Development and validation of an improved RT-PCR assay with nested universal primers for detection of hepatitis E virus strains with significant sequence divergence

Jun Inoue, Masaharu Takahashi, Yasuyuki Yazaki, Fumio Tsuda, Hiroaki Okamoto

Research output: Contribution to journalArticlepeer-review

81 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)325-333
Number of pages9
JournalJournal of Virological Methods
Volume137
Issue number2
DOIs
Publication statusPublished - 2006 Nov
Externally publishedYes

Keywords

  • Genotype
  • HEV RNA
  • Hepatitis E virus
  • Phylogenetic tree
  • RT-PCR

ASJC Scopus subject areas

  • Virology

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