crRNA-engineered CRISPR/Cas12a system coupled with RPA for ultrasensitive detection of Lactiplantibacillus plantarum

The growing probiotic industry requires rapid and precise strain detection methods. Here, a one-pot fluorescence platform integrating RPA with an enhanced CRISPR/Cas12a system (termed RPA-ECas12a) was developed for the detection of Lactiplantibacillus plantarum. Through rational 5'end DNA extension of the crRNA, an optimal variant (5'crRNA10) was identified, which increased the trans-cleavage catalytic efficiency of Cas12a by 33% (3.6 × 10⁸ M^-1 s^-1) compared to the wild-type crRNA. The resulting RPA-ECas12a platform detected L. plantarum with a limit of detection of 1.3 CFU/mL, a linear range from 10¹ to 10⁷ CFU/mL, and excellent precision (CVs < 10%). The entire detection was completed within 45 min. The platform demonstrated high selectivity and robustness when applied to commercial probiotic powders, yogurts and other complex food matrices. This work not only provides a sensitive and rapid detection tool for probiotic authentication but also offers a generalizable crRNA-engineering strategy to enhance the performance of CRISPR/Cas12a in diagnostic.