Restriction fragment length polymorphism analysis for rapid gag subtype determination of human immunodeficiency virus Type 1 in South Africa
Identifieur interne : 002728 ( Istex/Corpus ); précédent : 002727; suivant : 002729Restriction fragment length polymorphism analysis for rapid gag subtype determination of human immunodeficiency virus Type 1 in South Africa
Auteurs : Joanne Van Harmelen ; Elna Van Der Ryst ; Robin Wood ; Susan F. Lyons ; Carolyn WilliamsonSource :
- Journal of Virological Methods [ 0166-0934 ] ; 1999.
English descriptors
- KwdEn :
- Acci, African samples, Alamos database, Alui, Alui digestion, Boehringer mannheim, Cape town, Common subtype, Consensus sequences, Democratic republic, Digestion, Elsevier science, Endonuclease, European workshop report, Fragment, Fragment sizes, Genetic diversity, Harmelen, Heteroduplex mobility assay, Heterosexual infections, Heterosexual population, Human immunodeficiency virus type-1 (HIV-1), Human lymphotrophic virus type, Human virus type, Infectious bursal disease virus, Inner primers, Inner reactions, Limited diversity, Molecular weight markers, Other parts, Outer reaction, Polymerase chain reaction, Polymerase chain reaction (PCR), Rapid method, Rapid screening, Recombinant, Recombinant viruses, Resa, Resa patterns, Restriction endonuclease sites, Restriction endonucleases, Restriction enzyme sites, Restriction fragment length polymorphism, Restriction fragment length polymorphism (RFLP), Restriction fragment length polymorphism analysis, Restriction sites, Rflp, Rflp analysis, Rflp strategy, Rflp subtypes, Sequence analysis, Sequence subtypes, Subtype, Subtype designation, Subtypes, Subtyping, Swai digestion, Unknown samples, Unpublished data, Virological, Virological methods, Williamson, Xmni, gag gene.
- Teeft :
- Acci, African samples, Alamos database, Alui, Alui digestion, Boehringer mannheim, Cape town, Common subtype, Consensus sequences, Democratic republic, Digestion, Elsevier science, Endonuclease, European workshop report, Fragment, Fragment sizes, Genetic diversity, Harmelen, Heteroduplex mobility assay, Heterosexual infections, Heterosexual population, Human lymphotrophic virus type, Human virus type, Infectious bursal disease virus, Inner primers, Inner reactions, Limited diversity, Molecular weight markers, Other parts, Outer reaction, Polymerase chain reaction, Rapid method, Rapid screening, Recombinant, Recombinant viruses, Resa, Resa patterns, Restriction endonuclease sites, Restriction endonucleases, Restriction enzyme sites, Restriction fragment length polymorphism, Restriction fragment length polymorphism analysis, Restriction sites, Rflp, Rflp analysis, Rflp strategy, Rflp subtypes, Sequence analysis, Sequence subtypes, Subtype, Subtype designation, Subtypes, Swai digestion, Unknown samples, Unpublished data, Virological, Virological methods, Williamson, Xmni.
Abstract
Abstract: A rapid method for identification of human immunodeficiency virus Type 1 (HIV-1) gag subtypes was developed based on restriction fragment length polymorphism (RFLP) analysis of 400 or 650 bp long polymerase chain reaction (PCR) fragments encompassing the start of the p17 (400 bp) and part of the p24 (650bp) regions. The consensus sequences of subtypes A–D, the only subtypes identified in South Africa, were analyzed to detect restriction endonucleases which generate unique patterns for each subtype. Four restriction endonucleases were identified: AluI, AccI, SwaI and XmnI. Digestion of a 400 bp fragment with AluI allowed identification of subtype C. Samples not identified were then reamplified, and a 650 bp fragment digested with AccI to identify subtype B, followed by SwaI and XmnI to distinguish between subtypes A and D. This strategy was applied to 87 samples previously subtyped by either sequence analysis of the gag p17 region (n=33); or heteroduplex mobility assay (HMA) based on the env gene (n=75); or both (n=21). Out of the 87 samples, RFLP identified two samples as subtype A, 28 as subtype B, 56 as subtype C and one as a subtype D virus. No discrepancies were found between RFLP gag subtypes and gag sequence subtypes demonstrating the reliability of this method. There was also no discordance between gag RFLP subtypes and env HMA subtypes, suggesting that there were no recombinant viruses detected relating to the genomic regions analyzed. RFLP is an effective technique for the rapid screening in an HIV epidemic of limited diversity, such as in South Africa.
