Quantitation of hepatitis B virus dna (HBV DNA) in serum using the spot hybridization technique and scintillation counting
The simple spot hybridization technique to detect HBV DNA in serum was modified to concentrate Dane particles by pelleting. This minimised interference by serum components and allowed larger volumes of sera (up to 500 μl) to be used in serial dilution on borderline positive samples and increased the...
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| Veröffentlicht in: | Journal of virological methods 1985-12, Vol.12 (3), p.251-262 |
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| container_title | Journal of virological methods |
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| creator | Fagan, Elizabeth A. Guarner, Pancho Perera, Solangaratchige D.K. Trowbridge, Rachel Rolando, Nancy Davison, Fergus Williams, Roger |
| description | The simple spot hybridization technique to detect HBV DNA in serum was modified to concentrate Dane particles by pelleting. This minimised interference by serum components and allowed larger volumes of sera (up to 500 μl) to be used in serial dilution on borderline positive samples and increased the efficiency of filtering through a ‘Hybri.Dot’ system. Quantification of HBV DNA by
32P-scintillation Cerenkov counting based on serial standards of cloned HBV DNA processed through the ‘Hybri.dot’ was easy to perform and the assay had good sensitivity (detection limit 2 pg, mode 6.25 pg), precision and accuracy. The pellet-simple spot quantitative assay proved more sensitive than those involving lengthier extraction procedures or direct application of serum to hybridization filters. Scintillation counting curves were linear over a wider range (0–800 pg HBV DNA) than optical densitometry measurements (0–50 pg) of autoradiographs. There was an excellent correlation with DNA polymerase activity (r = 0.948;
P 45 pg cloned equivalents) was detected in 4 out of 14 DNA polymerase negative sera and in 7 of 7 borderline (DNA polymerase cpm range 116–303) samples. This sensitive, quantitative HBV DNA assay should be of considerable value in studies on infectivity and effectiveness of antiviral therapies. |
| doi_str_mv | 10.1016/0166-0934(85)90136-3 |
| format | Article |
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32P-scintillation Cerenkov counting based on serial standards of cloned HBV DNA processed through the ‘Hybri.dot’ was easy to perform and the assay had good sensitivity (detection limit 2 pg, mode 6.25 pg), precision and accuracy. The pellet-simple spot quantitative assay proved more sensitive than those involving lengthier extraction procedures or direct application of serum to hybridization filters. Scintillation counting curves were linear over a wider range (0–800 pg HBV DNA) than optical densitometry measurements (0–50 pg) of autoradiographs. There was an excellent correlation with DNA polymerase activity (r = 0.948;
P <0.001) but the assay proved more sensitive since HBV DNA (> 45 pg cloned equivalents) was detected in 4 out of 14 DNA polymerase negative sera and in 7 of 7 borderline (DNA polymerase cpm range 116–303) samples. This sensitive, quantitative HBV DNA assay should be of considerable value in studies on infectivity and effectiveness of antiviral therapies.</description><identifier>ISSN: 0166-0934</identifier><identifier>EISSN: 1879-0984</identifier><identifier>DOI: 10.1016/0166-0934(85)90136-3</identifier><identifier>PMID: 3833870</identifier><identifier>CODEN: JVMEDH</identifier><language>eng</language><publisher>London: Elsevier B.V</publisher><subject>32P-scintillation (Cerenkov) counting ; Biological and medical sciences ; Densitometry ; DNA polymerase ; DNA, Viral - blood ; DNA-Directed DNA Polymerase - metabolism ; Fundamental and applied biological sciences. Psychology ; Hepatitis B e Antigens - analysis ; hepatitis B virus ; Hepatitis B virus - enzymology ; Hepatitis B virus - genetics ; Humans ; Microbiology ; Nucleic Acid Hybridization ; quantitative serum HBV DNA ; Scintillation Counting ; Techniques used in virology ; Virology</subject><ispartof>Journal of virological methods, 1985-12, Vol.12 (3), p.251-262</ispartof><rights>1985</rights><rights>1986 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c417t-b6c2ea8b000bbb5d6edcdf42aa44a6bb05caa74bf23f0454daf78aedd9df09e93</citedby><cites>FETCH-LOGICAL-c417t-b6c2ea8b000bbb5d6edcdf42aa44a6bb05caa74bf23f0454daf78aedd9df09e93</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/0166093485901363$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3536,27903,27904,65309</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=8753586$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/3833870$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Fagan, Elizabeth A.