Development and application of a quantitative RT-PCR potency assay for a pentavalent rotavirus vaccine (RotaTeq ®)
A sensitive and reproducible method to determine the in vitro infectious potency of a pentavalent reassortant rotavirus vaccine (RotaTeq ®) has been developed as an alternative to classical potency assays. Potency was determined based on cell-based viral replication followed by quantitative reverse-...
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| Veröffentlicht in: | Journal of virological methods 2006-02, Vol.131 (2), p.193-201 |
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| creator | Ranheim, Todd Mathis, Pamela K. Joelsson, Daniel B. Smith, Mary Ellen Campbell, Kathryn M. Lucas, Gwendolyn Barmat, Scott Melissen, Erin Benz, Robert Lewis, John A. Chen, Jie Schofield, Timothy Sitrin, Robert D. Hennessey, John P. |
| description | A sensitive and reproducible method to determine the in vitro infectious potency of a pentavalent reassortant rotavirus vaccine (RotaTeq
®) has been developed as an alternative to classical potency assays. Potency was determined based on cell-based viral replication followed by quantitative reverse-transcription polymerase chain reaction (RT-QPCR) analysis. In the assay, confluent Vero cell monolayers in 96-well plates were inoculated with serial dilutions of test samples, a pentavalent reassortant rotavirus reference standard and assay controls, followed by incubation for 24
h. The cells were lysed with a Triton X-100 solution and the lysates assayed by RT-QPCR to quantitate viral nucleic acid produced during replication. The RT-QPCR utilizes primer/probe sets specific to each virus reassortant and the potencies of each sample were determined relative to the reference standard. This assay, hereafter referred to as the Multivalent QPCR-Based Potency Assay (M-QPA), permits the specific quantitation of each individual reassortant virus in the presence of the other four reassortant viruses. In addition, the assay was demonstrated to be concordant with a traditional method (plaque assay) for the quantitation of infectious virus particles. It is anticipated that assays of this type will become a valuable tool in the assignment of potency values and in the monitoring of stability of live virus vaccines. |
| doi_str_mv | 10.1016/j.jviromet.2005.08.013 |
| format | Article |
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®) has been developed as an alternative to classical potency assays. Potency was determined based on cell-based viral replication followed by quantitative reverse-transcription polymerase chain reaction (RT-QPCR) analysis. In the assay, confluent Vero cell monolayers in 96-well plates were inoculated with serial dilutions of test samples, a pentavalent reassortant rotavirus reference standard and assay controls, followed by incubation for 24
h. The cells were lysed with a Triton X-100 solution and the lysates assayed by RT-QPCR to quantitate viral nucleic acid produced during replication. The RT-QPCR utilizes primer/probe sets specific to each virus reassortant and the potencies of each sample were determined relative to the reference standard. This assay, hereafter referred to as the Multivalent QPCR-Based Potency Assay (M-QPA), permits the specific quantitation of each individual reassortant virus in the presence of the other four reassortant viruses. In addition, the assay was demonstrated to be concordant with a traditional method (plaque assay) for the quantitation of infectious virus particles. It is anticipated that assays of this type will become a valuable tool in the assignment of potency values and in the monitoring of stability of live virus vaccines.</description><identifier>ISSN: 0166-0934</identifier><identifier>EISSN: 1879-0984</identifier><identifier>DOI: 10.1016/j.jviromet.2005.08.013</identifier><identifier>PMID: 16214228</identifier><identifier>CODEN: JVMEDH</identifier><language>eng</language><publisher>London: Elsevier B.V</publisher><subject>Animals ; Biological and medical sciences ; Cercopithecus aethiops ; Fundamental and applied biological sciences. Psychology ; Microbiology ; Potency assay ; Quantitative RT-PCR ; Reverse Transcriptase Polymerase Chain Reaction - methods ; RNA, Viral - analysis ; Rotavirus - genetics ; Rotavirus - physiology ; Rotavirus vaccine ; Rotavirus Vaccines - genetics ; Techniques used in virology ; Vaccines, Attenuated - genetics ; Vero Cells ; Viral Plaque Assay ; Virology ; Virus Replication</subject><ispartof>Journal of virological methods, 2006-02, Vol.131 (2), p.193-201</ispartof><rights>2005 Elsevier B.V.