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).
Clin Chem
; 66(8): 1047-1054, 2020 08 01.
Article
in English
| MEDLINE | ID: covidwho-209847
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.
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Pneumonia, Viral
/
RNA, Viral
/
Coronavirus Infections
/
Nucleic Acid Amplification Techniques
/
Betacoronavirus
Type of study:
Diagnostic study
/
Observational study
/
Prognostic study
/
Randomized controlled trials
Limits:
Humans
Language:
English
Journal:
Clin Chem
Journal subject:
Chemistry, Clinical
Year:
2020
Document Type:
Article
Affiliation country:
Clinchem
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