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Hot start reverse transcriptase: an approach for improved real-time RT-PCR performance.

Nils Rutschke1,2, Jan Zimmermann1, Ronny Möller1,2

  • 1altona Diagnostics GmbH, Moerkenstr. 12, 22767 Hamburg, Germany.

Journal of Analytical Science and Technology
|April 1, 2020
PubMed
Summary

A novel aptamer enhances real-time reverse transcriptase polymerase chain reaction (RT-PCR) by enabling a temperature-dependent "hot start" RT. This improves diagnostic assay sensitivity for detecting viruses like MERS-CoV.

Keywords:
Hot startMERS-CoVReal-time RT-PCRReverse transcriptase

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Diagnostic Assays

Background:

  • Real-time reverse transcriptase polymerase chain reaction (RT-PCR) is crucial for RNA detection and quantification in molecular diagnostics.
  • A significant challenge in RT-PCR is preventing non-specific amplification at temperatures below optimal annealing.
  • This study explores aptamers for temperature-dependent control of reverse transcriptase activity.

Purpose of the Study:

  • To investigate the potential of an aptamer for achieving a "hot start" reverse transcriptase (RT) activity.
  • To evaluate the aptamer's ability to regulate RT activity based on temperature.
  • To enhance the specificity and sensitivity of diagnostic RT-PCR assays.

Main Methods:

  • Development and application of an aptamer targeting reverse transcriptase.
  • Implementation of a one-step real-time RT-PCR assay for Middle East respiratory syndrome coronavirus (MERS-CoV) detection.
  • Assessment of RT activity at varying temperatures in the presence and absence of the aptamer.

Main Results:

  • The aptamer demonstrated temperature-dependent control, reducing RT activity at low temperatures and maintaining full activity at 55°C.
  • The presence of the aptamer led to a twofold increase in the sensitivity (limit of detection) of the MERS-CoV assay.
  • Non-specific product formation was effectively minimized by the aptamer-mediated hot start.

Conclusions:

  • Aptamer-mediated "hot start" reverse transcriptase offers a promising strategy for improving diagnostic real-time RT-PCR.
  • This approach enhances assay sensitivity and specificity, crucial for accurate molecular diagnostics.
  • The findings support the use of aptamers in optimizing existing and developing new diagnostic tools.