Url:
DOI: 10.1016/S0166-0934(98)00163-3
Links to Exploration step
ISTEX:78626E611136FBCFEE6068DC821F7A83FA111144Le document en format XML
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methods</term><term>Williamson</term><term>Xmni</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: A rapid method for identification of human immunodeficiency virus Type 1 (HIV-1) gag subtypes was developed based on restriction fragment length polymorphism (RFLP) analysis of 400 or 650 bp long polymerase chain reaction (PCR) fragments encompassing the start of the p17 (400 bp) and part of the p24 (650bp) regions. The consensus sequences of subtypes A–D, the only subtypes identified in South Africa, were analyzed to detect restriction endonucleases which generate unique patterns for each subtype. Four restriction endonucleases were identified: AluI, AccI, SwaI and XmnI. Digestion of a 400 bp fragment with AluI allowed identification of subtype C. Samples not identified were then reamplified, and a 650 bp fragment digested with AccI to identify subtype B, followed by SwaI and XmnI to distinguish between subtypes A and D. This strategy was applied to 87 samples previously subtyped by either sequence analysis of the gag p17 region (n=33); or heteroduplex mobility assay (HMA) based on the env gene (n=75); or both (n=21). Out of the 87 samples, RFLP identified two samples as subtype A, 28 as subtype B, 56 as subtype C and one as a subtype D virus. No discrepancies were found between RFLP gag subtypes and gag sequence subtypes demonstrating the reliability of this method. There was also no discordance between gag RFLP subtypes and env HMA subtypes, suggesting that there were no recombinant viruses detected relating to the genomic regions analyzed. RFLP is an effective technique for the rapid screening in an HIV epidemic of limited diversity, such as in South Africa.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>rflp</json:string><json:string>subtypes</json:string><json:string>subtype</json:string><json:string>harmelen</json:string><json:string>endonuclease</json:string><json:string>recombinant</json:string><json:string>resa</json:string><json:string>acci</json:string><json:string>alui</json:string><json:string>digestion</json:string><json:string>cape town</json:string><json:string>virological methods</json:string><json:string>rflp analysis</json:string><json:string>virological</json:string><json:string>human virus type</json:string><json:string>xmni</json:string><json:string>restriction endonucleases</json:string><json:string>williamson</json:string><json:string>genetic diversity</json:string><json:string>recombinant viruses</json:string><json:string>subtype designation</json:string><json:string>heteroduplex mobility assay</json:string><json:string>restriction fragment length polymorphism</json:string><json:string>fragment</json:string><json:string>polymerase chain reaction</json:string><json:string>limited diversity</json:string><json:string>consensus sequences</json:string><json:string>european workshop report</json:string><json:string>heterosexual population</json:string><json:string>boehringer mannheim</json:string><json:string>unpublished data</json:string><json:string>fragment sizes</json:string><json:string>alui digestion</json:string><json:string>heterosexual infections</json:string><json:string>rflp subtypes</json:string><json:string>rapid method</json:string><json:string>human lymphotrophic virus type</json:string><json:string>infectious bursal disease virus</json:string><json:string>restriction fragment length polymorphism analysis</json:string><json:string>inner primers</json:string><json:string>restriction enzyme sites</json:string><json:string>sequence subtypes</json:string><json:string>inner reactions</json:string><json:string>elsevier science</json:string><json:string>molecular weight markers</json:string><json:string>sequence analysis</json:string><json:string>alamos database</json:string><json:string>restriction sites</json:string><json:string>resa patterns</json:string><json:string>democratic republic</json:string><json:string>rapid screening</json:string><json:string>other parts</json:string><json:string>rflp strategy</json:string><json:string>common subtype</json:string><json:string>swai digestion</json:string><json:string>unknown samples</json:string><json:string>restriction endonuclease sites</json:string><json:string>african samples</json:string><json:string>outer reaction</json:string></teeft></keywords><author><json:item><name>Joanne van Harmelen</name><affiliations><json:string>Department of Medical Microbiology, University of Cape Town, Observatory, Cape Town 7925, South Africa</json:string></affiliations></json:item><json:item><name>Elna van der Ryst</name><affiliations><json:string>Department of Virology, University of the Free State, Bloemfontein 