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)
| Autor: | Ahmed Abd El Wahed, Gerhard Dobler, Iris Bachmann, Frank T. Hufert, Marina Schramm, Ole Behrmann, Gregory Dame, Martin Spiegel |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Exonuclease Exonucleases Clinical Biochemistry Pneumonia Viral Recombinase Polymerase Amplification Amplification Real-Time Polymerase Chain Reaction 01 natural sciences Sensitivity and Specificity Article POCT 03 medical and health sciences Betacoronavirus Humans Pandemics Biochemistry medical biology Chemistry SARS-CoV-2 010401 analytical chemistry Biochemistry (medical) RNA COVID-19 Molecular biology Reverse transcriptase In vitro 0104 chemical sciences 3. Good health 030104 developmental biology Real-time polymerase chain reaction Point-of-Care Testing Point-of-care Nucleic acid biology.protein RNA Viral Primer (molecular biology) Coronavirus Infections DNA Probes Nucleic Acid Amplification Techniques RPA |
| Zdroj: | Clinical Chemistry |
| ISSN: | 1530-8561 0009-9147 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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