Abstract
A rapid isothermal method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, is reported. The procedure uses an unprecedented reverse transcription-free (RTF) approach for converting genomic RNA into DNA. This involves the formation of an RNA/DNA heteroduplex whose selective cleavage generates a short DNA trigger strand, which is then rapidly amplified using the exponential amplification reaction (EXPAR). Deploying the RNA-to-DNA conversion and amplification stages of the RTF-EXPAR assay in a single step results in the detection, via a fluorescence read-out, of single figure copy numbers per microliter of SARS-CoV-2 RNA in under 10 min. In direct three-way comparison studies, the assay has been found to be faster than both RT-qPCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP), while being just as sensitive. The assay protocol involves the use of standard laboratory equipment and is readily adaptable for the detection of other RNA-based pathogens.
Original language | English |
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Article number | e2100347118 |
Journal | Proceedings of the National Academy of Sciences of the United States of America |
Volume | 118 |
Issue number | 35 |
Early online date | 16 Aug 2021 |
DOIs | |
Publication status | Published - 31 Aug 2021 |
Bibliographical note
Funding Information:ACKNOWLEDGMENTS. We thank Dr A. Lovering for providing experimental equipment. We also thank Dr, Christine Bruce, High Containment Microbiology of PHE, Porton Down, for supplying samples from which SARS-CoV-2 RNA was purified. This research was funded by the Midlands Integrative Bioscience Training Partnership funded by the Biotechnology and Bioscience Research Council (BB/R506175/1).
Funding Information:
We thank Dr A. Lovering for providing experimental equipment. We also thank Dr, Christine Bruce, High Containment Microbiology of PHE, Porton Down, for supplying samples from which SARS-CoV-2 RNA was purified. This research was funded by the Midlands Integrative Bioscience Training Partnership funded by the Biotechnology and Bioscience Research Council (BB/R506175/1).
Keywords
- COVID-19/diagnosis
- COVID-19 Testing/methods
- Humans
- Nucleic Acid Amplification Techniques/methods
- RNA, Viral/genetics
- Reverse Transcription
- SARS-CoV-2/genetics
- Sensitivity and Specificity