Efficient magnetic enrichment cascade single-step RPA-CRISPR/Cas12a assay for rapid and ultrasensitive detection of Staphylococcus aureus in food samples

Staphylococcus aureus (S. aureus) is a prevalent foodborne pathogen that could cause severe food poisoning. Thus, rapid, efficient, and ultrasensitive detection of S. aureus in food samples is urgently needed. Here, we report an efficient magnetic enrichment cascade single-step recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay for the ultrasensitive detection of S. aureus. Magnetic beads (MBs) functionalized with S. aureus-specific antibodies were initially used for S. aureus enrichment from the complex matrix, with 98% capture efficiency in 5 min and 100 -fold sensitivity improvement compared with unenriched S. aureus. Next, a single-step RPACRISPR/Cas12a-based diagnostic system with optimized extraction-free bacteria lysis was constructed. This assay could detect as low as 1 copy/mu L (five copies/reaction) of extracted DNA template and 10 CFU/mL of S. aureus within 40 min. Furthermore, the assay could effectively detect S. aureus in real food samples such as lake water, orange juice, pork, and lettuce, with concordant results to qPCR assays. The proposed cascade signalamplification assay eliminates the need for lengthy bacterial culture and complex sample preparation steps. Hence, the proposed assay shows great application potential for rapid, efficient, and ultrasensitive detection of pathogens in real food samples.