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)

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 RT-RPA primer and 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 by means of 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 crossreactivity to any other screened coronaviruses as well as respiratory viruses of clinical significance. The developed RT-RPA assay produced 100% diagnostic sensitivity and specificity when compared to RT-qPCR (n=20). Conclusions: 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. Downloaded from https://academic.oup.com/clinchem/advance-article-abstract/doi/10.1093/clinchem/hvaa116/5834714 by guest on 09 June 2020

Suitcase Lab for Rapid Detection of SARS-CoV-2 Based on Recombinase Polymerase Amplification Assay

“…The E gene RNA was also used as a positive control for the RT-PCR. Oligonucleotides for the RdRP and E genes were updated from our previous design for SARS-CoV and MERS CoV (unpublished data), whereas the N gene amplicon was modified from a previously published article 20 ( Table 1 ). All oligonucleotides were synthesized by TIB MOLBIOL GmbH (Berlin, Germany).…”

Development of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

“…(2020) [8], where 8 positive and 11 negative SARS-CoV-2 nasopharyngeal swab samples were tested with 100% sensitivity and specificity. However, an isothermal fluorescence reader was necessary to interpret the results.…”

“…While in our study, the RT-RPA end product was detected by observing the color changes and lateral flow strip which omitted the need of a real-time PCR machine. This would save on assay costs and facilitate onsite diagnosis in the field [8].…”

Rapid detection of SARS-CoV-2 by pulse-controlled amplification (PCA)

“…In recent months, however, a number of faster alternatives based on different platforms and technologies have come onto the market that deliver results within an hour or less. On the one hand there are isothermal NAAT (nucleic acid amplification technology) methods like LAMP (loop-mediated amplification) ( Dao Thi et al, 2020 ; Park et al, 2020 ; Yang et al, 2020 ; Zhang et al, 2020 ) or RPA (recombinase polymerase amplification) ( Behrmann et al, 2020 ), some of them in combination with a CRISPR-cas-based detection ( Broughton et al, 2020 ; Ding et al, 2020 ; Joung et al, 2020 ; Lucia et al, 2020 ). On the other hand, there are fully integrated cartridge systems like Cepheid’s GeneXpert (Cepheid, Sunnyvale, USA) and Abbott’s IDnow (Abbott, North Chicago, USA).…”