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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)
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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)
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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)
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Journal Article
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)
2020
Overview
Abstract
Background
The current outbreak of SARS-CoV-2 has spread to almost every country with more than 5 million confirmed cases and over 300,000 deaths as of May 26, 2020. Rapid first-line testing protocols are needed for outbreak control and surveillance.
Methods
We used computational and manual designs to generate a suitable set of reverse transcription recombinase polymerase amplification (RT-RPA) primer and exonuclease probe, internally quenched (exo-IQ), 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).
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.
Publisher
Oxford University Press,American Association for Clinical Chemistry, Inc
Subject
/ Assaying
/ Betacoronavirus - isolation & purification
/ Coronavirus Infections - diagnosis
/ Coronavirus Infections - virology
/ COVID-19
/ Ethylenediaminetetraacetic acid
/ Genes
/ Humans
/ N gene
/ Nucleic Acid Amplification Techniques - methods
/ Pneumonia, Viral - diagnosis
/ Real-Time Polymerase Chain Reaction
/ RNA
/ Severe acute respiratory syndrome coronavirus 2
/ Viruses
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