Development and application of a SYBR Green Ⅱ real-time RT-PCR for detection of swine hepatitis E virus / 中国人兽共患病学报

ABSTRACT

In order to establish a real-time RT-PCR based on SYBR Green Ⅱ for detection of hepatitis E virus (HEV),a pair of special primers was designed according to the conserved sequences of ORF2 in GenBank.Result showed that the standard curve of established SYBR Green Ⅱ real-time RT-PCR had a wide dynamic range from 4.10 × 102-4.10 × 108 copies/μL with a linear correlation(r2) of 0.996.The sensitivity could reach 1.00 × 102 copies/μL.The melting curve analysis using SYBR Green Ⅱ dye showed one specific peak with a melting temperature(Tm) of 84.0 C ±0.1 C.No amplification was detected from the RNA samples of porcine reproductive and respiratory syndrome virus,classial swine fever virus,transmissible gastroenteritis virus,porcine bocavirus,porcine epidemic dearrhoea virus porcine kobuvirus and porcine rotavirus by this PCR,respectively.Excellent reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay of 0.83 ％-0.94 ％ and inter-assay of 0.83％-0.94％.Further detection of 61 specimens showed that 9 of them were HEV positive,and the results of the quantitative RT-PCR were the same as that of the conventional RT-PCR.In conclusion,the real-time quantitative RT-PCR for HEV is feasible,the real-time RT-PCR established in this study will be useful for earlier rapid laboratory diagnosis and pathogenesis of HEV.