9300, South Africa</json:string></affiliations></json:item><json:item><name>Robin Wood</name><affiliations><json:string>Department of Medicine University of Cape Town, Observatory, Cape Town 7925, South Africa</json:string></affiliations></json:item><json:item><name>Susan F. Lyons</name><affiliations><json:string>National Institute for Virology, Private Bag X4, Sandringham 2131, South Africa</json:string></affiliations></json:item><json:item><name>Carolyn Williamson</name><affiliations><json:string>Department of Medical Microbiology, University of Cape Town, Observatory, Cape Town 7925, South Africa</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Human immunodeficiency virus type-1 (HIV-1)</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>gag gene</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Restriction fragment length polymorphism (RFLP)</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Polymerase chain reaction (PCR)</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Subtyping</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Recombinant viruses</value></json:item></subject><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>A rapid method for identification of human immunodeficiency virus Type 1 (HIV-1) gag subtypes was developed based on restriction fragment length polymorphism (RFLP) analysis of 400 or 650 bp long polymerase chain reaction (PCR) fragments encompassing the start of the p17 (400 bp) and part of the p24 (650bp) regions. The consensus sequences of subtypes A–D, the only subtypes identified in South Africa, were analyzed to detect restriction endonucleases which generate unique patterns for each subtype. Four restriction endonucleases were identified: AluI, AccI, SwaI and XmnI. Digestion of a 400 bp fragment with AluI allowed identification of subtype C. Samples not identified were then reamplified, and a 650 bp fragment digested with AccI to identify subtype B, followed by SwaI and XmnI to distinguish between subtypes A and D. This strategy was applied to 87 samples previously subtyped by either sequence analysis of the gag p17 region (n=33); or heteroduplex mobility assay (HMA) based on the env gene (n=75); or both (n=21). Out of the 87 samples, RFLP identified two samples as subtype A, 28 as subtype B, 56 as subtype C and one as a subtype D virus. No discrepancies were found between RFLP gag subtypes and gag sequence subtypes demonstrating the reliability of this method. There was also no discordance between gag RFLP subtypes and env HMA subtypes, suggesting that there were no recombinant viruses detected relating to the genomic regions analyzed. RFLP is an effective technique for the rapid screening in an HIV epidemic of limited diversity, such as in South Africa.</abstract><qualityIndicators><score>7.352</score><pdfVersion>1.2</pdfVersion><pdfPageSize>535 x 675 pts</pdfPageSize><refBibsNative>true</refBibsNative><keywordCount>6</keywordCount><abstractCharCount>1588</abstractCharCount><pdfWordCount>4352</pdfWordCount><pdfCharCount>25845</pdfCharCount><pdfPageCount>9</pdfPageCount><abstractWordCount>255</abstractWordCount></qualityIndicators><title>Restriction fragment length polymorphism analysis for rapid gag subtype determination of human immunodeficiency virus Type 1 in South Africa</title><pii><json:string>S0166-0934(98)00163-3</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Journal of Virological Methods</title><language><json:string>unknown</json:string></language><publicationDate>1999</publicationDate><issn><json:string>0166-0934</json:string></issn><pii><json:string>S0166-0934(00)X0050-X</json:string></pii><volume>78</volume><issue>1–2</issue><pages><first>51</first><last>59</last></pages><genre><json:string>journal</json:string></genre></host><categories><wos><json:string>science</json:string><json:string>virology</json:string><json:string>biotechnology & applied microbiology</json:string><json:string>biochemical research methods</json:string></wos><scienceMetrix><json:string>health sciences</json:string><json:string>biomedical research</json:string><json:string>virology</json:string></scienceMetrix><inist><json:string>sciences appliquees, technologies et medecines</json:string><json:string>sciences biologiques et medicales</json:string><json:string>sciences biologiques fondamentales et appliquees. psychologie</json:string><json:string>biotechnologie</json:string></inist></categories><publicationDate>1999</publicationDate><copyrightDate>1999</copyrightDate><doi><json:string>10.1016/S0166-0934(98)00163-3</json:string></doi><id>78626E611136FBCFEE6068DC821F7A83FA111144</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/document/78626E611136FBCFEE6068DC821F7A83FA111144/fulltext/pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/document/78626E611136FBCFEE6068DC821F7A83FA111144/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/document/78626E611136FBCFEE6068DC821F7A83FA111144/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Restriction fragment length polymorphism analysis for rapid gag subtype determination of human immunodeficiency virus Type 1 in South Africa</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>ELSEVIER</publisher><availability><p>©1999 Elsevier Science B.V.