Design and application of a set of universal PCR primers for genotyping of hepatitis E virus / 病毒学报
Chinese Journal of Virology
; (6): 9-16, 2009.
Article
em Zh
| WPRIM
| ID: wpr-334770
Biblioteca responsável:
WPRO
ABSTRACT
To improve the reliability and credibility of genotyping hepatitis E virus (HEV) and to explore the possibility of unifying standards of HEV genotyping by designing HEV universal primers for amplification of a long genomic fragment of different HEV genotypes. A set of universal primers (HEVuPrimer) was designed based on conserved regions determined by alignment analysis of 82 HEV strains with complete genome in GenBank. HEVuPrimer was compared with a set of previously used primers (MXJ primers) for their sequence-matching to different HEV strains and applied to amplify HEV genomic fragments from HEV reference strains with known different genotypes and clinical serum samples with anti-HEV-IgM by RT-nPCR. HEV genotyping based on the fragments amplified with HEVuPrimer was compared and validated with that based on HEV full genome and fragments obtained with MXJ primers. HEV genotyping by the phylogenetic analysis supplemented with the percent of nucleotide identity of the HEVuPrimer-determined fragments showed good correspondence with that based on HEV full-length genome. In addition, HEVuPrimer was much better than MXJ primers in matching sequences of HEV strains available from GenBank, and was able to amplify all the reference HEV strains with different genotypes. Among 124 samples with anti-HEV-IgM, 60 were positive for HEV RNA determined by a 644bp amplicon of RT-nPCR with the HEVuPrimenr. All the positive isolates belonged to HEV genotype 4 with nucleotide homology of 80.0%-99.9%, and could be further divided into 4 subgenotypes. Moreover, a novel subtype was identified with 6 HEV strains isolated very recently. The RT-nPCR using the HEVuPrimer and phylogenetic analysis of the amplified region provided strong evidences for its feasibility in HEV genetic classification. Our data have new implication for the consensus of genotype classification of HEV.
Texto completo:
1
Base de dados:
WPRIM
Assunto principal:
Vírus da Hepatite E
/
Genoma Viral
/
Primers do DNA
/
Reação em Cadeia da Polimerase Via Transcriptase Reversa
/
Genética
/
Genótipo
/
Métodos
Tipo de estudo:
Guideline
Idioma:
Zh
Revista:
Chinese Journal of Virology
Ano de publicação:
2009
Tipo de documento:
Article