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. 2020 May 8;hvaa116.
doi: 10.1093/clinchem/hvaa116. Online ahead of print.

Rapid Detection of SARS-CoV-2 by Low Volume Real-Time Single Tube Reverse Transcription Recombinase Polymerase Amplification Using an Exo Probe With an Internally Linked Quencher (exo-IQ)

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Rapid Detection of SARS-CoV-2 by Low Volume Real-Time Single Tube Reverse Transcription Recombinase Polymerase Amplification Using an Exo Probe With an Internally Linked Quencher (exo-IQ)

Ole Behrmann et al. Clin Chem. .
Free PMC article

Abstract

Background: The current outbreak of SARS-CoV-2 has spread to almost every country with more than three million confirmed cases and over two hundred thousand deaths as of April 28, 2020. Rapid first-line testing protocols are needed for outbreak control and surveillance.

Methods: We used computational and manual design to generate a suitable set of reverse transcription recombinase polymerase amplification (RT-RPA) primer and exonuclease probe, internally quenched (exo-IQ) probe sequences targeting the SARS-CoV-2 N gene. RT-RPA sensitivity was determined by amplification of in vitro transcribed RNA standards. Assay selectivity was demonstrated with a selectivity panel of 32 nucleic acid samples derived from common respiratory viruses. To validate the assay against full-length SARS-CoV-2 RNA, total viral RNA derived from cell culture supernatant and 19 nasopharyngeal swab samples (8 positive and 11 negative for SARS-CoV-2) were screened. All results were compared to established RT-qPCR assays.

Results: The 95% detection probability of the RT-RPA assay was determined to be 7.74 (95% CI: 2.87 - 27.39) RNA copies per reaction. The assay showed no cross-reactivity to any other screened coronaviruses or respiratory viruses of clinical significance. The developed RT-RPA assay produced 100% diagnostic sensitivity and specificity when compared to RT-qPCR (n=20).

Conclusion: With a run time of 15 to 20 minutes and first results being available in under 7 minutes for high RNA concentrations, the reported assay constitutes one of the fastest nucleic acid based detection methods for SARS-CoV-2 to date and may provide a simple to use alternative to RT-qPCR for first-line screening at the point of need.

Keywords: Amplification; COVID-19; POCT; Point-of-care; RPA; SARS-CoV-2.

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