</p></availability><date>1999</date></publicationStmt><notesStmt><note type="content">Fig. 1: (a) The RESA of the partial gag consensus sequences for subtypes A, B, C and D showing where the identifying restriction endonucleases digested and the fragment sizes in base pairs which are produced. (b) The strategy for RFLP, starting with AluI digestion of the 400 bp gag PCR product resulting in either three fragments (subtype C) or five fragments (subtypes A, B or D), allowing identification of subtype C samples. AccI digestion of the 400 or 650 bp PCR fragment for remaining unidentified samples should result in two fragments (subtype B) or one fragment (subtype A and D). SwaI digestion of the 650 bp fragment should result in either two fragments (subtype A) or one fragment (subtype D). Finally, XmnI digestion, predicted to digest only subtype D, of either the 400 or 650 bp fragment of any unidentified samples should result in two fragments. The predicted fragments from the consensus sequence analysis are shown in brackets.</note><note type="content">Fig. 2: RFLP analysis of the 400 or 650 bp fragment from unknown samples, allowing the identification of subtype B (a) and C (b) respectively. (a) The 650 bp fragment digested with AccI, yields two bands for subtype B (±410 bp; ±240 bp), whereas subtypes A, C and D are not digested (650 bp). An additional band can be seen for sample B3, possibly due to the presence of two subtype B variants, one with a deletion in the gag gene. (b) Digestion by AluI produced five fragments for subtypes A, B and D, but due to the similarity in size of two pairs of fragments, only two or three bands were usually visualised in the 4% agarose gel. Although three fragments were produced by AluI digestion of subtype C, only two fragments were visualised. The length of the upper fragment produced after AluI digestion (±240 bp) is larger for subtype C than subtypes A, B and D (±160 bp), allowing the identification of subtype C. The molecular weight markers used were markers VI and VIII (Boehringer Mannheim, Germany).</note><note type="content">Table 1: Gag subtype designation by RFLP analysis and RESAa, compared to subtype designation by genetic sequencing</note><note type="content">Table 2: A comparison of HIV-1 subtype designation using RFLP in the gag region and HMA in the env region</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Restriction fragment length polymorphism analysis for rapid gag subtype determination of human immunodeficiency virus Type 1 in South Africa</title><author xml:id="author-0000"><persName><forename type="first">Joanne</forename><surname>van Harmelen</surname></persName><note type="correspondence"><p>Corresponding author. Tel.: +27-21-406-6458; fax: +27-21-448-8153</p></note><affiliation>Department of Medical Microbiology, University of Cape Town, Observatory, Cape Town 7925, South Africa</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Elna</forename><surname>van der Ryst</surname></persName><affiliation>Department of Virology, University of the Free State, Bloemfontein 9300, South Africa</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Robin</forename><surname>Wood</surname></persName><affiliation>Department of Medicine University of Cape Town, Observatory, Cape Town 7925, South Africa</affiliation></author><author xml:id="author-0003"><persName><forename type="first">Susan F.</forename><surname>Lyons</surname></persName><affiliation>National Institute for Virology, Private Bag X4, Sandringham 2131, South Africa</affiliation></author><author xml:id="author-0004"><persName><forename type="first">Carolyn</forename><surname>Williamson</surname></persName><affiliation>Department of Medical Microbiology, University of Cape Town, Observatory, Cape Town 7925, South Africa</affiliation></author><idno type="istex">78626E611136FBCFEE6068DC821F7A83FA111144</idno><idno type="DOI">10.1016/S0166-0934(98)00163-3</idno><idno type="PII">S0166-0934(98)00163-3</idno></analytic><monogr><title level="j">Journal of Virological Methods</title><title level="j" type="abbrev">VIRMET</title><idno type="pISSN">0166-0934</idno><idno type="PII">S0166-0934(00)X0050-X</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1999"></date><biblScope unit="volume">78</biblScope><biblScope unit="issue">1–2</biblScope><biblScope unit="page" from="51">51</biblScope><biblScope unit="page" to="59">59</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1999</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>A