title: Development and validation of an improved RT-PCR assay with nested universal primers for detection of hepatitis E virus strains with significant sequence divergence.
abstract: Recent studies revealed that hepatitis E virus (HEV) genomes are more variable than previously thought and well-conserved regions suitable for designing universal primers are limited. In this study, based on alignment of 70 full-length HEV sequences of genotypes 1-4, a part of the ORF2/ORF3 overlapping region was found to be the best target region for PCR amplification of various HEV strains. Using the newly designed primers, an RT-PCR method (ORF2/3-137 PCR) that amplifies a 137-nucleotide (nt) sequence within the ORF2/ORF3 overlapping region and is capable of amplifying all known HEV sequences was developed. When compared with the previous RT-PCR method (ORF2-457 PCR) that amplifies a 457 nt ORF2 sequence, ORF2/3-137 PCR was two to three times more sensitive than ORF2-457 PCR upon testing serial dilutions of three HEV RNA-positive serum samples. The ORF2/3-137 PCR assay could detect viraemia in five patients with acute or fulminant hepatitis E 3-14 days longer than ORF2-457 PCR after disease onset. All 41 ORF2-457 PCR-positive serum samples of various genotypes tested positive for HEV RNA by the ORF2/3-137 PCR assay. Since the amplicons of ORF2/3-137 PCR contain variable sequences, a phylogenetic tree of the ORF2/3-137 products could clearly distinguish the different HEV genotypes.
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi-Ken 329-0498, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Inoue
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takahashi
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Yazaki
authors.forename: Fumio
authors.initials: F
authors.lastname: Tsuda
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Okamoto
mesh_terms: D017124:Conserved Sequence; D017931:DNA Primers; D005838:Genotype; D016751:Hepatitis E / Q000175:diagnosis* / Q000821:virology; D016752:Hepatitis E virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014764:Viral Proteins / Q000235:genetics; D014766:Viremia / Q000821:virology
chemical_list: D017931:DNA Primers; C400971:ORF2 protein, Hepatitis E virus; C400973:ORF3 protein, Hepatitis E virus; D012367:RNA, Viral; D014764:Viral Proteins