Study of TaqMan assay of real-time fluorescence quantitative PCR to detect Staphylococcus aureus in food

ObjectiveTo establish a rapid, sensitive and specific method to detect IStaphylococcus aureus/I in food by using real-time fluorescence quantitative PCR. MethodsA pair of primers and probe were designed by taking FemB gene of IStaphylococcus aureus/I as the target sequence. The DNA of IStaphylococcus aureus/I strain were extracted as the template to optimize the concentration ratio of primers and probe and the concentration of Mgsup2+/sup. The sensitivity, stability and specificity of the assay were tested by using IStaphylococcus aureus/I and other 10 related bacteria strains. The assay was also used to detect the samples. ResultsThe assay had high specificity and sensitivity at the primer concentration of 0.8 mol/L, probe concentration of 0.6 mol/L and Mgsup2+/supconcentration of 3.5 mmol/L. The results of 10 related bacteria strains detection was negative, but the detection of IStaphylococcus aureus/I had a positive result. The quantitive detection limit was 44 cfu/ml in pure cultured broth. The stability analysis indicated that the variation coefficient of IC/It was always less than 5% for three times detection of same sample. The sample detection indicated that TaqMan assay is more sensitive, rapid and simple compared with traditional. ConclusionBecause TaqMan assay is highly specific and stable, simple and rapid, it can be used in microbiological detection of food.