rapid method for identification of human immunodeficiency virus Type 1 (HIV-1) gag subtypes was developed based on restriction fragment length polymorphism (RFLP) analysis of 400 or 650 bp long polymerase chain reaction (PCR) fragments encompassing the start of the p17 (400 bp) and part of the p24 (650bp) regions. The consensus sequences of subtypes A–D, the only subtypes identified in South Africa, were analyzed to detect restriction endonucleases which generate unique patterns for each subtype. Four restriction endonucleases were identified: AluI, AccI, SwaI and XmnI. Digestion of a 400 bp fragment with AluI allowed identification of subtype C. Samples not identified were then reamplified, and a 650 bp fragment digested with AccI to identify subtype B, followed by SwaI and XmnI to distinguish between subtypes A and D. This strategy was applied to 87 samples previously subtyped by either sequence analysis of the gag p17 region (n=33); or heteroduplex mobility assay (HMA) based on the env gene (n=75); or both (n=21). Out of the 87 samples, RFLP identified two samples as subtype A, 28 as subtype B, 56 as subtype C and one as a subtype D virus. No discrepancies were found between RFLP gag subtypes and gag sequence subtypes demonstrating the reliability of this method. There was also no discordance between gag RFLP subtypes and env HMA subtypes, suggesting that there were no recombinant viruses detected relating to the genomic regions analyzed. RFLP is an effective technique for the rapid screening in an HIV epidemic of limited diversity, such as in South Africa.</p></abstract><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>Human immunodeficiency virus type-1 (HIV-1)</term></item><item><term>gag gene</term></item><item><term>Restriction fragment length polymorphism (RFLP)</term></item><item><term>Polymerase chain reaction (PCR)</term></item><item><term>Subtyping</term></item><item><term>Recombinant viruses</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1998-11-11">Modified</change><change when="1999">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/document/78626E611136FBCFEE6068DC821F7A83FA111144/fulltext/txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1a" NDATA="IMAGE" name="gr1a"></istex:entity><istex:entity SYSTEM="gr1b" NDATA="IMAGE" name="gr1b"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>VIRMET</jid><aid>2543</aid><ce:pii>S0166-0934(98)00163-3</ce:pii><ce:doi>10.1016/S0166-0934(98)00163-3</ce:doi><ce:copyright type="full-transfer" year="1999">Elsevier Science B.V.</ce:copyright></item-info><head><ce:title>Restriction fragment length polymorphism analysis for rapid <ce:italic>gag</ce:italic> subtype determination of human immunodeficiency virus Type 1 in South Africa</ce:title><ce:author-group><ce:author><ce:given-name>Joanne</ce:given-name><ce:surname>van Harmelen</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="CORR1">*</ce:cross-ref></ce:author><ce:author><ce:given-name>Elna</ce:given-name><ce:surname>van der Ryst</ce:surname><ce:cross-ref refid="AFF2"><ce:sup>b</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Robin</ce:given-name><ce:surname>Wood</ce:surname><ce:cross-ref refid="AFF3"><ce:sup>c</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Susan F.</ce:given-name><ce:surname>Lyons</ce:surname><ce:cross-ref refid="AFF4"><ce:sup>d</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Carolyn</ce:given-name><ce:surname>Williamson</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="AFF5"><ce:sup>e</ce:sup></ce:cross-ref></ce:author><ce:affiliation id="AFF1"><ce:label>a</ce:label><ce:textfn>Department of Medical Microbiology, University of Cape Town, Observatory, Cape Town 7925, South Africa</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>b</ce:label><ce:textfn>Department of Virology, University of the Free State, Bloemfontein 9300, South Africa</ce:textfn></ce:affiliation><ce:affiliation id="AFF3"><ce:label>c</ce:label><ce:textfn>Department of Medicine University of Cape Town, Observatory, Cape Town 7925, South Africa</ce:textfn></ce:affiliation><ce:affiliation id="AFF4"><ce:label>d</ce:label><ce:textfn>National Institute for Virology, Private Bag X4, Sandringham 2131, South Africa</ce:textfn></ce:affiliation><ce:affiliation id="AFF5"><ce:label>e</ce:label><ce:textfn>Department of SAIMR Virology, University of Cape Town, Observatory, Cape Town 7925, South Africa</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Corresponding author. Tel.: +27-21-406-6458; fax: +27-21-448-8153</ce:text></ce:correspondence></ce:author-group><ce:date-received day="29" month="7" year="1998"></ce:date-received><ce:date-revised day="11" month="11" year="1998"></ce:date-revised><ce:date-accepted day="13" month="11" year="1998"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>A