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.
© 2020 American Association for Clinical Chemistry.
A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2.Int J Mol Sci. 2020 Apr 18;21(8):2826. doi: 10.3390/ijms21082826. Int J Mol Sci. 2020. PMID: 32325642 Free PMC article.
Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification.BMC Vet Res. 2017 Aug 15;13(1):241. doi: 10.1186/s12917-017-1180-7. BMC Vet Res. 2017. PMID: 28810858 Free PMC article.
Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV-2.Emerg Microbes Infect. 2020 Dec;9(1):998-1007. doi: 10.1080/22221751.2020.1756698. Emerg Microbes Infect. 2020. PMID: 32306853
"Rapid diagnosis of Cucumber mosaic virus in banana plants using a fluorescence-based real-time isothermal reverse transcription-recombinase polymerase amplification assay".J Virol Methods. 2019 Aug;270:52-58. doi: 10.1016/j.jviromet.2019.04.024. Epub 2019 Apr 29. J Virol Methods. 2019. PMID: 31047971
Testing for SARS-CoV-2: the day the world turned its attention to the clinical laboratory.Clin Transl Sci. 2020 May 31. doi: 10.1111/cts.12828. Online ahead of print. Clin Transl Sci. 2020. PMID: 32475012 Review.