猪戊型肝炎病毒TaqMan荧光定量PCR检测方法的建立

Establishment of a TaqMan real-time PCR assay for detecting swine hepatitis E virus

为建立猪戊型肝炎病毒(SHEV)的TaqMan荧光定量PCR检测方法,本研究根据SHEV基因ORF2保守区设计引物及探针,并对PCR反应条件进行优化。结果表明,该方法扩增的相关系数可达0.999,在102拷贝/μL^109拷贝/μL内具有良好的线性关系。对猪圆环病毒2型、猪瘟病毒、猪繁殖与呼吸综合征病毒和伪狂犬病毒的RNA或DNA进行检测,结果均为阴性。该方法可以检出最低拷贝数为10拷贝/μL。重复性试验结果表明该方法的批内和批间变异系数均小于3.0%。本研究使用该荧光定量PCR检测方法对黑龙江省临床收集的131份样品(肝脏45份、粪便86份)进行检测,总阳性率为11.45%,其中肝脏阳性率为22.22%,粪便阳性率为5.81%。该方法的建立为SHEV的快速检出及绝对定量奠定了良好的基础。

To establish a rapid and sensitive detection method of swine hepatitis E virus（SHEV）, the Taq Man real-time PCR was developed with a pair of primers and the probe targeting the conserved region of SHEV ORF2. With the optimized reaction conditions, the standard curve showed a linear relationship from 102copies/μL to 109copies/μL with the correlation coefficient（R2） of 0.999. Specificity tests showed the assay had no cross reaction with porcine circovirus type 2, classical swine fever virus,porcine reproductive respiratory syndrome virusand pseudorabies virus. Sensitivity tests showed that the detection limit was 10copies/μL. Repeatability tests showed that thethe coefficient of variations of threshold cycles were than 3.0% for both intra- and intro-assay. The 131 clinical samples collected from Heilongjiang province（45 liver samples and 86 feces samples of pigs） were detected by the assay, and the total positive rate was 11.45%（22.22 % from liver samples and 5.81 % from feces samples）. The established real-time PCR assay had a potential application for rapid and quantitative detections of SHEV.