A reverse transcription-free CRISPR/Cas12a biosensor for ultrasensitive detection of SARS-CoV-2 variants.

In this work, we developed a reverse transcription-free self-primer isothermal exponential amplification reaction (RTF-SP-EXPAR) combined with a CRISPR/Cas12a biosensor for the simultaneous detection of single- and double-stranded amplification products of SARS-CoV-2 mutant variants. SP-EXPAR can recognize RNA targets directly, simultaneously generate double-stranded DNA products containing PAM sequences, and single-stranded DNA products without PAM sequences. Both types of SP-EXPAR products can be recognized by the crRNA and produce fluorescent signals, thereby enhancing detection sensitivity. This RTF-SP-EXPAR-CRISPR/Cas12a biosensor enables the detection of SARS-CoV-2 mutations within 1 h, achieving a detection limit of 7.49 aM and a dynamic range of 10 aM to 10 pM. This method shows high specificity in differentiating mutant variants from wild-type sequences. For the detection of 106 clinical samples, this RTF-SP-EXPAR-CRISPR/Cas12a assay demonstrates 100 % sensitivity and 100 % specificity compared with DNA sequencing results. These findings highlight our proposed assay's strong applicability for the application of RNA samples without reverse transcription.