Multiplex PCR assay and lyophilization for detection of Salmonella spp., Staphylococcus aureus and Bacillus cereus in pork products

Multiplex PCR (m-PCR) has the potential for more rapid detection of pathogens compared to simple PCR through the simultaneous amplification of multiple gene targets using several sets of specific primers. Here, we developed an m-PCR assay which combined dry reagent mixtures for ready-to-use simultaneous detection of Salmonella spp., Bacillus cereus, and Staphylococcus aureus. The assay did not show cross-reactivity with several common bacterial pathogens and the detection limit was 10(3) CFU/mL for mixed genomic DNA in pure culture. Lyophilized m-PCR reagents are stable for 2 months stored at 4 A degrees C and for 1 month stored at 25 A degrees C. Detection sensitivities of both dry and fresh mixes were able to simultaneously detect 10 CFU/mL of each pathogen in artificially inoculated samples after enrichment for 6 and 12 h. Results demonstrated that this method is both sensitive and specific and can be used for rapid detection and differentiation of foodborne diseases.