rapid method for identification of human immunodeficiency virus Type 1 (HIV-1) <ce:italic>gag</ce:italic> subtypes was developed based on restriction fragment length polymorphism (RFLP) analysis of 400 or 650 bp long polymerase chain reaction (PCR) fragments encompassing the start of the p17 (400 bp) and part of the p24 (650bp) regions. The consensus sequences of subtypes A–D, the only subtypes identified in South Africa, were analyzed to detect restriction endonucleases which generate unique patterns for each subtype. Four restriction endonucleases were identified: <ce:italic>Alu</ce:italic>I, <ce:italic>Acc</ce:italic>I, <ce:italic>Swa</ce:italic>I and <ce:italic>Xmn</ce:italic>I. Digestion of a 400 bp fragment with <ce:italic>Alu</ce:italic>I allowed identification of subtype C. Samples not identified were then reamplified, and a 650 bp fragment digested with <ce:italic>Acc</ce:italic>I to identify subtype B, followed by <ce:italic>Swa</ce:italic>I and <ce:italic>Xmn</ce:italic>I to distinguish between subtypes A and D. This strategy was applied to 87 samples previously subtyped by either sequence analysis of the <ce:italic>gag</ce:italic> p17 region (<ce:italic>n</ce:italic>=33); or heteroduplex mobility assay (HMA) based on the <ce:italic>env</ce:italic> gene (<ce:italic>n</ce:italic>=75); or both (<ce:italic>n</ce:italic>=21). Out of the 87 samples, RFLP identified two samples as subtype A, 28 as subtype B, 56 as subtype C and one as a subtype D virus. No discrepancies were found between RFLP <ce:italic>gag</ce:italic> subtypes and <ce:italic>gag</ce:italic> sequence subtypes demonstrating the reliability of this method. There was also no discordance between <ce:italic>gag</ce:italic> RFLP subtypes and <ce:italic>env</ce:italic> HMA subtypes, suggesting that there were no recombinant viruses detected relating to the genomic regions analyzed. RFLP is an effective technique for the rapid screening in an HIV epidemic of limited diversity, such as in South Africa.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords class="keyword"><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>Human immunodeficiency virus type-1 (HIV-1)</ce:text></ce:keyword><ce:keyword><ce:text>gag gene</ce:text></ce:keyword><ce:keyword><ce:text>Restriction fragment length polymorphism (RFLP)</ce:text></ce:keyword><ce:keyword><ce:text>Polymerase chain reaction (PCR)</ce:text></ce:keyword><ce:keyword><ce:text>Subtyping</ce:text></ce:keyword><ce:keyword><ce:text>Recombinant viruses</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Restriction fragment length polymorphism analysis for rapid gag subtype determination of human immunodeficiency virus Type 1 in South Africa</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Restriction fragment length polymorphism analysis for rapid</title></titleInfo><name type="personal"><namePart type="given">Joanne</namePart><namePart type="family">van Harmelen</namePart><affiliation>Department of Medical Microbiology, University of Cape Town, Observatory, Cape Town 7925, South Africa</affiliation><description>Corresponding author. Tel.: +27-21-406-6458; fax: +27-21-448-8153</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Elna</namePart><namePart type="family">van der Ryst</namePart><affiliation>Department of Virology, University of the Free State, Bloemfontein 9300, South Africa</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Robin</namePart><namePart type="family">Wood</namePart><affiliation>Department of Medicine University of Cape Town, Observatory, Cape Town 7925, South Africa</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Susan F.</namePart><namePart type="family">Lyons</namePart><affiliation>National Institute for Virology, Private Bag X4, Sandringham 2131, South Africa</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Carolyn</namePart><namePart type="family">Williamson</namePart><affiliation>Department of Medical Microbiology, University of Cape Town, Observatory, Cape Town 7925, South Africa</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1999</dateIssued><dateModified encoding="w3cdtf">1998-11-11</dateModified><copyrightDate encoding="w3cdtf">1999</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: A rapid method for identification of human immunodeficiency virus Type 1 (HIV-1) gag subtypes was developed based on restriction fragment length polymorphism (RFLP) analysis of 400 or 650 bp long polymerase chain reaction (PCR) fragments encompassing the start of the p17 (400 bp) and part of the p24 (650bp) regions. The consensus sequences of subtypes A–D, the only subtypes identified in South Africa, were analyzed to detect restriction endonucleases which generate unique patterns for each subtype. Four restriction endonucleases were identified: AluI, AccI, SwaI and XmnI. Digestion of a 400 bp fragment with