三重LAMP法检测食品中沙门氏菌、单增李斯特菌和金黄色葡萄球菌  

Development of Multiplex LAMP Method for the Detection of Salmonella spp., Staphylococcus aureus and Listeria monocytogenes in Foods

作　　者：姜侃 吕沁风[2] 汪新 夏琳 张峥 陈小珍 

机构地区：[1]浙江省质量检测科学研究院,浙江杭州310013 [2]浙江国际旅行卫生保健中心,浙江杭州310003

出　　处：《食品科学》2013年第24期182-187,共6页Food Science

基　　金：浙江省科技厅优先主题重点发展项目(2009C13013);浙江省质监系统科研计划项目(20120101);质检公益性行业科研专项(201010043)

摘　　要：为能一步快速检测沙门氏菌、金黄色葡萄球菌和单增李斯特菌3种食源性致病菌,建立了三重环介导等温扩增(LAMP)检测方法。针对沙门氏菌的侵袭蛋白(invA)基因、金黄色葡萄球菌的耐热核酸酶(nuc)基因、单增李斯特菌(hly)基因3个高度保守的基因,设计出3套LAMP引物。以8株代表目标菌株,三重LAMP反应体系中Mg2+浓度4.6mmol/L、扩增温度61℃。通过电泳检测、离心观察沉淀和荧光检测证明该方法能够在90min内一次性检出以上3种致病菌。3种菌的检测灵敏度均约为10fg/μL,是PCR检测灵敏度(10pg/μL)的1000倍。以无害李斯特菌等常见的20株非目标菌株为对照,证明了此方法的特异性。LAMP产物DNA测序结果与预期一致,证明了该方法的正确性。该三重LAMP检测方法具有快速、特异、灵敏度高的优点,适用于食品中沙门氏菌、金黄色葡萄球菌和单增李斯特菌的快速检测。A rapid one-step method to detect foodborne pathogens such as Salmonella spp., Staphylococcus aureus and Listeria monocytogenes in foods was developed multiple LAMP （loop-mediated isothermal amplification）. According to the highly conservative gene invA of Salmonella spp., the nuc gene of S. aureus and the hly gene of L. monocytogenes, three sets of primers were designed and synthesized. Eight representative test strains were used in LAMP development. The optimized LAMP reaction system was as follows： Mg2＋ concentration of 4.6 mmol/L and reaction temperature of 61 ℃. Electrophoresis of LAMP products, observation of the centrifugal precipitate and fluorescent staining with SYBR Green I indicated that three pathogens could be detected within 90 min in one step by using this LAMP system. The LAMP assay showed a sensitivity of 10 fg/μL, which is 1000 times higher than that of conventional PCR detection （10 pg/μL）. The specificity was verified by using 20 strains of non-targeting bacteria including L. innocua as negative controls. The sequence analysis of LAMP products confirmed that this assay could correctly amplify the target gene in accordance with the design presented in this study. Overall, the novel multiple LAMP method reported herein is characteristics of rapid detection, high specificity and excellent sensitivity, which can be applied in the rapid detection of Salmonella spp., S. aureus and L. monocytogenes.

关 键 词：LAMP 沙门氏菌 金黄色葡萄球菌 单增李斯特菌 快速检测 

分 类 号：Q781[生物学—分子生物学]