One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses
A one-tube reverse transcription-polymerase chain reaction (RT-PCR) for absolute feline coronavirus (FCoV) quantitation was developed. The assay is based on the 5′ nuclease activity of the Thermus flavus (Tfl) polymerase and a fluorogenic probe which generates fluorescence when it is cleaved. The fl...
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Veröffentlicht in: | Journal of virological methods 1999, Vol.77 (1), p.37-46 |
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description | A one-tube reverse transcription-polymerase chain reaction (RT-PCR) for absolute feline coronavirus (FCoV) quantitation was developed. The assay is based on the 5′ nuclease activity of the
Thermus flavus (Tfl) polymerase and a fluorogenic probe which generates fluorescence when it is cleaved. The fluorogenic probe, also called TaqMan™ probe (Perkin Elmer, Foster City, USA), is an oligonucleotide designed to bind between the two PCR primers to the target cDNA and is labeled with a reporter and a quencher dye. In the intact probe, the quencher dye suppresses the fluorescence of the reporter dye by Förster-type energy transfer. During the polymerase extension steps the
Tfl exonuclease activity cleaves the hybridised probe resulting in the generation of fluorescent emission of the reporter dye. The threshold cycle (
C
T value) indicates the increase of reporter fluorescence and is directly related to the initial amount of target cDNA or RNA, respectively. Fluorescence is monitored in real time after each cycle by a Perkin-Elmer ABI Prism® 7700 Sequence Detector. After completion of amplification, the
C
T values of the samples are calculated back to a standard curve, generated by amplification of diluted standard molecules. The one-tube RT-PCR described below allows precise quantitation, is highly sensitive, rapid (no separate reverse transcription step and no post-amplification steps), easy to handle, allows for a high sample throughput, shows a very good reproducibility, and can be executed with a low risk of contamination. The design of the primers–probe combination enables the detection of all known FCoV strains and is also useful for the detection of canine coronavirus, transmissible gastroenteritis virus and porcine respiratory coronavirus. |
doi_str_mv | 10.1016/S0166-0934(98)00129-3 |
format | Article |
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Thermus flavus (Tfl) polymerase and a fluorogenic probe which generates fluorescence when it is cleaved. The fluorogenic probe, also called TaqMan™ probe (Perkin Elmer, Foster City, USA), is an oligonucleotide designed to bind between the two PCR primers to the target cDNA and is labeled with a reporter and a quencher dye. In the intact probe, the quencher dye suppresses the fluorescence of the reporter dye by Förster-type energy transfer. During the polymerase extension steps the
Tfl exonuclease activity cleaves the hybridised probe resulting in the generation of fluorescent emission of the reporter dye. The threshold cycle (
C
T value) indicates the increase of reporter fluorescence and is directly related to the initial amount of target cDNA or RNA, respectively. Fluorescence is monitored in real time after each cycle by a Perkin-Elmer ABI Prism® 7700 Sequence Detector. After completion of amplification, the
C
T values of the samples are calculated back to a standard curve, generated by amplification of diluted standard molecules. The one-tube RT-PCR described below allows precise quantitation, is highly sensitive, rapid (no separate reverse transcription step and no post-amplification steps), easy to handle, allows for a high sample throughput, shows a very good reproducibility, and can be executed with a low risk of contamination. The design of the primers–probe combination enables the detection of all known FCoV strains and is also useful for the detection of canine coronavirus, transmissible gastroenteritis virus and porcine respiratory coronavirus.</description><identifier>ISSN: 0166-0934</identifier><identifier>EISSN: 1879-0984</identifier><identifier>DOI: 10.1016/S0166-0934(98)00129-3</identifier><identifier>PMID: 10029323</identifier><identifier>CODEN: JVMEDH</identifier><language>eng</language><publisher>London: Elsevier B.V</publisher><subject>Animals ; Base Sequence ; Biological and medical sciences ; Cat Diseases - virology ; Cats ; Coronavirus ; Coronavirus - genetics ; Coronavirus - isolation & purification ; Coronavirus Infections - veterinary ; Coronavirus Infections - virology ; DNA Primers ; DNA Probes ; DNA, Complementary ; Dogs ; Exodeoxyribonucleases - metabolism ; FCoV ; Feline coronavirus ; Fluorescent Dyes ; Fluorogenic 5′ nuclease assay ; Fundamental and applied biological sciences. Psychology ; Humans ; Microbiology ; Molecular Sequence Data ; One-tube RT-PCR ; Quantitation ; Reverse Transcriptase Polymerase Chain Reaction - methods ; RNA, Viral - analysis ; Sensitivity and Specificity ; Taq Polymerase ; TaqMan ; Techniques used in virology ; Thermus flavus ; Viral RNA ; Virology</subject><ispartof>Journal of virological methods, 1999, Vol.77 (1), p.37-46</ispartof><rights>1999 Elsevier Science B.V.