CHARACTERIZATION OF A DUPLEX POLYMERASE CHAIN REACTION ASSAY FOR THE DETECTION OF ENTEROTOXIGENIC STRAINS OF STAPHYLOCOCCUS AUREUS

ABSTRACT Sequence analysis demonstrated that all reported primers based on the thermonuclease (TNase) gene ( nuc ) used in the detection of the foodborne pathogen Staphylococcus aureus were derived from the nuclease gene, instead of the thermonuclease gene. In this study, two pairs of primers targeting the sequences of the nuc and 16S ribosomal RNA genes were designed in a duplex polymerase chain reaction (PCR) assay for the detection of S. aureus , particularly those of the enterotoxigenic strains. After optimization of the amplification conditions by uniform design, two specific products were amplified in the PCR detection system: a 223‐bp nuc fragment and a 565‐bp 16S rDNA fragment. Evaluation tests demonstrated that this duplex PCR method has high specificity for only S. aureus . The detection limit of the assay was 9.35 pg/µL using S. aureus genomic DNA. The sensitivity for direct detection of milk samples sparked with S. aureus was 10 4 –10 5 cfu/mL, and the sensitivity was 1 cfu/mL as an initial concentration when an enrichment process was used. A PCR‐based detection assay for enterotoxin gene was performed in parallel with the detection of the nuc ‐encoded TNase activity in several S. aureus strains, and a good correlation between TNase production and enterotoxigenicity was demonstrated in all the strains tested.