Hepatitis E virus (HEV) sequences including four major genotypes representative strains available in GenBank were aligned with the DNAMAN software. The highly conserved internal region of ORF2 was then subjected to design primers and a probe. Furthermore, a 0.3 kb fragment of HEV ORF2 containing the amplification region was transcribed in vitro to synthese cRNA standard and a universal real-time TaqMan PCR assay was optimized and developed to detect and quantify main genotypes RNA of HEV. The specificity and reliability of the real-time RT-PCR was confirmed by testing genotype I HEV, genotype IV HEV and clinical samples. The detection limit of real-time RT-PCR was found 2.0 x 10(1) copies per reaction using in vitro transcribed cRNA. Compared with nested RT-PCR in diagnosis of HEV, the real-time RT-PCR developed was 10 to 100-fold more sensitive than the nested RT-PCR. The detection results from 54 clinical specimens indicated real-time RT-PCR was a rapid, sensitive and reproducible diagnostic method for HEV. This assay will be useful as an early and rapid diagnostic assay for HEV.
Base Sequence ; Fluorescence ; Hepatitis E ; virology ; Hepatitis E virus ; isolation & purification ; Humans ; Molecular Sequence Data ; RNA, Viral ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Viral Proteins ; analysis ; genetics