RT-qPCR Detection of SARS-CoV-2: No Need for a Dedicated Reverse Transcription Step

• Published in: International journal of molecular sciences Vol. 23; no. 3; p. 1303
• Main Authors: Bustin, Stephen A, Shipley, Gregory L, Kirvell, Sara, Mueller, Reinhold, Nolan, Tania
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 24.01.2022; MDPI
• Subjects: Assaying; Clinical Laboratory Techniques; Complementary DNA; Coronaviruses; COVID-19; COVID-19 - diagnosis; COVID-19 Nucleic Acid Testing - methods; Diagnostic tests; DNA Primers - chemistry; DNA Primers - genetics; Enzymes; Experiments; Fluorescence; High temperature; Humans; Infectious diseases; Molecular Diagnostic Techniques - methods; molecular diagnostics; Polymerase chain reaction; Protocol; Reagents; Real-Time Polymerase Chain Reaction - methods; Reverse transcription; Reverse Transcription - physiology; RNA; RNA, Viral - genetics; Room temperature; RT-qPCR; SARS-CoV-2; SARS-CoV-2 - genetics; Sensitivity analysis; Sensitivity and Specificity; Severe acute respiratory syndrome coronavirus 2; Temperature; Temperature requirements; COVID-19; molecular diagnostics; SARS-CoV-2; RNA; RT-qPCR; reverse transcription
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms23031303

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.