Genotyping of hepatitis E virus by PCR combining with single restriction endonuclease analysis.
- Author:
Ning PAN
1
;
Xing DAI
;
Ji-hong MENG
;
She-lan LIU
Author Information
- Publication Type:Journal Article
- MeSH: DNA Restriction Enzymes; metabolism; DNA, Complementary; genetics; metabolism; Deoxyribonucleases, Type II Site-Specific; metabolism; Genotype; Hepatitis E; blood; genetics; immunology; Hepatitis E virus; genetics; Humans; Polymerase Chain Reaction; methods; RNA, Viral; genetics; Reverse Transcriptase Polymerase Chain Reaction
- From: Chinese Journal of Experimental and Clinical Virology 2005;19(2):179-181
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo develop a simple method for genotyping of hepatitis E virus (HEV) and to investigate HEV genotype distribution in Nanjing area.
METHODSTwenty-seven full HEV sequences currently-available in GenBank were analyzed with MegAlign and MapDraw programs of DNA STAR software. Degenerate primers were designed and applied to amplify a fragment in HEV ORF1 region. HEV genotypes were determined by the size of the PCR products and by single restriction endonuclease analysis.
RESULTSThe PCR products of HEV genotype 1 and 2 were 275 bp and 269 bp in size. Distinctively, the PCR products of genotype 3 and 4 were 317 bp and 314 bp in size. Moreover, the PCR products of genotype 1 could be digested by Nae 1, but the products of genotype 2 could not. Distinctively, the PCR products of HEV genotype 3 could be digested by Not 1, but the products of genotype 4 could not. Six HEV reference strains standing for different HEV genotypes were clustered into their own types as predicted. Within 43 HEV IgM-positive clinical specimens collected in Nanjing, 19 were HEV PCR-positive and identified as genotype 4.
CONCLUSIONA simple method of PCR combined with single restriction endonuclease analysis is developed for HEV genotyping. This assay allows rapid identification of a large number of HEV isolates directly from clinical specimens. Among patients with hepatitis E in Nanjing, most were infected with HEV genotype 4.
