Establishment of a real time quantitative reverse-transcription PCR method for detection of hepatitis E virus based on standard plasmid

Abstract

Objective To establish a real-time quantitative reverse-transcription PCR (qRT-PCR) method for detection of hepatitis E virus (HEV) of different genotypes based on standard HEV DNA plasmid in order to promote its application in clinical laboratory. Methods Specific primers and probe of HEV were designed based on the conserved open reading frame 3 (ORF3) regions. HEV DNA plasmids were constructed and 10-fold serial dilutions of the plasmids were prepared and used as standards to establish one-step qRT-PCR. The established method was compared with HEV antigen, antibody and RT-nPCR assays. Some positive samples were sequenced and analyzed by evolutionary tree. Results The one-step qRT-PCR method for HEV detection in serum or feces samples was successfully establish. It could reach a sensitivity of 25 copies/test and 77.8% of its results were consistent with those by HEV antigen assay. Nine patients were infected with HEV of genotypes 4a, 4d or 4n as indicated by evolutionary tree. Conclusion The HEV qRT-PCR method based on its standard plasmid is successfully established, which paves the way for commercialization of clinical applications. Key words: HEV; Standard plasmid; qRT-PCR; RT-nPCR