Utilizing Supercoiled Plasmids in CRISPR-Dx for Viral Gene Detection.

Having sensitive, specific, and simple diagnostic tools is essential for a well-functioning healthcare system. Among the prevalent diagnostic methodologies, CRISPR-Dx, a technique heavily reliant on costly equipment for fluorescent signaling, has garnered substantial attention. In this paper, we develop less expensive and non-optical CRISPR-based pathogen diagnostic systems using supercoiled ds-plasmid DNA (ФХ174). This new method employs activation of Cas12a protein within a CRISPR-based system that leads to post-nicking relaxation of supercoiled ФХ174 plasmid. We also utilize this new method to detect viral DNA in AVV samples up to 0.2 pM concentration of target DNA. Gel electrophoresis serves to assess the proportion of supercoiled to the relaxed plasmid. The findings indicate that plasmid reporting systems are specific and inexpensive compared to conventional fluorescence reporters.