Development of a RPA-CRISPR-Cas12a Assay for Rapid, Simple, and Sensitive Detection of Mycoplasma hominis

Graphical Abstract Schematic of Mycoplasma hominis nucleic acid detection based on the CRISPR-Cas12a system. Clinical samples from cervical or urethral swabs were collected and incubated with lysis buffer to release nucleic acid (10 min). Extracted DNA (1 μl) is subjected to the RPA reaction with specific primers at 37°C. After 20 min, RPA product was subjected to the CRISPR-Cas12a reaction for cleavage. The collateral nuclease activity of Cas12a (250 nM) proteins were activated upon specific binding to crRNA (crRNA3, 62.5 nM) and the DNA product; thus, Cas12a cut the quenched fluorescent ssDNA reporter (125 nM) (30 min). The generated fluorescence signal would be measured by a fluorescence plate reader or visualized by lateral flow strips.