Simultaneous enumeration of Cronobacter sakazakii and Staphylococcus aureus in powdered infant foods through duplex TaqMan real-time PCR

Abstract Cronobacter sakazakii and Staphylococcus aureus are important pathogens. Targeting the species-specific macromolecular synthesis (MMS) operon and thermostable nuclease gene (nuc), a duplex TaqMan real-time PCR (dRT-PCR) assay was developed for their simultaneous enumeration within two kinds of powdered infant foods (PIFs). The assay was highly specific, producing fluorescent signals exclusively from target bacteria. The quantification limits were 29.10 and 8.97 copies μL−1 for standard plasmids containing MMS and nuc fragments. Without enrichment, the dRT-PCR could quantify as low as 102 and 101 cfu mL−1 C. sakazakii and S. aureus in both pure culture and spiked PIFs. Quantitative linearity ranged from 102 to 107 cfu mL−1 for C. sakazakii and from 101 to 107 cfu mL−1 for S. aureus. Bacterial counts derived from dRT-PCR were consistent with those calculated from plating for randomly spiked PIFs. The dRT-PCR assay provides a promising culture-independent alternative enabling direct, simultaneous and robust pathogen quantification.