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.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
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Validation Study
MeSH terms
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Conserved Sequence
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DNA Primers
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Genotype
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Hepatitis E / diagnosis*
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Hepatitis E / virology
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Hepatitis E virus / genetics
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Hepatitis E virus / isolation & purification*
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Humans
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Molecular Sequence Data
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Phylogeny
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RNA, Viral / analysis*
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RNA, Viral / genetics
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Reverse Transcriptase Polymerase Chain Reaction / methods*
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Sensitivity and Specificity
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Sequence Analysis, DNA
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Viral Proteins / genetics
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Viremia / virology
Substances
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DNA Primers
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ORF2 protein, Hepatitis E virus
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ORF3 protein, Hepatitis E virus
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RNA, Viral
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Viral Proteins
Associated data
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GENBANK/AB259173
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GENBANK/AB259174
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GENBANK/AB259175
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GENBANK/AB259176
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GENBANK/AB259177
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GENBANK/AB259178
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GENBANK/AB259179
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GENBANK/AB259180
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GENBANK/AB259181
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GENBANK/AB259182
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GENBANK/AB259183
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GENBANK/AB259184
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GENBANK/AB259185
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GENBANK/AB259186
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GENBANK/AB259187
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GENBANK/AB259188
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GENBANK/AB259189
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GENBANK/AB259190
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GENBANK/AB259191
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GENBANK/AB259192
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GENBANK/AB259193
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GENBANK/AB259194
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GENBANK/AB259195
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GENBANK/AB259196
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GENBANK/AB259197
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GENBANK/AB259198
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GENBANK/AB259199
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GENBANK/AB259200
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GENBANK/AB259201
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GENBANK/AB259202
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GENBANK/AB259203
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GENBANK/AB259204
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GENBANK/AB259205
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GENBANK/AB259206
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GENBANK/AB259207
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GENBANK/AB259208
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GENBANK/AB259209
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GENBANK/AB259210
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GENBANK/AB259211
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GENBANK/AB259212
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GENBANK/AB259213