Hot start reverse transcriptase: an approach for improved real-time RT-PCR performance
Identifieur interne : 001928 ( Main/Exploration ); précédent : 001927; suivant : 001929Hot start reverse transcriptase: an approach for improved real-time RT-PCR performance
Auteurs : Nils Rutschke [Allemagne] ; Jan Zimmermann [Allemagne] ; Ronny Möller [Allemagne] ; Gerd Klöck [Allemagne] ; Mathias Winterhalter [Allemagne] ; Annika Leune [Allemagne]Source :
- Journal of Analytical Science and Technology [ 2093-3134 ] ; 2015.
Abstract
Reverse transcriptase is an indispensable enzyme for real-time reverse transcriptase (RT)-PCR, a standard method in molecular diagnostics for detection and quantification of defined RNA molecules. The prevention of non-specific products due to elongation of misprimed oligonucleotides by the enzyme at temperatures beneath the specific annealing temperature is one of the biggest challenges in real-time RT-PCR.
In the present study, an aptamer directed against the reverse transcriptase was analyzed for its potential to attain a temperature-dependent reverse transcriptase (“hot start” RT).
The hot start effect was investigated in a one-step real-time RT-PCR assay for the detection of Middle East respiratory syndrome coronavirus (MERS-CoV). Results with aptamer revealed a reduced RT activity at low temperatures while achieving full activity at the specific annealing temperature of 55 °C. Sensitivity (limit of detection (LoD) 95 %) of the MERS-CoV assay was increased by about two times in the presence of aptamer.
The study demonstrates the potential of aptamer-dependent hot start RT for the improvement of diagnostic real-time RT-PCR assays.
Url:
DOI: 10.1186/s40543-015-0063-4
PubMed: 32226638
PubMed Central: 7099353
Affiliations:
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Hamburg</wicri:noRegion><placeName><settlement type="city">Hambourg</settlement><region type="land" nuts="2">Hambourg</region></placeName></affiliation></author></analytic><series><title level="j">Journal of Analytical Science and Technology</title><idno type="ISSN">2093-3134</idno><idno type="eISSN">2093-3371</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p id="Par1">Reverse transcriptase is an indispensable enzyme for real-time reverse transcriptase (RT)-PCR, a standard method in molecular diagnostics for detection and quantification of defined RNA molecules. The prevention of non-specific products due to elongation of misprimed oligonucleotides by the enzyme at temperatures beneath the specific annealing temperature is one of the biggest challenges in real-time RT-PCR.</p><p id="Par2">In the present study, an aptamer directed against the reverse transcriptase was analyzed for its potential to attain a temperature-dependent reverse transcriptase (“hot start” RT).</p></sec><sec><title>Findings</title><p id="Par3">The hot start effect was investigated in a one-step real-time RT-PCR assay for the detection of Middle East respiratory syndrome coronavirus (MERS-CoV). Results with aptamer revealed a reduced RT activity at low temperatures while achieving full activity at the specific annealing temperature of 55 °C. Sensitivity (limit of detection (LoD) 95 %) of the MERS-CoV assay was increased by about two times in the presence of aptamer.</p></sec><sec><title>Conclusions</title><p id="Par4">The study demonstrates the potential of aptamer-dependent hot start RT for the improvement of diagnostic real-time RT-PCR assays.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Birch, De" uniqKey="Birch D">DE Birch</name></author><author><name sortKey="Kolmodin, L" uniqKey="Kolmodin L">L Kolmodin</name></author><author><name sortKey="Laird, Wj" uniqKey="Laird W">WJ Laird</name></author><author><name sortKey="Mckinney, N" uniqKey="Mckinney N">N McKinney</name></author><author><name sortKey="Wong, J" uniqKey="Wong J">J Wong</name></author><author><name sortKey="Young, Kky" uniqKey="Young K">KKY Young</name></author><author><name sortKey="Zangenberg, Ga" uniqKey="Zangenberg G">GA Zangenberg</name></author><author><name sortKey="Zoccoli, Ma" 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sortKey="Rutschke, Nils" sort="Rutschke, Nils" uniqKey="Rutschke N" first="Nils" last="Rutschke">Nils Rutschke</name><name sortKey="Winterhalter, Mathias" sort="Winterhalter, Mathias" uniqKey="Winterhalter M" first="Mathias" last="Winterhalter">Mathias Winterhalter</name><name sortKey="Zimmermann, Jan" sort="Zimmermann, Jan" uniqKey="Zimmermann J" first="Jan" last="Zimmermann">Jan Zimmermann</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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