Abstract
Reverse transcription of RNA coupled to amplification of the resulting cDNA by the polymerase chain reaction (RT-PCR) is one of the principal molecular technologies in use today, with applications across all areas of science and medicine. In its real-time, fluorescence-based usage (RT-qPCR), it has long been a core technology driving the accurate, rapid and sensitive laboratory diagnosis of infectious diseases. However, RT-qPCR protocols have changed little over the past 30 years, with the RT step constituting a significant percentage of the time taken to complete a typical RT-qPCR assay. When applied to research investigations, reverse transcription has been evaluated by criteria such as maximum yield, length of transcription, fidelity, and faithful representation of an RNA pool. Crucially, however, these are of less relevance in a diagnostic RT-PCR test, where speed and sensitivity are the prime RT imperatives, with specificity contributed by the PCR component. We propose a paradigm shift that omits the requirement for a separate high-temperature RT step at the beginning of an RT-qPCR assay. This is achieved by means of an innovative protocol that incorporates suitable reagents with a revised primer and amplicon design and we demonstrate a proof of principle that incorporates the RT step as part of the PCR assay setup at room temperature. Use of this modification as part of a diagnostic assay will of course require additional characterisation, validation and optimisation of the PCR step. Combining this revision with our previous development of fast qPCR protocols allows completion of a 40 cycle RT-qPCR run on a suitable commercial instrument in approximately 15 min. Even faster times, in combination with extreme PCR procedures, can be achieved.
Highlights
All box and whiskers plots show the ∆Cqs for the independent reverse transcription (RT)-qPCR reaction, with the whiskers delineating the minimum and maximum values
The ability of reverse transcriptase enzymes (RTases) to transcribe at even lower temperatures was tested by carrying out the following reactions in duplicate: RNA samples in RT reaction buffer were split in two, one being reverse transcribed with SS4 or Ultrascript 2.0 (US2) and random hexamers at 50 ◦ C, the other set left on ice for 5 min, followed by amplification and detection of assays N1, N2 and N3
These have been shaped by the role of RNA-dependent DNA polymerases in cDNA synthesis, cloning, expression analysis and sequencing
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Whilst early protocols included 60 min incubation times for the RT step [9], most contemporary protocols tend to be more rapid at between 15 [10] and 30 min [11], markedly longer RT polymerisation times continue to be used [12]. This is generally not a problem for research applications but becomes an issue when RT-PCR is used in the context of rapid diagnostic testing. We anticipate that this innovation will provide a ground-breaking new standard for future RT-qPCR protocols and result in reduced assay times and simpler POC instrumentation
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