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

Abstract

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.