Rapid and simple detection of Pediococcus using ARMS-CRISPR/Cas12a method

Pediococcus spp. are lactic acid bacteria, which are prevalent in various environments, including plants and animals. Notably, they constitute a significant component of the intestinal microbiota in both humans and animals. Despite this, numerous obstacles remain in developing tools that are both highly sensitive and specific for distinguishing this genus. This study established a fluorescent detection system using an amplification-resistant mutation system-based polymerase chain reaction (ARMS-CRISPR/Cas12a) for identifying 16S rRNA gene containing single nucleotide polymorphism (SNP) in Pediococcus spp. By aligning the sequences of Pediococcus spp. with those of other genera, we performed a comprehensive statistical analysis of SNP sites within Pediococcus spp. and designed specific primers using the 16S rRNA gene sequence of Pediococcus pentosaceus STS-6. The results demonstrated that, under optimised conditions (a Cas12a:crRNA ratio of 1:1 at 37 °C), the dual recognition process combining ARMS-PCR with CRISPR/Cas12a achieved high specificity and sensitivity in the detection of Pediococcus spp. The detection limit for genomic DNA was 8.15 × 10^-5 ng/μL, demonstrating significantly higher sensitivity than gel electrophoresis. The entire detection process took approximately 1.5 h. In summary, the ARMS-CRISPR/Cas12a detection system established in this study provided a rapid and effective method to detect the 16S rRNA gene of clinically relevant Pediococcus spp. probiotics, meeting the requirements for food production detection.