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【目的】开发一种同时对食品中沙门氏菌、志贺氏菌和金黄色葡萄球菌快速、灵敏、准确的检测方法。【方法】利用特异性免疫磁球,在37°C条件下从250 m L猪肉增菌液体系中边富集边循环捕获目标菌。快速提取DNA后,利用特异性的引物与探针,对3种食源性致病菌进行三重荧光定量PCR检测。【结果】针对沙门氏菌、志贺氏菌和金黄色葡萄球菌的检测限分别达到2.0、6.8和9.6 CFU/g。方法总体灵敏度、特异性和准确度达到99.2%、100%及99.5%。对151份实际样品进行检测,与国标(GB/T 4789.4-2010、GB 4789.5-2012和GB/T4789.10-2010)方法的检测结果相比,金黄色葡萄球菌有一例阴性偏差。【结论】开发的基于免疫磁分离的三重荧光定量PCR方法,能够在8 h内完成对食品中3种致病菌检测,并且灵敏度高、特异性好、检测准确,可以作为快速应对此类食品安全突发事件的检测手段。
【Objective】 To develop a rapid, sensitive and accurate method for simultaneous detection of Salmonella, Shigella and Staphylococcus aureus in food. 【Method】 Target bacteria were harvested from the 250 mL pork enrichment broth at 37 ° C by using specific immunomagnetic beads. After rapid DNA extraction, three kinds of foodborne pathogenic bacteria were detected by triplex fluorescence quantitative PCR using specific primers and probes. 【Result】 The detection limits against Salmonella, Shigella and Staphylococcus aureus reached 2.0, 6.8 and 9.6 CFU / g, respectively. The overall sensitivity, specificity and accuracy of the method were 99.2%, 100% and 99.5%. 151 actual samples were tested. Compared with the test results of the national standard (GB / T 4789.4-2010, GB 4789.5-2012 and GB / T4789.10-2010), there was a negative bias for Staphylococcus aureus. 【Conclusion】 The developed triplex fluorescence quantitative PCR method based on immune magnetic separation can detect three kinds of pathogenic bacteria in food within 8 h with high sensitivity and specificity and accurate detection and can be used as a rapid response to such foods Security incident detection means.