The Development and Application of a Method for the Detection of Viable but Nonculturable <i>Staphylococcus aureus</i> by Magnetic Bead Separation Combined With PMA‐qPCR

ABSTRACT Staphylococcus aureus is a major food‐borne pathogen and easy to be induced into a viable but nonculturable state under stress conditions from food processing, causing a significant and potential risk to public health. In this study, the primers for femB gene were examined to be specific to S . aureus . 10 μM of PMA was determined as the optimal treatment concentration to minimize the PCR amplification signal produced by dead bacterial DNA. When ∼10 7 CFU/mL of S . aureus was treated with sorbic acid, about 85% of S . aureus ATCC 6538 cells and 76% S . aureus ‐1 cells were induced into VBNC state after 6 days, respectively. 1 mg/mL of Fe 3 O 4 nanomagnetic beads, at 37°C for 10 min were determined as the optimal condition to separate VBNC S . aureus cells. The detection capability of Fe 3 O 4 ‐PMA‐qPCR for VBNC S . aureus in rice, chicken, and milk was comparable with DNeasy PowerFood Microbial Kit‐PMA‐qPCR, and the detection efficiency of Fe 3 O 4 ‐PMA‐qPCR was comparatively more excellent. Compared to commercial Kit‐PMA‐qPCR, the established Fe 3 O 4 ‐PMA‐qPCR combined with plating assay was applied to quantify VBNC S . aureus inoculated into food samples, with advantages of specificity to S . aureus , short time, low cost, and sensitive results.