Development of Triplex PMA-qPCR for Rapid Quantification of Viable Total and Sea-positive Staphylococcus aureus in Quick-Frozen Flour and Rice Products
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    Abstract:

    The objective of this study is to develop a triplex PMA-qPCR method for rapid quantification of viable total and sea-positive Staphylococcus aureus in quick-frozen flour and rice products, simultaneously indicating the possible false negative detection results. Firstly, primers and probes targeting the conserved region of RpiR and sea gene were designed, and a competitive internal amplification control was constructed. Then triplex qPCR method was developed by optimizing reaction conditions and evaluating the specificity and detection limit. Food sample preparation combining propidium monoazide (PMA) was used to eliminate DNA of dead cells. The standard curves for quantification of viable total and sea-positive Staphylococcus aureus in pure culture and frozen foods were established, respectively. Artificial contamination assays and plate count methods were used to evaluate the accuracy of triplex PMA-qPCR method developed in this study. It was shown that triplex PMA-qPCR method had good amplification efficiency and specificity; when quick-frozen flour and rice products were contaminated above 103 CFU/g Staphylococcus aureus, the target gene RpiR could be effectively detected, especially, the contamination of Staphylococcus aureus was between 103 CFU/g and 107 CFU/g, the cycle threshold values (Ct) and the log number of bacterial concentration presented the satisfactory linear, the correlation coefficient R2 approached 0.994, and the quantification result obtained by PMA-qPCR method showed good consistency with plate count methods; when quick-frozen flour and rice products were contaminated above 103 CFU/g sea-positive Staphylococcus aureus, the contamination risk of enterotoxin A could be effectively evaluated. Taken all together, triplex PMA-qPCR method developed in this study could accurately quantify viable Staphylococcus aureus and evaluate the contamination risk of enterotoxin A in quick-frozen flour and rice products.

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  • Online: July 31,2020
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