Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 118, Issue 35, Pages -Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2100347118
Keywords
RNA detection COVID-19 assay nucleic acids EXPAR isothermal amplification
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Funding
- Midlands Integrative Bioscience Training Partnership by the Biotechnology and Bioscience Research Council [BB/R506175/1]
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A rapid isothermal method for detecting SARS-CoV-2 has been developed, using a reverse transcription-free approach to convert RNA into DNA and rapidly amplifying it. The assay is faster than traditional methods like RT-qPCR and RT-LAMP, while maintaining sensitivity, making it a promising tool for detecting RNA-based pathogens.
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 threeway 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.
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