PCR-based procedures for detection and quantification of Staphylococcus aureus and their application in food.
OBJECTIVE
To evaluate the specificity of nuc targeted primers for PCR detection of Staphylococcus aureus in different food matrices and to establish a RTQ-PCR procedure suitable for the routine detection and quantification of this pathogen in food.


RESULTS
Specificity of nuc targeted primers (Pri1-Pri2 and the newly designed RTQ-PCR primers) was tested on a total of 157 strains of genetically confirmed identity, including reference and food isolates. PCR detection on artificially inoculated beef samples by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) showed a sensitivity value around 10(3) CFU g(-1). The two RTQ-PCR systems, incorporating SYBR-Green I and TaqMan, respectively, applied in the present work improved the sensitivity of conventional PCR by lowering the detection level to 10 and 100 cells, respectively. Out of 164 naturally contaminated foods tested for the presence of Staph. aureus, 74 were positive by conventional PCR and 69 by the traditional culture method with a high degree of result agreement between both methodologies (93.3%).


CONCLUSIONS
PCR approaches, using nuc targeted primers, have proved specific and combined with growth techniques may improve detection of Staph. aureus in different types of food.


CONCLUSIONS
The SYBR-Green I real-time PCR approach established allows the sensitive, automated and quantitative detection of Staph. aureus for routine analysis at a reasonable cost.
Alarcón
B
B
Departamento de Microbiología y Ecología, Universitat de València, Spain.
Vicedo
B
B
Aznar
R
R
D000818:Animals; D002417:Cattle; D015169:Colony Count, Microbial / Q000379:methods; D046508:Culture Techniques / Q000379:methods; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D005516:Food Microbiology; D008460:Meat / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D004269:DNA, Bacterial