</creatorcontrib><creatorcontrib>Guarner, Pancho</creatorcontrib><creatorcontrib>Perera, Solangaratchige D.K.</creatorcontrib><creatorcontrib>Trowbridge, Rachel</creatorcontrib><creatorcontrib>Rolando, Nancy</creatorcontrib><creatorcontrib>Davison, Fergus</creatorcontrib><creatorcontrib>Williams, Roger</creatorcontrib><title>Quantitation of hepatitis B virus dna (HBV DNA) in serum using the spot hybridization technique and scintillation counting</title><title>Journal of virological methods</title><addtitle>J Virol Methods</addtitle><description>The simple spot hybridization technique to detect HBV DNA in serum was modified to concentrate Dane particles by pelleting. This minimised interference by serum components and allowed larger volumes of sera (up to 500 μl) to be used in serial dilution on borderline positive samples and increased the efficiency of filtering through a ‘Hybri.Dot’ system. Quantification of HBV DNA by
32P-scintillation Cerenkov counting based on serial standards of cloned HBV DNA processed through the ‘Hybri.dot’ was easy to perform and the assay had good sensitivity (detection limit 2 pg, mode 6.25 pg), precision and accuracy. The pellet-simple spot quantitative assay proved more sensitive than those involving lengthier extraction procedures or direct application of serum to hybridization filters. Scintillation counting curves were linear over a wider range (0–800 pg HBV DNA) than optical densitometry measurements (0–50 pg) of autoradiographs. There was an excellent correlation with DNA polymerase activity (r = 0.948;
P <0.001) but the assay proved more sensitive since HBV DNA (> 45 pg cloned equivalents) was detected in 4 out of 14 DNA polymerase negative sera and in 7 of 7 borderline (DNA polymerase cpm range 116–303) samples. This sensitive, quantitative HBV DNA assay should be of considerable value in studies on infectivity and effectiveness of antiviral therapies.</description><subject>32P-scintillation (Cerenkov) counting</subject><subject>Biological and medical sciences</subject><subject>Densitometry</subject><subject>DNA polymerase</subject><subject>DNA, Viral - blood</subject><subject>DNA-Directed DNA Polymerase - metabolism</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Hepatitis B e Antigens - analysis</subject><subject>hepatitis B virus</subject><subject>Hepatitis B virus - enzymology</subject><subject>Hepatitis B virus - genetics</subject><subject>Humans</subject><subject>Microbiology</subject><subject>Nucleic Acid Hybridization</subject><subject>quantitative serum HBV DNA</subject><subject>Scintillation Counting</subject><subject>Techniques used in virology</subject><subject>Virology</subject><issn>0166-0934</issn><issn>1879-0984</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1985</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkVtrVDEUhYModaz-A4U8iLQPR5PJ5eS8CG2tViiKoL6GXHY6kZmcaZJTaH-9mZ5hHvUh5LK_vbJYG6HXlLynhMoPbcmODIyfKHE6EMpkx56gBVX90J4Vf4oWB-Q5elHKH0KI6Bk7QkdMMaZ6skAPPyaTaqymxjHhMeAVbNu5xoLP8V3MU8E-GXxydf4bf_p2dopjwgXytMFTiekG1xXgsh0rXt3bHH18mIUquFWKtxNgkzwuLrY_1uu55sap3dLNS_QsmHWBV_v9GP36fPnz4qq7_v7l68XZdec47WtnpVuCUbaZt9YKL8E7H_jSGM6NtJYIZ0zPbViyQLjg3oReGfB-8IEMMLBj9G7W3eaxOSpVb2Jx0OwkGKeieym5oAP5L0g5J4ouVQP5DLo8lpIh6G2OG5PvNSV6Nxu9C17vgtdK6MfZaNba3uz1J7sBf2jaD6PV3-7rpjizDtkkF8sBU71gQsmGfZwxaKHdRci65QvJgY8ZXNV-jP_28Re6Rq0f</recordid><startdate>19851201</startdate><enddate>19851201</enddate><creator>Fagan, Elizabeth A.</creator><creator>Guarner, Pancho</creator><creator>Perera, Solangaratchige D.K.