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Hot start reverse transcriptase: an approach for improved real-time RT-PCR performance
BACKGROUND: 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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Korea Basic Science Institute
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099353/ https://www.ncbi.nlm.nih.gov/pubmed/32226638 http://dx.doi.org/10.1186/s40543-015-0063-4 |
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author | Rutschke, Nils Zimmermann, Jan Möller, Ronny Klöck, Gerd Winterhalter, Mathias Leune, Annika |
author_facet | Rutschke, Nils Zimmermann, Jan Möller, Ronny Klöck, Gerd Winterhalter, Mathias Leune, Annika |
author_sort | Rutschke, Nils |
collection | PubMed |
description | BACKGROUND: 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). FINDINGS: 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. CONCLUSIONS: The study demonstrates the potential of aptamer-dependent hot start RT for the improvement of diagnostic real-time RT-PCR assays. |
format | Online Article Text |
id | pubmed-7099353 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Korea Basic Science Institute |
record_format | MEDLINE/PubMed |
spelling | pubmed-70993532020-03-27 Hot start reverse transcriptase: an approach for improved real-time RT-PCR performance Rutschke, Nils Zimmermann, Jan Möller, Ronny Klöck, Gerd Winterhalter, Mathias Leune, Annika J Anal Sci Technol Short Report BACKGROUND: 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). FINDINGS: 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. CONCLUSIONS: The study demonstrates the potential of aptamer-dependent hot start RT for the improvement of diagnostic real-time RT-PCR assays. Korea Basic Science Institute 2015-06-21 2015 /pmc/articles/PMC7099353/ /pubmed/32226638 http://dx.doi.org/10.1186/s40543-015-0063-4 Text en © Rutschke et al. 2015 This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0), which permits use, duplication, adaptation, distribution, and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. |
spellingShingle | Short Report Rutschke, Nils Zimmermann, Jan Möller, Ronny Klöck, Gerd Winterhalter, Mathias Leune, Annika Hot start reverse transcriptase: an approach for improved real-time RT-PCR performance |
title | Hot start reverse transcriptase: an approach for improved real-time RT-PCR performance |
title_full | Hot start reverse transcriptase: an approach for improved real-time RT-PCR performance |
title_fullStr | Hot start reverse transcriptase: an approach for improved real-time RT-PCR performance |
title_full_unstemmed | Hot start reverse transcriptase: an approach for improved real-time RT-PCR performance |
title_short | Hot start reverse transcriptase: an approach for improved real-time RT-PCR performance |
title_sort | hot start reverse transcriptase: an approach for improved real-time rt-pcr performance |
topic | Short Report |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099353/ https://www.ncbi.nlm.nih.gov/pubmed/32226638 http://dx.doi.org/10.1186/s40543-015-0063-4 |
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