牛奶中活沙门氏菌BCAC-EMA-Rti-LAMP快速检测方法的研究  

Development of a BCAC-EMA-Rti-LAMP Assay for Rapid Detection of low Viable Salmonella in Milk without Enrichment

作　　者：张天添 陈鹄 吴国平 舒梅 林丽萍 钟婵 Zhang Tian-tian;Chen Hu;Wu Guo-ping;Shu Mei;Lin Li-ping;Zhong Chan(College of Food Science and Engineering,Key Lab for Agricultural Products Processing and Quality Control of Nanchang City,Jiangxi Agricultural University,Nanchang 330045,China)

机构地区：[1]江西农业大学食品科学与工程学院/南昌市农产品加工与质量控制重点实验室

出　　处：《江西农业大学学报》2019年第6期1192-1199,共8页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然科学基金项目（31560480,31760483）;江西省自然科学基金项目（20171ACB20013）~~

摘　　要：研究以沙门氏菌的invA基因序列设计特异性引物,结合蒙脱石封闭的活性炭(BCAC)吸附、叠氮溴化乙锭(EMA)前处理牛奶样品,建立了一种基于实时荧光环介导等温扩增法(Rti-LAMP)非预增菌快速检测牛奶中低浓度活沙门氏菌的方法。以每份25 mL牛奶样品,人工模拟不同浓度沙门氏菌污染,用4.0 g蒙脱石封闭的活性炭吸附处理样品,然后对回收精制的细菌样品用4.0μg/mL EMA终浓度作用处理,提取细菌样品DNA用于RtiLAMP扩增检测,该方法检测灵敏度为1×10^1CFU/mL活沙门氏菌,整个检测过程约5 h。以沙门氏菌国标检测法为参照,采用BCAC-EMA-Rti-LAMP检测生鲜牛奶样品27份,结果显示:2种方法均检测到同一份沙门氏菌阳性牛奶样品,而BCAC-EMA-Rti-LAMP另检测到3份牛奶样品沙门氏菌阳性。研究表明,建立的BCAC-EMARti-LAMP检测方法较国标检测法更灵敏、快速,可用于牛奶沙门氏菌污染的快速检测。In this study,a real-time loop-mediated isothermal amplified(Rti-LAMP)DNA assay system targeting the invA gene was developed for the rapid detection of viable Salmonella in milk without enrichment.The Rti-LAMP assay was combined with bentonite coated activated carbon(BCAC)adsorption for removing DNA amplification inhibitors of milk and ethidium bromide monoazide(EMA)treatment.The assay involved seeding 25 mL portions of milk with various numbers of Salmonella cells.Four grams of BCAC was used to treat milk samples for removing DNA amplification inhibitor.Then four micrograms per milliliter of EMA was chosen as the final concentration for discriminating viable Salmonella from the dead Salmonella cells in Rti-LAMP assay.The detection limit of viable Salmonella was 1×10^1 CFU/mL in milk within 5 h.Twenty-seven raw milk samples were detected by the BCAC-EMA-Rti-LAMP,using Salmonella culture as the control. The results indicated that only one same positive sample was detected by the two methods,but other three Salmonella positive samples were also detected by the BCAC-EMA-Rti-LAMP assay.These data suggested that the BCACEMA-Rti-LAMP assay was much sensitive and rapid than Salmonella culture,which is expected to be used in the rapid detection of milk contaminated Salmonella without enrichment.

关 键 词：牛奶 沙门氏菌 实时荧光环介导等温扩增法 蒙脱石封闭的活性炭 叠氮溴化乙锭 

分 类 号：TS201.3[轻工技术与工程—食品科学]