</rights><rights>1999 INIST-CNRS</rights><rights>Copyright © 1999 Elsevier Science B.V. All rights reserved. 1999 Elsevier Science B.V.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c589t-64685254011e098256a4a92b9339046ee6584556b51eaaddd36e7e1948ebab853</citedby><cites>FETCH-LOGICAL-c589t-64685254011e098256a4a92b9339046ee6584556b51eaaddd36e7e1948ebab853</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/S0166-0934(98)00129-3$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>230,314,780,784,885,3550,4024,27923,27924,27925,45995</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1673660$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10029323$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Gut, Marco</creatorcontrib><creatorcontrib>M. Leutenegger, Christian</creatorcontrib><creatorcontrib>B. Huder, Jon</creatorcontrib><creatorcontrib>C. Pedersen, Niels</creatorcontrib><creatorcontrib>Lutz, Hans</creatorcontrib><title>One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses</title><title>Journal of virological methods</title><addtitle>J Virol Methods</addtitle><description>A one-tube reverse transcription-polymerase chain reaction (RT-PCR) for absolute feline coronavirus (FCoV) quantitation was developed. The assay is based on the 5′ nuclease activity of the
Thermus flavus (Tfl) polymerase and a fluorogenic probe which generates fluorescence when it is cleaved. The fluorogenic probe, also called TaqMan™ probe (Perkin Elmer, Foster City, USA), is an oligonucleotide designed to bind between the two PCR primers to the target cDNA and is labeled with a reporter and a quencher dye. In the intact probe, the quencher dye suppresses the fluorescence of the reporter dye by Förster-type energy transfer. During the polymerase extension steps the
Tfl exonuclease activity cleaves the hybridised probe resulting in the generation of fluorescent emission of the reporter dye. The threshold cycle (
C
T value) indicates the increase of reporter fluorescence and is directly related to the initial amount of target cDNA or RNA, respectively. Fluorescence is monitored in real time after each cycle by a Perkin-Elmer ABI Prism® 7700 Sequence Detector. After completion of amplification, the
C
T values of the samples are calculated back to a standard curve, generated by amplification of diluted standard molecules. The one-tube RT-PCR described below allows precise quantitation, is highly sensitive, rapid (no separate reverse transcription step and no post-amplification steps), easy to handle, allows for a high sample throughput, shows a very good reproducibility, and can be executed with a low risk of contamination. The design of the primers–probe combination enables the detection of all known FCoV strains and is also useful for the detection of canine coronavirus, transmissible gastroenteritis virus and porcine respiratory coronavirus.</description><subject>Animals</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Cat Diseases - virology</subject><subject>Cats</subject><subject>Coronavirus</subject><subject>Coronavirus - genetics</subject><subject>Coronavirus - isolation & purification</subject><subject>Coronavirus Infections - veterinary</subject><subject>Coronavirus Infections - virology</subject><subject>DNA Primers</subject><subject>DNA Probes</subject><subject>DNA, Complementary</subject><subject>Dogs</subject><subject>Exodeoxyribonucleases - metabolism</subject><subject>FCoV</subject><subject>Feline coronavirus</subject><subject>Fluorescent Dyes</subject><subject>Fluorogenic 5′ nuclease assay</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Humans</subject><subject>Microbiology</subject><subject>Molecular Sequence Data</subject><subject>One-tube RT-PCR</subject><subject>Quantitation</subject><subject>Reverse Transcriptase Polymerase Chain Reaction - methods</subject><subject>RNA, Viral - analysis</subject><subject>Sensitivity and Specificity</subject><subject>Taq Polymerase</subject><subject>TaqMan</subject><subject>Techniques used in virology</subject><subject>Thermus flavus</subject><subject>Viral RNA</subject><subject>Virology</subject><issn>0166-0934</issn><issn>1879-0984</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1999</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU9v1DAQxS0EokvhI4ByQBUcAnYcO_YFVFVtQarUA3C2Js6ka5S1d21npX57vH9U2lMvtjTvN88zfoS8Z_QLo0x-_VUOWVPN209afaaUNbrmL8iCqU6XsmpfksUDckLepPSXUio6zl-TE0Zpo3nDF2Rz67HOc4_VOM0hhjv0zlYRtxgTVjmCTza6dXbB1-sw3a8wQhHsEpwvGNidUo0hVnmJ1WYGn12GfTGM1YiT84Uuxh62Ls4J01vyaoQp4bvjfUr-XF3-vvhR39xe_7w4v6mtUDrXspVKNKKljGFZpxESWtBNrznXtJWIUqhWCNkLhgDDMHCJHTLdKuyhV4Kfkm8H3_Xcr3Cw6Ms2k1lHt4J4bwI481TxbmnuwtZ0TAnRNsXg7GgQw2bGlM3KJYvTBB7DnIzUZVBJxbMg6xiXnaQFFAfQxpBSxPFhGkbNLlWzT9XsIjNamX2qhpe-D49XedR1iLEAH48AJAvTWGKzLv3nZMfl_v3vBwzLv28dRpOsQ29xcBFtNkNwz0zyD9uBwas</recordid><startdate>1999</startdate><enddate>1999</enddate><creator>Gut, Marco</creator><creator>M. Leutenegger, Christian</creator><creator>B. Huder, Jon</creator><creator>C. Pedersen, Niels</creator><creator>Lutz, Hans</creator><general>Elsevier B.V</general><general>Elsevier</general><general>Elsevier Science