</creator><creator>Trowbridge, Rachel</creator><creator>Rolando, Nancy</creator><creator>Davison, Fergus</creator><creator>Williams, Roger</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7T7</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>19851201</creationdate><title>Quantitation of hepatitis B virus dna (HBV DNA) in serum using the spot hybridization technique and scintillation counting</title><author>Fagan, Elizabeth A. ; Guarner, Pancho ; Perera, Solangaratchige D.K. ; Trowbridge, Rachel ; Rolando, Nancy ; Davison, Fergus ; Williams, Roger</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c417t-b6c2ea8b000bbb5d6edcdf42aa44a6bb05caa74bf23f0454daf78aedd9df09e93</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1985</creationdate><topic>32P-scintillation (Cerenkov) counting</topic><topic>Biological and medical sciences</topic><topic>Densitometry</topic><topic>DNA polymerase</topic><topic>DNA, Viral - blood</topic><topic>DNA-Directed DNA Polymerase - metabolism</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Hepatitis B e Antigens - analysis</topic><topic>hepatitis B virus</topic><topic>Hepatitis B virus - enzymology</topic><topic>Hepatitis B virus - genetics</topic><topic>Humans</topic><topic>Microbiology</topic><topic>Nucleic Acid Hybridization</topic><topic>quantitative serum HBV DNA</topic><topic>Scintillation Counting</topic><topic>Techniques used in virology</topic><topic>Virology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Fagan, Elizabeth A.</creatorcontrib><creatorcontrib>Guarner, Pancho</creatorcontrib><creatorcontrib>Perera, Solangaratchige D.K.</creatorcontrib><creatorcontrib>Trowbridge, Rachel</creatorcontrib><creatorcontrib>Rolando, Nancy</creatorcontrib><creatorcontrib>Davison, Fergus</creatorcontrib><creatorcontrib>Williams, Roger</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of virological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Fagan, Elizabeth A.</au><au>Guarner, Pancho</au><au>Perera, Solangaratchige D.K.</au><au>Trowbridge, Rachel</au><au>Rolando, Nancy</au><au>Davison, Fergus</au><au>Williams, Roger</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Quantitation of hepatitis B virus dna (HBV DNA) in serum using the spot hybridization technique and scintillation counting</atitle><jtitle>Journal of virological methods</jtitle><addtitle>J Virol Methods</addtitle><date>1985-12-01</date><risdate>1985</risdate><volume>12</volume><issue>3</issue><spage>251</spage><epage>262</epage><pages>251-262</pages><issn>0166-0934</issn><eissn>1879-0984</eissn><coden>JVMEDH</coden><abstract>The simple spot hybridization technique to detect HBV DNA in serum was modified to concentrate Dane particles by pelleting. This minimised interference by serum components and allowed larger volumes of sera (up to 500 μl) to be used in serial dilution on borderline positive samples and increased the efficiency of filtering through a ‘Hybri.Dot’ system. Quantification of HBV DNA by
32P-scintillation Cerenkov counting based on serial standards of cloned HBV DNA processed through the ‘Hybri.dot’ was easy to perform and the assay had good sensitivity (detection limit 2 pg, mode 6.25 pg), precision and accuracy. The pellet-simple spot quantitative assay proved more sensitive than those involving lengthier extraction procedures or direct application of serum to hybridization filters. Scintillation counting curves were linear over a wider range (0–800 pg HBV DNA) than optical densitometry measurements (0–50 pg) of autoradiographs. There was an excellent correlation with DNA polymerase activity (r = 0.948;
P <0.001) but the assay proved more sensitive since HBV DNA (> 45 pg cloned equivalents) was detected in 4 out of 14 DNA polymerase negative sera and in 7 of 7 borderline (DNA polymerase cpm range 116–303) samples. This sensitive, quantitative HBV DNA assay should be of considerable value in studies on infectivity and effectiveness of antiviral therapies.</abstract><cop>London</cop><cop>Amsterdam</cop><cop>New York, NY</cop><pub>Elsevier B.V</pub><pmid>3833870</pmid><doi>10.1016/0166-0934(85)90136-3</doi><tpages>12</tpages></addata></record> |
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| ispartof | Journal of virological methods, 1985-12, Vol.12 (3), p.251-262 |
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| subjects | 32P-scintillation (Cerenkov) counting Biological and medical sciences Densitometry DNA polymerase DNA, Viral - blood DNA-Directed DNA Polymerase - metabolism Fundamental and applied biological sciences. Psychology Hepatitis B e Antigens - analysis hepatitis B virus Hepatitis B virus - enzymology Hepatitis B virus - genetics Humans Microbiology Nucleic Acid Hybridization quantitative serum HBV DNA Scintillation Counting Techniques used in virology Virology |
| title | Quantitation of hepatitis B virus dna (HBV DNA) in serum using the spot hybridization technique and scintillation counting |
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