Detection and quantitation of group A rotaviruses by competitive and real-time reverse transcription-polymerase chain reaction
A competitive reverse transcription-polymerase chain reaction (RT-PCR) was developed to detect and to quantitate the RNA of group A rotaviruses. In the assay, a 433 bp fragment is amplified by a one-tube RT-PCR protocol using primers with binding sites located in a highly conserved region of segment...
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| Veröffentlicht in: | Journal of virological methods 2002-09, Vol.105 (2), p.277-285 |
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| container_title | Journal of virological methods |
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| creator | Schwarz, Bernd-Andreas Bange, Reinhard Vahlenkamp, Thomas W Johne, Reimar Müller, Hermann |
| description | A competitive reverse transcription-polymerase chain reaction (RT-PCR) was developed to detect and to quantitate the RNA of group A rotaviruses. In the assay, a 433 bp fragment is amplified by a one-tube RT-PCR protocol using primers with binding sites located in a highly conserved region of segment 6 of the rotavirus genome. An in vitro synthesized RNA with a 43-base deletion with respect to the wild-type sequence of this fragment was used as an internal control. Using these transcripts as templates, 10 RNA molecules were amplified reproducibly and detected in ethidium bromide-stained agarose gels or by fluorimetry using the SYBR Green I dye in a real-time RT-PCR assay. The efficiency of the protocol was confirmed by the detection of small amounts of viral RNA of group A rotaviruses in clinical samples obtained from various animal species and man. |
| doi_str_mv | 10.1016/S0166-0934(02)00118-0 |
| format | Article |
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In the assay, a 433 bp fragment is amplified by a one-tube RT-PCR protocol using primers with binding sites located in a highly conserved region of segment 6 of the rotavirus genome. An in vitro synthesized RNA with a 43-base deletion with respect to the wild-type sequence of this fragment was used as an internal control. Using these transcripts as templates, 10 RNA molecules were amplified reproducibly and detected in ethidium bromide-stained agarose gels or by fluorimetry using the SYBR Green I dye in a real-time RT-PCR assay. 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In the assay, a 433 bp fragment is amplified by a one-tube RT-PCR protocol using primers with binding sites located in a highly conserved region of segment 6 of the rotavirus genome. An in vitro synthesized RNA with a 43-base deletion with respect to the wild-type sequence of this fragment was used as an internal control. Using these transcripts as templates, 10 RNA molecules were amplified reproducibly and detected in ethidium bromide-stained agarose gels or by fluorimetry using the SYBR Green I dye in a real-time RT-PCR assay. The efficiency of the protocol was confirmed by the detection of small amounts of viral RNA of group A rotaviruses in clinical samples obtained from various animal species and man.</description><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Cell Line</subject><subject>Competitive RT-PCR</subject><subject>DNA Primers</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Group A rotavirus</subject><subject>Humans</subject><subject>Microbiology</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Real-time RT-PCR</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>RNA, Viral - genetics</subject><subject>RNA, Viral - isolation & purification</subject><subject>Rotavirus - classification</subject><subject>Rotavirus - genetics</subject><subject>Rotavirus - isolation & purification</subject><subject>Sensitivity and Specificity</subject><subject>Sequence Deletion</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>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1v1DAQhi0EokvhJ4ByAcEhMP6K4xOqyqdUiQNwtrzOBIySOLWdlfbCb8fJruixF9saPzPvzLyEPKfwlgJt3n0vR1OD5uI1sDcAlLY1PCA72ipdwq14SHb_kQvyJKU_ACAV54_JBWVMQdPAjvz9gBld9mGq7NRVt4udss92C4S--hXDMldXVQzZHnxcEqZqf6xcGGcsnD_glhbRDnX2I5bXAWPCKkc7JRf9vFaq5zAcR4y2fLjf1k9rwib6lDzq7ZDw2fm-JD8_ffxx_aW--fb56_XVTe0EY7mWCigXDW9ZJ7Ww2DbOKb2XXSOwE1zQVirVCo1S9GUy2VhHmQPY61bxzjX8krw61Z1juF0wZTP65HAY7IRhSUYxyqjkcC9YlLRWghVQnkAXQ0oRezNHP9p4NBTM6pDZHDLr-g0wszlkVoEXZ4FlP2J3l3W2pAAvz4BNzg59WaTz6Y7jGkRptnDvTxyWvR08RpOcx8lh52Nx1HTB39PKP7e_rl0</recordid><startdate>20020901</startdate><enddate>20020901</enddate><creator>Schwarz, Bernd-Andreas</creator><creator>Bange, Reinhard</creator><creator>Vahlenkamp, Thomas W</creator><creator>Johne, Reimar</creator><creator>Müller, Hermann</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>7U9</scope><scope>H94</scope><scope>7X8</scope></search><sort><creationdate>20020901</creationdate><title>Detection and quantitation of group A rotaviruses by competitive and real-time reverse transcription-polymerase chain reaction</title><author>Schwarz, Bernd-Andreas ; Bange, Reinhard ; Vahlenkamp, Thomas W ; Johne, Reimar ; Müller, Hermann</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c422t-5701346382d594ae86cc79b5d64ed43418577849e54f27056ac12c00b9873dc63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Cell Line</topic><topic>Competitive RT-PCR</topic><topic>DNA Primers</topic><topic>Fundamental and applied biological sciences. 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| ispartof | Journal of virological methods, 2002-09, Vol.105 (2), p.277-285 |
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| subjects | Base Sequence Biological and medical sciences Cell Line Competitive RT-PCR DNA Primers Fundamental and applied biological sciences. Psychology Group A rotavirus Humans Microbiology Polymerase Chain Reaction - methods Real-time RT-PCR Reverse Transcriptase Polymerase Chain Reaction RNA, Viral - genetics RNA, Viral - isolation & purification Rotavirus - classification Rotavirus - genetics Rotavirus - isolation & purification Sensitivity and Specificity Sequence Deletion Techniques used in virology Virology |
| title | Detection and quantitation of group A rotaviruses by competitive and real-time reverse transcription-polymerase chain reaction |
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