</rights><rights>2006 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c396t-ae0526e9e8b904a671669df6750856464e6b06942f22614c018438c6565f2c1f3</citedby><cites>FETCH-LOGICAL-c396t-ae0526e9e8b904a671669df6750856464e6b06942f22614c018438c6565f2c1f3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jviromet.2005.08.013$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,777,781,3537,27905,27906,45976</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=17461835$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16214228$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ranheim, Todd</creatorcontrib><creatorcontrib>Mathis, Pamela K.</creatorcontrib><creatorcontrib>Joelsson, Daniel B.</creatorcontrib><creatorcontrib>Smith, Mary Ellen</creatorcontrib><creatorcontrib>Campbell, Kathryn M.</creatorcontrib><creatorcontrib>Lucas, Gwendolyn</creatorcontrib><creatorcontrib>Barmat, Scott</creatorcontrib><creatorcontrib>Melissen, Erin</creatorcontrib><creatorcontrib>Benz, Robert</creatorcontrib><creatorcontrib>Lewis, John A.</creatorcontrib><creatorcontrib>Chen, Jie</creatorcontrib><creatorcontrib>Schofield, Timothy</creatorcontrib><creatorcontrib>Sitrin, Robert D.</creatorcontrib><creatorcontrib>Hennessey, John P.</creatorcontrib><title>Development and application of a quantitative RT-PCR potency assay for a pentavalent rotavirus vaccine (RotaTeq ®)</title><title>Journal of virological methods</title><addtitle>J Virol Methods</addtitle><description>A sensitive and reproducible method to determine the in vitro infectious potency of a pentavalent reassortant rotavirus vaccine (RotaTeq
®) has been developed as an alternative to classical potency assays. Potency was determined based on cell-based viral replication followed by quantitative reverse-transcription polymerase chain reaction (RT-QPCR) analysis. In the assay, confluent Vero cell monolayers in 96-well plates were inoculated with serial dilutions of test samples, a pentavalent reassortant rotavirus reference standard and assay controls, followed by incubation for 24
h. The cells were lysed with a Triton X-100 solution and the lysates assayed by RT-QPCR to quantitate viral nucleic acid produced during replication. The RT-QPCR utilizes primer/probe sets specific to each virus reassortant and the potencies of each sample were determined relative to the reference standard. This assay, hereafter referred to as the Multivalent QPCR-Based Potency Assay (M-QPA), permits the specific quantitation of each individual reassortant virus in the presence of the other four reassortant viruses. In addition, the assay was demonstrated to be concordant with a traditional method (plaque assay) for the quantitation of infectious virus particles. It is anticipated that assays of this type will become a valuable tool in the assignment of potency values and in the monitoring of stability of live virus vaccines.</description><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Cercopithecus aethiops</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Microbiology</subject><subject>Potency assay</subject><subject>Quantitative RT-PCR</subject><subject>Reverse Transcriptase Polymerase Chain Reaction - methods</subject><subject>RNA, Viral - analysis</subject><subject>Rotavirus - genetics</subject><subject>Rotavirus - physiology</subject><subject>Rotavirus vaccine</subject><subject>Rotavirus Vaccines - genetics</subject><subject>Techniques used in virology</subject><subject>Vaccines, Attenuated - genetics</subject><subject>Vero Cells</subject><subject>Viral Plaque Assay</subject><subject>Virology</subject><subject>Virus Replication</subject><issn>0166-0934</issn><issn>1879-0984</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkM9q3DAQh0VpaTZpXyHo0tIe7OifZfnWsm3aQiBl2Z6FVh6BFtvySrZhX6oPkSerlt2SY04zDN_8RvoQuqWkpITKu325X3wMPUwlI6QqiSoJ5a_Qiqq6KUijxGu0yqDMPRdX6DqlPclgzflbdEUlo4IxtULpGyzQhbGHYcJmaLEZx85bM_kw4OCwwYfZDJOf8mQBvNkWv9cbPIYJBnvEJiVzxC7EzI05wSymOwXFkFsf54QXY60fAH_a5NEWDvjp7-d36I0zXYL3l3qD_tx_365_Fg-PP36tvz4UljdyKgyQikloQO0aIoys82-a1sm6IqqSQgqQOyIbwRxjkgpLqBJcWVnJyjFLHb9BH8-5YwyHGdKke58sdJ0ZIMxJ10SqqlE8g_IM2hhSiuD0GH1v4lFTok-69V7_161PujVROuvOi7eXC_Ouh_Z57eI3Ax8ugEnWdC6awfr0zNVCUsWrzH05c5B9LB6iTtZnxdD6CHbSbfAvveUfB0aiEg</recordid><startdate>20060201</startdate><enddate>20060201</enddate><creator>Ranheim, Todd</creator><creator>Mathis, Pamela K.</creator><creator>Joelsson, Daniel B.</creator><creator>Smith, Mary Ellen</creator><creator>Campbell, Kathryn M.</creator><creator>Lucas, Gwendolyn</creator><creator>Barmat, Scott</creator><creator>Melissen, Erin</creator><creator>Benz, Robert</creator><creator>Lewis, John A.</creator><creator>Chen, Jie</creator><creator>Schofield, Timothy</creator><creator>Sitrin, Robert D.</creator><creator>Hennessey, John P.</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>7X8</scope></search><sort><creationdate>20060201</creationdate><title>Development and application of a quantitative RT-PCR potency assay for a pentavalent rotavirus vaccine (RotaTeq ®)</title><author>Ranheim, Todd ; Mathis, Pamela K. ; Joelsson, Daniel B. ; Smith, Mary Ellen ; Campbell, Kathryn M. ; Lucas, Gwendolyn ; Barmat, Scott ; Melissen, Erin ; Benz, Robert ; Lewis, John A. ; Chen, Jie ; Schofield, Timothy ; Sitrin, Robert D. ; Hennessey, John P.