关键词: 恒温隔绝式聚合酶链式反应；金黄色葡萄球菌；DNA提取；nuc基因；快速检测

Abstract: A method for detecting Staphylococcus aureus in foods was established by insulated isothermal polymerase chain reaction (iiPCR). In this study, specific primers and probes were designed based on the nuc gene, and the extraction of template DNA from S. aureus in food samples was explored. After optimizing the amount of primers, probes and templates, the specificity, sensitivity and stability of iiPCR were evaluated, and the application of iiPCR and traditional PCR was compared to detect S. aureus in artificially contaminated samples and actual food samples. The results showed that the water bath method was suitable for rapid extraction of template DNA due to its simple operation, short time consumption, and low instrument requirements. The iiPCR method had strong specificity, high sensitivity and good stability without cross-reaction with other bacteria. It took six hours to detect S. aureus in artificially contaminated pork samples and milk samples by the iiPCR method with a detection limit of 103 and 102 CFU/mL, respectively, while the traditional PCR method took eight hours with a detection limit of 105 CFU/mL. For actual food samples, the verification experiments showed that the results of the iiPCR method at 6 h were consistent with those of traditional PCR at 12 h and the traditional culture method at 16 h. It was proved that the iiPCR method could detect S. aureus in foods more quickly and accurately.

Key words: insulated isothermal polymerase chain reaction; Staphylococcus aureus; DNA extraction; nuc gene; rapid detection