B.V</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><scope>5PM</scope></search><sort><creationdate>1999</creationdate><title>One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses</title><author>Gut, Marco ; M. Leutenegger, Christian ; B. Huder, Jon ; C. Pedersen, Niels ; Lutz, Hans</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c589t-64685254011e098256a4a92b9339046ee6584556b51eaaddd36e7e1948ebab853</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1999</creationdate><topic>Animals</topic><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Cat Diseases - virology</topic><topic>Cats</topic><topic>Coronavirus</topic><topic>Coronavirus - genetics</topic><topic>Coronavirus - isolation & purification</topic><topic>Coronavirus Infections - veterinary</topic><topic>Coronavirus Infections - virology</topic><topic>DNA Primers</topic><topic>DNA Probes</topic><topic>DNA, Complementary</topic><topic>Dogs</topic><topic>Exodeoxyribonucleases - metabolism</topic><topic>FCoV</topic><topic>Feline coronavirus</topic><topic>Fluorescent Dyes</topic><topic>Fluorogenic 5′ nuclease assay</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Humans</topic><topic>Microbiology</topic><topic>Molecular Sequence Data</topic><topic>One-tube RT-PCR</topic><topic>Quantitation</topic><topic>Reverse Transcriptase Polymerase Chain Reaction - methods</topic><topic>RNA, Viral - analysis</topic><topic>Sensitivity and Specificity</topic><topic>Taq Polymerase</topic><topic>TaqMan</topic><topic>Techniques used in virology</topic><topic>Thermus flavus</topic><topic>Viral RNA</topic><topic>Virology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Gut, Marco</creatorcontrib><creatorcontrib>M. Leutenegger, Christian</creatorcontrib><creatorcontrib>B. Huder, Jon</creatorcontrib><creatorcontrib>C. Pedersen, Niels</creatorcontrib><creatorcontrib>Lutz, Hans</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><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of virological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Gut, Marco</au><au>M. Leutenegger, Christian</au><au>B. Huder, Jon</au><au>C. Pedersen, Niels</au><au>Lutz, Hans</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses</atitle><jtitle>Journal of virological methods</jtitle><addtitle>J Virol Methods</addtitle><date>1999</date><risdate>1999</risdate><volume>77</volume><issue>1</issue><spage>37</spage><epage>46</epage><pages>37-46</pages><issn>0166-0934</issn><eissn>1879-0984</eissn><coden>JVMEDH</coden><abstract>A one-tube reverse transcription-polymerase chain reaction (RT-PCR) for absolute feline coronavirus (FCoV) quantitation was developed. The assay is based on the 5′ nuclease activity of the
Thermus flavus (Tfl) polymerase and a fluorogenic probe which generates fluorescence when it is cleaved. The fluorogenic probe, also called TaqMan™ probe (Perkin Elmer, Foster City, USA), is an oligonucleotide designed to bind between the two PCR primers to the target cDNA and is labeled with a reporter and a quencher dye. In the intact probe, the quencher dye suppresses the fluorescence of the reporter dye by Förster-type energy transfer. During the polymerase extension steps the
Tfl exonuclease activity cleaves the hybridised probe resulting in the generation of fluorescent emission of the reporter dye. The threshold cycle (
C
T value) indicates the increase of reporter fluorescence and is directly related to the initial amount of target cDNA or RNA, respectively. Fluorescence is monitored in real time after each cycle by a Perkin-Elmer ABI Prism® 7700 Sequence Detector. After completion of amplification, the
C
T values of the samples are calculated back to a standard curve, generated by amplification of diluted standard molecules. The one-tube RT-PCR described below allows precise quantitation, is highly sensitive, rapid (no separate reverse transcription step and no post-amplification steps), easy to handle, allows for a high sample throughput, shows a very good reproducibility, and can be executed with a low risk of contamination. The design of the primers–probe combination enables the detection of all known FCoV strains and is also useful for the detection of canine coronavirus, transmissible gastroenteritis virus and porcine respiratory coronavirus.</abstract><cop>London</cop><cop>Amsterdam</cop><cop>New York, NY</cop><pub>Elsevier B.V</pub><pmid>10029323</pmid><doi>10.1016/S0166-0934(98)00129-3</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Animals Base Sequence Biological and medical sciences Cat Diseases - virology Cats Coronavirus Coronavirus - genetics Coronavirus - isolation & purification Coronavirus Infections - veterinary Coronavirus Infections - virology DNA Primers DNA Probes DNA, Complementary Dogs Exodeoxyribonucleases - metabolism FCoV Feline coronavirus Fluorescent Dyes Fluorogenic 5′ nuclease assay Fundamental and applied biological sciences. Psychology Humans Microbiology Molecular Sequence Data One-tube RT-PCR Quantitation Reverse Transcriptase Polymerase Chain Reaction - methods RNA, Viral - analysis Sensitivity and Specificity Taq Polymerase TaqMan Techniques used in virology Thermus flavus Viral RNA Virology |
title | One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses |
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