Hot start reverse transcriptase: an approach for improved real-time RT-PCR performance
Identifieur interne : 002672 ( Ncbi/Curation ); précédent : 002671; suivant : 002673Hot 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
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<series><title level="j">Journal of Analytical Science and Technology</title>
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<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>
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<author><name sortKey="Atwood, Sm" uniqKey="Atwood S">SM Atwood</name>
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<author><name sortKey="Daiss, Jl" uniqKey="Daiss J">JL Daiss</name>
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<author><name sortKey="Van Boheemen, S" uniqKey="Van Boheemen S">S Van Boheemen</name>
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<author><name sortKey="Bestebroer, Tm" uniqKey="Bestebroer T">TM Bestebroer</name>
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<author><name sortKey="Osterhaus, Adme" uniqKey="Osterhaus A">ADME Osterhaus</name>
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<author><name sortKey="Fouchier, Ram" uniqKey="Fouchier R">RAM Fouchier</name>
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