Session: Analytical Division Poster:

The pandemic of coronavirus disease of 2019 (COVID-19) is challenging the capacity of molecular diagnosis worldwide. We achieved the detection of SARS-CoV-2 RNA in one tube by coupling reverse transcription-recombinase polymerase amplification (RT-RPA) with the CRISPR-Cas12a system. RNA of SARS-CoV-2 is first reverse transcribed into complementary DNA (cDNA). The cDNA is exponentially amplified using RPA. Specific DNA amplicons are recognized by the ribonucleoprotein, consisting of CRISPR RNA and Cas12a, through the base pairing between the CRISPR RNA and the target. The interaction of the DNA amplicon with the ribonucleoprotein activates the trans-cleavage activity of the Cas12a to cleave multiple short single-stranded DNA reporters. The conservative target site chosen for RPA amplification ensures the coverage of SARS-CoV-2 variants, and the CRISPR-based detection improves the assay specificity. The assay was able to detect as low as a few hundred copies of SARS-CoV-2 RNA extracted from Vero E6 cell culture. We applied our assay to the detection of RNA extracts prepared from 46 upper respiratory swabs samples. These samples were originally collected for COVID-19 diagnosis. Twenty-one samples are SARS-CoV-2 positive identified by RT-PCR, with a threshold cycle (Ct) value ranging from 19 to 34. Our assay identified 18/21 positive samples to be positive, with 100% positive for samples with Ct below 32. Our method identified all 25 RT-PCR negative samples negative. Our assay operates at a single temperature (40 °C) in a single tube and uses different reagents from those of RT-RCR, and thus holds the promise to complement current tests for COVID-19 screening.

Kanti Pabbaraju

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