Amplification-free quantification of Lactiplantibacillus plantarum in food fermentations based on CRISPR/Cas12a with multi-crRNAs

Food fermentations involve numerous microorganisms that are crucial for the quality of the fermented food. Quantifying microorganisms would be beneficial for regulating food fermentations. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems have emerged as efficient methods for detecting microorganisms. However, it shows great limitations in detection sensitivity and quantitative capability. Herein, we developed a novel amplification-free CRISPR/Cas12a-based quantification method (AFCQ) to effectively quantify Lactiplantibacillus plantarum by adding multiple crRNAs and chemical additives. The transcleavage rate using a combination of 5 CRISPR RNAs (crRNAs) and dithiothreitol (DTT) was 89.4 times that of single crRNA. Under the optimized condition, AFCQ allows for quantifying L. plantarum genome from 1.563 x 107 copies/reaction to 1.000 x 109 copies/reaction (R2 = 0.995) with the limit of detection (LOD) at 3.142 x 106 copies/reaction. Finally, quantitative performances of AFCQ were well consistent with quantitative real-time PCR by detecting L. plantarum spiked in 7 real fermented food samples. We expected that AFCQ could be widely used to quantify various target microbes in food fermentations and standardize relevant microbial detection sectors in the food field in the near future.