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

作　　者：马凯[1] 李宝明[1] 白羽[1] 陈尔凝[1] 刘杰[1] 武会娟[1] 高丽娟[1] 

机构地区：[1]北京市理化分析测试中心,北京100089

出　　处：《微生物学通报》2014年第11期2369-2377,共9页Microbiology China

基　　金：北京市科技新星项目(No.2008B31)

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 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] 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 specific antibodies were used to capture target pathogens from 250 m L 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.

关 键 词：免疫磁分离 多重荧光定量PCR 沙门氏菌 志贺氏菌 金黄色葡萄球菌 

分 类 号：TS207.3[轻工技术与工程—食品科学]