Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A multi-site nested reverse transcription and polymerase chain reaction (RT-PCR) followed by restriction endonuclease analysis (REA) was developed to identify hepatitis E virus (HEV) in clinical specimens. Four sets of primers were selected to amplify regions in the HEV genome supposed to encode the helicase, polymerase, and parts of the viral capsid protein. Digestion of the nested PCR products with HinfI, HaeII, AvaII, BglI, KpnI, SmaI, or EcoRI generated readily recognizable profiles that confirm the HEV sequences and/or distinguish the unique Mexico genotype (our positive control) from all other isolates (Asian genotype). In addition, the hydroxyapatite (HA) adsorption method was compared to other adsorption and extraction methods widely used to purify viral RNA from clinical specimens for RT-PCR. All methods presented the same sensitivity of recovery of HEV RNA, but only the adsorption methods efficiently removed fecal enzymatic inhibitors. The HA method gave the best results and was the most economic in terms of time, cost, manipulations and reagents. The method was validated by screening a small number of serum and fecal specimens available from patients with acute non-A,B,C hepatitis in Nepal. HEV RNA was identified in half (5/11) of the fecal specimens obtained from patients with evidence of recent HEV infection, but in none of the 14 patients without a serological marker for hepatitis E.
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PMID:Identification of hepatitis E virus in clinical specimens: amplification of hydroxyapatite-purified virus RNA and restriction endonuclease analysis. 950 51

The genomic variability of hepatitis E virus (HEV) was examined by restriction endonuclease analysis (REA) of four genomic cDNA copies comprising a 499 bp segment of the putative polymerase gene, a 264 bp segment of the helicase gene, and two, 680 bp and 448 bp, segments of the capsid gene. Analysis of the deduced restriction sites of all 27 HEV sequences currently available in the GenBank, and digestion of reverse-transcribed and nested PCR amplified segments obtained from six Nepali isolates were used to devise and test a REA genotyping assay. The assay allowed easy discrimination between the Mexico and Asian genotypes, and the classification of the Asian genotypes into three, or perhaps four subgenotypes. In addition, endonucleases identifiers of individual isolate or clusters of isolates were found. This assay permits rapid identification of a large number of HEV isolates directly from clinical specimens for studies on the molecular epidemiology and evolution of HEV.
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PMID:Genotyping of hepatitis E virus in clinical specimens by restriction endonuclease analysis. 950 14

Hepatitis E virus (HEV) is the major agent of acute hepatitis in developing countries where the infection occurs sporadically or in large waterborne epidemics. HEV, classified in the Caliciviridae, is not culturable. The detection of HEV RNA by RT-PCR in serum and stool samples is reliable during the 7 to 15 days following the onset of the disease. Restriction endonuclease analysis, cloning and sequencing of PCR products allow a phylogenetic analysis of HEV isolates. Although they belong to a single serotype, strains recovered from different geographical regions display a significant genetic heterogeneity. Sequencing data from ORF1 and ORF2 regions has led to the characterization of 3 distinct genotypes: genotype I gathering the Asian and African subgenotypes; genotype II gathering swine and human US strains; genotype III limited to the Mexico prototype. Novel variants are currently described from Africa (Nigeria), China and Europe (Greece and Italy). Each genotype appears to be related to a well defined geographical area. Nevertheless, a genetic variability is observed within endemic regions such as Asia or Africa. Nigerian endemic isolates especially could represent an intermediate stage in the evolutionary process towards genetic diversity. The animal reservoir, proved by the detection of HEV sequences by PCR among pigs in Nepal and in the USA, could help to resolve unanswered questions about the origin of HEV genotypes, their spread and evolution.
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PMID:[Geographical distribution of hepatitis E virus genotypes]. 1057 64