Rapid On-Site Detection of Lacticaseibacillus paracasei Using a Portable MIRA-CRISPR/Cas12a Naked-Eye Fluorescence Assay.

Probiotics play an important role in maintaining human gastrointestinal health and immune function, driving a rapidly expanding global market for probiotic-fortified foods. Lacticaseibacillus paracasei is one of the most widely applied probiotic strains in functional foods and dairy formulations, with well-documented health-promoting properties. For probiotic foods, authenticity requires rigorous verification for efficacy and compliance, which depends on rapid strain-specific identification. However, conventional culture-based methods are time-consuming and cannot distinguish closely related Lacticaseibacillus species, while PCR-based assays require expensive thermal cyclers and trained personnel, barring deployment in resource-limited settings. Here, we report the development and validation of a MIRA-CRISPR/Cas12a fluorescence assay for rapid, species-specific, and highly sensitive on-site detection of Lacticaseibacillus paracasei. This integrated workflow combines 8 min rapid crude DNA extraction, 30 min isothermal MIRA pre-amplification, and 20 min CRISPR/Cas12a signal amplification with naked-eye fluorescence readout under blue light excitation, and requires only miniaturized portable equipment. The assay achieved a reliable limit of detection of 100 CFU/mL in milk-matrix spiked samples, showing no cross-reactivity across 17 tested lactic acid bacteria strains. In a validation set of 48 food samples, our method demonstrated 100% concordance with the gold-standard species-specific qPCR assay. This field-deployable method is appropriate for on-site quality control in probiotic manufacturing, label compliance verification, and frontline market regulatory inspections.