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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
3
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pubmed:dateCreated |
1997-6-2
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pubmed:abstractText |
Amplification of a 3-kb genome region from the RNA polymerase gene to the 3' poly(A) tail of small round-structured virus (SRSV) by reverse transcription-PCR (RT-PCR) has been difficult to achieve because of a stable secondary structure in a region between the RNA polymerase gene and the 5' end of the second open reading frame. We have developed a one-tube RT-PCR method to efficiently amplify this region. The method comprises three procedures: purification of poly(A)+ RNA from a starting RNA solution by oligo(dT)30 covalently linked to latex particles, buffer exchange, and continuous RT and PCR in a single tube containing all reaction components. The key elements of this method are (i) first-strand cDNA synthesis with the Superscript II version of RNase H- Moloney murine leukemia virus reverse transcriptase at 50 degrees C for 10 min by using the RNA-oligo(dT)30 hybrid on the latex particles as the template and primer, and (ii) PCR by Taq and Pwo DNA polymerases mixed together with a mixture of 12 phased oligo(dT)25 antisense primers. The detection threshold of the one-tube RT-PCR method was as little as 0.2 ng of the crude RNA used as the source of the template. Using this method, we obtained 3-kb products from 24 SRSV strains previously characterized into four genetic groups. These included 5 P1-A, 4 P1-B, 5 P2-A, and 10 P2-B strains. Because SRSVs have not yet been cultivated in vitro, this novel method should facilitate molecular characterization of SRSVs to provide a firm scientific foundation for improvements and refinements of SRSV diagnostics.
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pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-10152948,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-1280649,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-1328679,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-1374554,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-1383265,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-1579465,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-1766870,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-2022317,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-2177224,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-2291473,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-2347496,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-2786130,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-3459152,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-6340109,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-7507181,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-7515226,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-7518525,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-7541215,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-7561776,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-7650166,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-7699068,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-7852972,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-7931187,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8046390,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8077710,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8128635,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8134376,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8195372,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8202550,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8341601,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8380940,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8391187,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8636696,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8636707,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8636708,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8642693,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8666079,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8678832,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8732863,
http://linkedlifedata.com/resource/pubmed/commentcorrection/9041391-8830118
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Mar
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pubmed:issn |
0095-1137
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:volume |
35
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
570-7
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pubmed:dateRevised |
2009-11-18
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pubmed:meshHeading |
pubmed-meshheading:9041391-Base Sequence,
pubmed-meshheading:9041391-Caliciviridae Infections,
pubmed-meshheading:9041391-DNA Primers,
pubmed-meshheading:9041391-DNA-Directed RNA Polymerases,
pubmed-meshheading:9041391-Evaluation Studies as Topic,
pubmed-meshheading:9041391-Gastroenteritis,
pubmed-meshheading:9041391-Genes, Viral,
pubmed-meshheading:9041391-Humans,
pubmed-meshheading:9041391-Moloney murine leukemia virus,
pubmed-meshheading:9041391-Norwalk virus,
pubmed-meshheading:9041391-Polymerase Chain Reaction,
pubmed-meshheading:9041391-RNA, Messenger,
pubmed-meshheading:9041391-RNA, Viral,
pubmed-meshheading:9041391-RNA-Directed DNA Polymerase,
pubmed-meshheading:9041391-Virology
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pubmed:year |
1997
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pubmed:articleTitle |
A one-tube method of reverse transcription-PCR to efficiently amplify a 3-kilobase region from the RNA polymerase gene to the poly(A) tail of small round-structured viruses (Norwalk-like viruses).
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pubmed:affiliation |
Viral Gastroenteritis Section, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA. txa5@CIDDVD1.em.cdc.gov
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pubmed:publicationType |
Journal Article
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