PMA-LAMP方法检测灭菌乳中金黄色葡萄球菌的研究  

Detecting of viable Staphylococcus aureus by loop-mediated isothermal amplification coupling with propidium monoazide in dairy products

作　　者：胡惠秩[1] 满朝新[2] 董鑫悦[1] 刘珊珊[1] 薛玉清[1] 杨士芹[1] 谢鲲昊[1] 刘颖[1] 卢雁[2] 姜毓君[1,2] 

机构地区：[1]东北农业大学乳品科学教育部重点实验室,黑龙江哈尔滨150030 [2]东北农业大学国家乳业工程技术研究中心,黑龙江哈尔滨150030

出　　处：《食品工业科技》2012年第21期300-304,308,共6页Science and Technology of Food Industry

基　　金：国家科技支撑计划项目(2012BAK17B04)

摘　　要：近年来,金黄色葡萄球菌引起的食品安全事件频发,这就需要建立一种快速准确地检测食品中金黄色葡萄球菌的方法。传统方法检测食品中金黄色葡萄球菌活菌存在很多缺点。由于细胞死亡后其DNA依然能够存活许久,所以传统方法不能有效区分DNA来自死菌还是活菌。通过荧光染料PMA与快速检测技术LAMP相结合的方法快速、灵敏的检测灭菌乳中金黄色葡萄球菌,并对死/活菌进行区分。根据金黄色葡萄球菌nuc基因设计引物,并对LAMP反应体系及PMA-LAMP方法进行优化,同时优化了灭菌乳中提取DNA的方法。在纯培养的金黄色葡萄球菌中,PMA-LAMP方法检测灵敏度为3.2CFU/mL,PMA-PCR方法的检测灵敏度为3.2×102CFU/mL;对人工污染灭菌乳中的金黄色葡萄球,PMA-LAMP方法检测灵敏度为5×101CFU/mL,而PMA-PCR方法检测灵敏度为5×103CFU/mL。整个反应需要大约6h,说明PMA-LAMP方法检测灭菌乳中金黄色葡萄球菌特异性强、灵敏度高、时间短且操作简便,有望成为快速检测金黄色葡萄球菌活菌的新方法。In recent years, the increasing number of Staphylococcus aureus outbreaks linked to food product highlighted the need to develop technique with rapid, simple and accurate.The conventional technologies for rapid and sensitive detection of S. aureus viable cells in produce had several limitations.The signal from viable versus dead ceils could not be distinguished due to the persistence of DNA after the cell death,resulting in overestimate the number of cells.In the present study,some problems had been solved by developing a new concept with DNA -intercalating dye propidium membrane（ PMA） combining with loop-mediated isothermal amplification（LAMP）. The test was used to analyse S. aureus and routine dairy products, a series of primers targeted six distinct sequences of nuc gene were designed, which was characteristic of S. aureus. Also, the LAMP assay and the performance of PMA-LAMP for detecting viable S.aureus were optimized.Moreover,they improved the method for extraction DNA from dairy samples. In pure culture, the detection limit of PMA- LAMP was 3.2CFU/mL, up to 100-fold more sensitive than PCR.In dariy products, PMA-LAMP assay could detect as less as 5×101CFU/mL compared to that of PCR-PMA was 5×103CFU/mL. The complete LAMP-PMA assay for took about 6h, demonstrating the method was rapid and convenient. In conclusion, PMA-LAMP offers a novel DNA-based detection method for distinction between viable and dead cells with wide application in food products.

关 键 词：PMA—LAMP 金黄色葡萄球菌 乳 检测 

分 类 号：TS207.3[轻工技术与工程—食品科学]