RPB0079

Pathogen Description

Target Pathogen Pathogen Name NCBI Taxonomy ID Order Family Genus Species Pathogen type
SARS-CoV-2 SARS-CoV-2, 2019-nCoV, COVID-19, COVID-19 virus, SARS2, Wuhan coronavirus, Human coronavirus 2019, COVID19, HCoV-19, SARS-2, SARS-CoV4 2697049 Nidovirales Coronaviridae Betacoronavirus Severe acute respiratory syndrome-related coronavirus virus

Primer Description

Primer Name Sequence(5'-3') Length(bp) Primer Final Concentration(μM） GC Content（%） Predicted Melting Temperature(℃） Molecular Weight(g/moles) Positions in GenBank accession number
RPA-SARS-2-FW CCTCTTCTCGTTCCTCATCACGTAGTCGCAAC 32 \ 53.1 65.2 9622.29 \
RPA-SARS-2-RV AGTGACAGTTTGGCCTTGTTGTTGTTGGCCTT 32 \ 46.9 65.69 9874.43 \
RPA-SARS-2-P CCTGCTAGAATGGCTGGCAATGGCGGTGAT(AP site)CTGCTCTTGCTTTGC 45 \ 55.6 73.48 13865.99 \

Gene Description

Target Gene GenBank ID
N gene \

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
\ \ \ 20 42 Exo Probe 1 copies/ul 95 100

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2020 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). Behrmann, Ole; Bachmann, Iris; Spiegel, Martin; Schramm, Marina; Abd El Wahed, Ahmed; Dobler, Gerhard; Dame, Gregory; Hufert, Frank T; CLIN CHEM 32384153 10.1093/clinchem/hvaa116

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).

Author(s):

Behrmann, Ole; Bachmann, Iris; Spiegel, Martin; Schramm, Marina; Abd El Wahed, Ahmed; Dobler, Gerhard; Dame, Gregory; Hufert, Frank T;

Journal:

CLIN CHEM

Year:

2020

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.