Ultrarapid detection of SARS-CoV-2 RNA using a reverse transcription-free exponential amplification reaction, RTF-EXPAR

Jake G Carter, Lorea Orueta Iturbe, Jean-Louis H A Duprey, Ian R Carter, Craig D Southern, Marium Rana, Celina M Whalley, Andrew Bosworth, Andrew D Beggs, Matthew R Hicks, James H R Tucker, Timothy R Dafforn

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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 languageEnglish
Article numbere2100347118
JournalProceedings of the National Academy of Sciences of the United States of America
Volume118
Issue number35
Early online date16 Aug 2021
DOIs
Publication statusPublished - 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

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