基于免疫磁分离的三重荧光定量PCR检测食品中沙门氏菌、志贺氏菌和金黄色葡萄球菌

Rapid and simultaneous detection of Salmonella, Shigella, and Staphylococcus aureus in food using a multiplex RT-PCR assay based on immunomagnetic separation

摘要:

【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 mL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/T 4789.10-2010)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。

Abstract:

[Objective] This study aimed to develop a rapid and simultaneous detection method for Salmonella, Shigella, and Staphylococcus (S.) aureus in food. [Methods] Magnetic beads coated with speci?c antibodies were used to capture target pathogens from 250 mL at 37 °C. After quick DNA extraction, multiplex RT-PCR was applied to detect the target pathogens with three sets of specific primers and probes. [Results] The limit of detections of immunomagnetic separation (IMS)-multiplex real-time PCR (RT-PCR) method were 2.0 CFU/g for Salmonella, 6.8 CFU/g for shigella and 9.6 CFU/g for S. aureus. The sensitivity, specificity, and accuracy of this method were 99.2%, 100%, and 99.5%, respectively. One hundred fifty-one samples were tested using the IMS-multiplex RT-PCR and GB methods, and only one negetive deviation was detected. [Conclusion] The IMS-multiplex RT-PCR method with high sensitivity, specificity and accuracy could enable the simultaneous detection of Salmonella, Shigella, and S. aureus in food within 8 h and offers the opportunity for a quick response in an emergency when these bacteria are detected.