.92×10 copies/μL. This method was used to test the clinical diarrhea feces samples of pig farms in some areas of Guangxi from 2017 to 2019,and the positive rate of porcine enterovirus G was 18.42%,indicating that pig farms in some areas of Guangxi had porcine enterovirus G infection. A real-time quantitative PCR assay for porcine enterovirus G was successfully established in this study,which can be used for clinical detection of porcine enterovirus G.