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c396t-ae0526e9e8b904a671669df6750856464e6b06942f22614c018438c6565f2c1f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Cercopithecus aethiops</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Microbiology</topic><topic>Potency assay</topic><topic>Quantitative RT-PCR</topic><topic>Reverse Transcriptase Polymerase Chain Reaction - methods</topic><topic>RNA, Viral - analysis</topic><topic>Rotavirus - genetics</topic><topic>Rotavirus - physiology</topic><topic>Rotavirus vaccine</topic><topic>Rotavirus Vaccines - genetics</topic><topic>Techniques used in virology</topic><topic>Vaccines, Attenuated - genetics</topic><topic>Vero Cells</topic><topic>Viral Plaque Assay</topic><topic>Virology</topic><topic>Virus Replication</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ranheim, Todd</creatorcontrib><creatorcontrib>Mathis, Pamela K.</creatorcontrib><creatorcontrib>Joelsson, Daniel B.</creatorcontrib><creatorcontrib>Smith, Mary Ellen</creatorcontrib><creatorcontrib>Campbell, Kathryn M.</creatorcontrib><creatorcontrib>Lucas, Gwendolyn</creatorcontrib><creatorcontrib>Barmat, Scott</creatorcontrib><creatorcontrib>Melissen, Erin</creatorcontrib><creatorcontrib>Benz, Robert</creatorcontrib><creatorcontrib>Lewis, John A.</creatorcontrib><creatorcontrib>Chen, Jie</creatorcontrib><creatorcontrib>Schofield, Timothy</creatorcontrib><creatorcontrib>Sitrin, Robert D.</creatorcontrib><creatorcontrib>Hennessey, John P.</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>MEDLINE - Academic</collection><jtitle>Journal of virological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ranheim, Todd</au><au>Mathis, Pamela K.</au><au>Joelsson, Daniel B.</au><au>Smith, Mary Ellen</au><au>Campbell, Kathryn M.</au><au>Lucas, Gwendolyn</au><au>Barmat, Scott</au><au>Melissen, Erin</au><au>Benz, Robert</au><au>Lewis, John A.</au><au>Chen, Jie</au><au>Schofield, Timothy</au><au>Sitrin, Robert D.</au><au>Hennessey, John P.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development and application of a quantitative RT-PCR potency assay for a pentavalent rotavirus vaccine (RotaTeq ®)</atitle><jtitle>Journal of virological methods</jtitle><addtitle>J Virol Methods</addtitle><date>2006-02-01</date><risdate>2006</risdate><volume>131</volume><issue>2</issue><spage>193</spage><epage>201</epage><pages>193-201</pages><issn>0166-0934</issn><eissn>1879-0984</eissn><coden>JVMEDH</coden><abstract>A sensitive and reproducible method to determine the in vitro infectious potency of a pentavalent reassortant rotavirus vaccine (RotaTeq
®) has been developed as an alternative to classical potency assays. Potency was determined based on cell-based viral replication followed by quantitative reverse-transcription polymerase chain reaction (RT-QPCR) analysis. In the assay, confluent Vero cell monolayers in 96-well plates were inoculated with serial dilutions of test samples, a pentavalent reassortant rotavirus reference standard and assay controls, followed by incubation for 24
h. The cells were lysed with a Triton X-100 solution and the lysates assayed by RT-QPCR to quantitate viral nucleic acid produced during replication. The RT-QPCR utilizes primer/probe sets specific to each virus reassortant and the potencies of each sample were determined relative to the reference standard. This assay, hereafter referred to as the Multivalent QPCR-Based Potency Assay (M-QPA), permits the specific quantitation of each individual reassortant virus in the presence of the other four reassortant viruses. In addition, the assay was demonstrated to be concordant with a traditional method (plaque assay) for the quantitation of infectious virus particles. It is anticipated that assays of this type will become a valuable tool in the assignment of potency values and in the monitoring of stability of live virus vaccines.</abstract><cop>London</cop><cop>Amsterdam</cop><cop>New York, NY</cop><pub>Elsevier B.V</pub><pmid>16214228</pmid><doi>10.1016/j.jviromet.2005.08.013</doi><tpages>9</tpages></addata></record> |
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| subjects | Animals Biological and medical sciences Cercopithecus aethiops Fundamental and applied biological sciences. Psychology Microbiology Potency assay Quantitative RT-PCR Reverse Transcriptase Polymerase Chain Reaction - methods RNA, Viral - analysis Rotavirus - genetics Rotavirus - physiology Rotavirus vaccine Rotavirus Vaccines - genetics Techniques used in virology Vaccines, Attenuated - genetics Vero Cells Viral Plaque Assay Virology Virus Replication |
| title | Development and application of a quantitative RT-PCR potency assay for a pentavalent rotavirus vaccine (RotaTeq ®) |
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