Rapid and simultaneous detection of viable Cronobacter sakazakii, Staphylococcus aureus, and Bacillus cereus in infant food products by PMA-mPCR assay with internal amplification control

• mPCR based method was developed to simultaneously detect viable Cronobacter sakazakii, Staphylococcus aureus, and Bacillus cereus.
• PMA was selected to eliminate the interference from dead cells.
• IAC was used to prevent false-negative results.
• The assay could detect 100–101 CFU/g viable cells in infant food samples after 12 h enrichment.

Cronobacter sakazakii, Staphylococcus aureus, and Bacillus cereus are important pathogens contaminating infant food products. In this study, we developed a specific, sensitive, and accurate technique for the simultaneous detection of viable C. sakazakii, S. aureus, and B. cereus in infant food products. An internal amplification control (IAC) was added to the multiplex PCR (mPCR) as indicator of false negative results that are mainly caused by the PCR inhibitors in food products. In addition, to detect only the viable bacteria, propidium monoazide (PMA) was applied to selectively suppress the PCR signal from dead cells. The limit of detection (LOD) for the viable cells with or without PMA treatment was 104 CFU/mL for C. sakazakii and B. cereus, and 102 CFU/mL for S. aureus in pure culture, showing that the PMA treatment did not influence the sensitivity. After 12 h enrichment, the PMA-mPCR-IAC assay could detect as low as 101 CFU/g for C. sakazakii and S. aureus, and 100 CFU/g for B. cereus in spiked infant food products (infant formula, noodles, milk and rice noodles). This PMA-mPCR-IAC assay we developed hold promise for the simultaneous detection of C. sakazakii, S. aureus, and B. cereus in infant food products.