AluI allowed identification of subtype C. Samples not identified were then reamplified, and a 650 bp fragment digested with AccI to identify subtype B, followed by SwaI and XmnI to distinguish between subtypes A and D. This strategy was applied to 87 samples previously subtyped by either sequence analysis of the gag p17 region (n=33); or heteroduplex mobility assay (HMA) based on the env gene (n=75); or both (n=21). Out of the 87 samples, RFLP identified two samples as subtype A, 28 as subtype B, 56 as subtype C and one as a subtype D virus. No discrepancies were found between RFLP gag subtypes and gag sequence subtypes demonstrating the reliability of this method. There was also no discordance between gag RFLP subtypes and env HMA subtypes, suggesting that there were no recombinant viruses detected relating to the genomic regions analyzed. RFLP is an effective technique for the rapid screening in an HIV epidemic of limited diversity, such as in South Africa.</abstract><note type="content">Fig. 1: (a) The RESA of the partial gag consensus sequences for subtypes A, B, C and D showing where the identifying restriction endonucleases digested and the fragment sizes in base pairs which are produced. (b) The strategy for RFLP, starting with AluI digestion of the 400 bp gag PCR product resulting in either three fragments (subtype C) or five fragments (subtypes A, B or D), allowing identification of subtype C samples. AccI digestion of the 400 or 650 bp PCR fragment for remaining unidentified samples should result in two fragments (subtype B) or one fragment (subtype A and D). SwaI digestion of the 650 bp fragment should result in either two fragments (subtype A) or one fragment (subtype D). Finally, XmnI digestion, predicted to digest only subtype D, of either the 400 or 650 bp fragment of any unidentified samples should result in two fragments. The predicted fragments from the consensus sequence analysis are shown in brackets.</note><note type="content">Fig. 2: RFLP analysis of the 400 or 650 bp fragment from unknown samples, allowing the identification of subtype B (a) and C (b) respectively. (a) The 650 bp fragment digested with AccI, yields two bands for subtype B (±410 bp; ±240 bp), whereas subtypes A, C and D are not digested (650 bp). An additional band can be seen for sample B3, possibly due to the presence of two subtype B variants, one with a deletion in the gag gene. (b) Digestion by AluI produced five fragments for subtypes A, B and D, but due to the similarity in size of two pairs of fragments, only two or three bands were usually visualised in the 4% agarose gel. Although three fragments were produced by AluI digestion of subtype C, only two fragments were visualised. The length of the upper fragment produced after AluI digestion (±240 bp) is larger for subtype C than subtypes A, B and D (±160 bp), allowing the identification of subtype C. The molecular weight markers used were markers VI and VIII (Boehringer Mannheim, Germany).</note><note type="content">Table 1: Gag subtype designation by RFLP analysis and RESAa, compared to subtype designation by genetic sequencing</note><note type="content">Table 2: A comparison of HIV-1 subtype designation using RFLP in the gag region and HMA in the env region</note><subject lang="en"><genre>Keywords</genre><topic>Human immunodeficiency virus type-1 (HIV-1)</topic><topic>gag gene</topic><topic>Restriction fragment length polymorphism (RFLP)</topic><topic>Polymerase chain reaction (PCR)</topic><topic>Subtyping</topic><topic>Recombinant viruses</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Virological Methods</title></titleInfo><titleInfo type="abbreviated"><title>VIRMET</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">199903</dateIssued></originInfo><identifier type="ISSN">0166-0934</identifier><identifier type="PII">S0166-0934(00)X0050-X</identifier><part><date>199903</date><detail type="volume"><number>78</number><caption>vol.</caption></detail><detail type="issue"><number>1–2</number><caption>no.</caption></detail><extent unit="issue-pages"><start>1</start><end>226</end></extent><extent unit="pages"><start>51</start><end>59</end></extent></part></relatedItem><identifier type="istex">78626E611136FBCFEE6068DC821F7A83FA111144</identifier><identifier type="ark">ark:/67375/6H6-20R3HXRR-L</identifier><identifier type="DOI">10.1016/S0166-0934(98)00163-3</identifier><identifier type="PII">S0166-0934(98)00163-3</identifier><accessCondition type="use and reproduction" contentType="copyright">©1999 Elsevier Science B.V.</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Elsevier Science B.V., ©1999</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/document/78626E611136FBCFEE6068DC821F7A83FA111144/metadata/json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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