Detection of hepatitis B pre-core mutant by allele specific polymerase chain reaction.
Identifieur interne : 000B70 ( Istex/Curation ); précédent : 000B69; suivant : 000B71Detection of hepatitis B pre-core mutant by allele specific polymerase chain reaction.
Auteurs : E S Lo [Royaume-Uni] ; Y M Lo [Royaume-Uni] ; C H Tse [Royaume-Uni] ; K A Fleming [Royaume-Uni]Source :
- Journal of Clinical Pathology [ 0021-9746 ] ; 1992-08.
English descriptors
- Teeft :
- Acute hepatitis, Allele, Allele specificity, Amplification, Amplification phase, Annealing temperature, Assay, Base pair, Chronic hepatitis, Clin pathol, Clinical samples, Clinical specimen, Diagnostic phase, Fulminant, Fulminant hepatitis, Hepatitis, Hong kong, Internal control, Liver biopsy specimens, Liver tissues, Mutagenesis primers, Mutant, Mutant sequence, Mutant sequences, Mutant template, Mutant virus, Mutant viruses, Mutation, Nuffield department, Paraffin, Polymerase, Polymerase chain reaction, Positive control, Precore region, Preliminary investigation, Primer, Queen elizabeth hospital, Relative locations, Sequence specificity, Severe hepatitis, Viral variants, Virus type, Wild type, Wild type template, Wild type virus.
Abstract
AIM: Development of a specific polymerase chain reaction (PCR) assay for detection of the pre-core, stop codon, mutant of hepatitis B virus (HBV). METHODS: PCR primers, specific at the 3'-end for nucleotide 1896 of either the pre-core, stop codon, mutant or wild type HBV, were synthesised using published sequence data. Positive control templates for both types of virus were synthesised by the PCR, incorporating sequences specific for each virus type at the appropriate position. These templates were used to optimise the specificity of the procedure. Formalin fixed, paraffin wax embedded human tissue from acute or fulminant HBV hepatitis from Hong Kong or Oxford was then investigated for presence of mutant or wild type virus. The HBV DNA was amplified from this tissue using a two step procedure, with an initial amplification phase followed by a second diagnostic phase on optimally diluted target DNA. RESULTS: Specific detection of mutant or wild type HBV was achieved. An important factor in determining specificity was the temperature of annealing, 70 degrees C proving to be highly specific. To overcome the inherent variation of target copy number in clinical samples and to provide an intrinsic positive control, it was important to generate and standardise the amount of target HBV used for the specific PCR. Two cases of fulminant hepatitis and four cases of acute hepatitis from Hong Kong, and one case of fulminant hepatitis from Oxford, contained only wild type HBV, with no evidence of a mutant virus. CONCLUSION: This method can be applied to FFPE tissues. It is rapid, non-radioactive, and specific for the stop codon mutation at nucleotide 1896 of HBV. Preliminary investigation of a small number of cases of fulminant hepatitis from Oxford and Hong Kong showed only wild type virus. The result differs from results published from Japan and Israel.
Url:
DOI: 10.1136/jcp.45.8.689
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type="university">Université d'Oxford</orgName><country>Royaume-Uni</country><placeName><settlement type="city">Oxford</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Oxfordshire</region></placeName></affiliation></author><author><name sortKey="Tse, C H" sort="Tse, C H" uniqKey="Tse C" first="C H" last="Tse">C H Tse</name><affiliation wicri:level="4"><mods:affiliation>University of Oxford, Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital.</mods:affiliation><orgName type="university">Université d'Oxford</orgName><country>Royaume-Uni</country><placeName><settlement type="city">Oxford</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Oxfordshire</region></placeName></affiliation></author><author><name sortKey="Fleming, K A" sort="Fleming, K A" uniqKey="Fleming K" first="K A" last="Fleming">K A Fleming</name><affiliation wicri:level="4"><mods:affiliation>University of Oxford, Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital.</mods:affiliation><orgName type="university">Université d'Oxford</orgName><country>Royaume-Uni</country><placeName><settlement type="city">Oxford</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Oxfordshire</region></placeName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:4B749ABE71DC3198C91B746E2E9DB5568F8D0B46</idno><date when="1992" year="1992">1992</date><idno type="doi">10.1136/jcp.45.8.689</idno><idno type="url">https://api.istex.fr/ark:/67375/NVC-GS61FN93-L/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000B70</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000B70</idno><idno type="wicri:Area/Istex/Curation">000B70</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Detection of hepatitis B pre-core mutant by allele specific polymerase chain reaction.</title><author><name sortKey="Lo, E S" sort="Lo, E S" uniqKey="Lo E" first="E S" last="Lo">E S Lo</name><affiliation wicri:level="4"><mods:affiliation>University of Oxford, Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital.</mods:affiliation><orgName type="university">Université d'Oxford</orgName><country>Royaume-Uni</country><placeName><settlement type="city">Oxford</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Oxfordshire</region></placeName></affiliation></author><author><name sortKey="Lo, Y M" sort="Lo, Y M" uniqKey="Lo Y" first="Y M" last="Lo">Y M Lo</name><affiliation wicri:level="4"><mods:affiliation>University of Oxford, Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital.</mods:affiliation><orgName type="university">Université d'Oxford</orgName><country>Royaume-Uni</country><placeName><settlement type="city">Oxford</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Oxfordshire</region></placeName></affiliation></author><author><name sortKey="Tse, C H" sort="Tse, C H" uniqKey="Tse C" first="C H" last="Tse">C H Tse</name><affiliation wicri:level="4"><mods:affiliation>University of Oxford, Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital.</mods:affiliation><orgName type="university">Université d'Oxford</orgName><country>Royaume-Uni</country><placeName><settlement type="city">Oxford</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Oxfordshire</region></placeName></affiliation></author><author><name sortKey="Fleming, K A" sort="Fleming, K A" uniqKey="Fleming K" first="K A" last="Fleming">K A Fleming</name><affiliation wicri:level="4"><mods:affiliation>University of Oxford, Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital.</mods:affiliation><orgName type="university">Université d'Oxford</orgName><country>Royaume-Uni</country><placeName><settlement type="city">Oxford</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Oxfordshire</region></placeName></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Clinical Pathology</title><title level="j" type="abbrev">J Clin Pathol</title><idno type="ISSN">0021-9746</idno><idno type="eISSN">1472-4146</idno><imprint><publisher>BMJ Publishing Group Ltd and Association of Clinical Pathologists</publisher><date type="published" when="1992-08">1992-08</date><biblScope unit="volume">45</biblScope><biblScope unit="issue">8</biblScope><biblScope unit="page" from="689">689</biblScope></imprint><idno type="ISSN">0021-9746</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0021-9746</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Acute hepatitis</term><term>Allele</term><term>Allele specificity</term><term>Amplification</term><term>Amplification phase</term><term>Annealing temperature</term><term>Assay</term><term>Base pair</term><term>Chronic hepatitis</term><term>Clin pathol</term><term>Clinical samples</term><term>Clinical specimen</term><term>Diagnostic phase</term><term>Fulminant</term><term>Fulminant hepatitis</term><term>Hepatitis</term><term>Hong kong</term><term>Internal control</term><term>Liver biopsy specimens</term><term>Liver tissues</term><term>Mutagenesis primers</term><term>Mutant</term><term>Mutant sequence</term><term>Mutant sequences</term><term>Mutant template</term><term>Mutant virus</term><term>Mutant viruses</term><term>Mutation</term><term>Nuffield department</term><term>Paraffin</term><term>Polymerase</term><term>Polymerase chain reaction</term><term>Positive control</term><term>Precore region</term><term>Preliminary investigation</term><term>Primer</term><term>Queen elizabeth hospital</term><term>Relative locations</term><term>Sequence specificity</term><term>Severe hepatitis</term><term>Viral variants</term><term>Virus type</term><term>Wild type</term><term>Wild type template</term><term>Wild type virus</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">AIM: Development of a specific polymerase chain reaction (PCR) assay for detection of the pre-core, stop codon, mutant of hepatitis B virus (HBV). METHODS: PCR primers, specific at the 3'-end for nucleotide 1896 of either the pre-core, stop codon, mutant or wild type HBV, were synthesised using published sequence data. Positive control templates for both types of virus were synthesised by the PCR, incorporating sequences specific for each virus type at the appropriate position. These templates were used to optimise the specificity of the procedure. Formalin fixed, paraffin wax embedded human tissue from acute or fulminant HBV hepatitis from Hong Kong or Oxford was then investigated for presence of mutant or wild type virus. The HBV DNA was amplified from this tissue using a two step procedure, with an initial amplification phase followed by a second diagnostic phase on optimally diluted target DNA. RESULTS: Specific detection of mutant or wild type HBV was achieved. An important factor in determining specificity was the temperature of annealing, 70 degrees C proving to be highly specific. To overcome the inherent variation of target copy number in clinical samples and to provide an intrinsic positive control, it was important to generate and standardise the amount of target HBV used for the specific PCR. Two cases of fulminant hepatitis and four cases of acute hepatitis from Hong Kong, and one case of fulminant hepatitis from Oxford, contained only wild type HBV, with no evidence of a mutant virus. CONCLUSION: This method can be applied to FFPE tissues. It is rapid, non-radioactive, and specific for the stop codon mutation at nucleotide 1896 of HBV. Preliminary investigation of a small number of cases of fulminant hepatitis from Oxford and Hong Kong showed only wild type virus. The result differs from results published from Japan and Israel.</div></front></TEI></record>Pour manipuler ce document sous Unix (Dilib)
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