BCAC-EMA-Rti-LAMP非预增菌快速检测鱼肉污染活的副溶血弧菌研究  

Rapid Detection of Viable Vibrio parahaemolyticus in Fish by BCAC-EMA-Rti-LAMP without Enrichment

作　　者：杨珺 陈鹄 吴国平 舒梅 李盛艳 钟婵 YANG Jun;CHEN Hu;WU Guo-ping;SHU Mei;LI Sheng-yan;ZHONG Chan(Key Lab for Agricultural Products Processing and Quality Control of Nanchang City,College of Food Science and Engineering,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,江西南昌330045

出　　处：《江西农业大学学报》2020年第5期1042-1049,共8页Acta Agriculturae Universitatis Jiangxiensis

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

摘　　要：【目的】随着人民物质生活水平的不断提高,对食品质量要求也愈来愈高,食品安全现如今已经成为影响人类生命健康财产安全的重大公共卫生问题。食源性致病菌是引起食品安全中毒事件的主要原因,而副溶血弧菌主要污染海产品从而引起急性肠胃炎。因此,快速有效地检测筛查污染的海产品,是有效防控副溶血弧菌食源性疾病暴发的必要条件。【方法】研究以副溶血弧菌特异性tlh基因设计合成引物,以Midori Green为核酸荧光染料,构建了一种副溶血弧菌实时荧光环介导等温扩增(Rti-LAMP)检测技术,并联合蒙脱石封闭的活性炭(BCAC)、叠氮溴化乙锭(EMA)对样品前处理,建立了一种非预增菌快速检测鱼肉污染活的副溶血弧菌方法。【结果】Rti-LAMP检测纯培养副溶血弧菌灵敏度达1 CFU/反应,1 h以内完成。模拟污染1 g鱼肉样品,经4 g BCAC吸附和6μg/mL EMA终浓度处理细菌样品,提取细菌DNA用于Rti-LAMP扩增反应,最低检出限为6 CFU/g活的副溶血弧菌,整个检测过程需约5 h。对市售33份新鲜海鱼样品检测,BCAC-EMARti-LAMP检测出3份阳性样品,BCAC-Rti-LAMP为6份阳性样品,而相应国标检测法为2份阳性样品。【结论】研究表明,BCAC-EMA-Rti-LAMP可以有效检测区分样品中活的副溶血弧菌污染,较国标检测法更快速、灵敏,有望作为鱼肉副溶血弧菌污染的一种快速筛查检测方法。[Objective]With the continuous improvement of human’s living standard,food quality requirements are increasingly high,food safety has become a major public health problem affecting the safety of human life,health and property.Foodborne pathogenic bacteria are the main causes of food safety poisoning incidents,and Vibrio parahaemolyticus is one of the important pathogenic bacteria causing acute gastroenteritis.Therefore,rapid and effective detection and screening of contaminated seafood is a necessary condition for effective prevention and control of foodborne disease outbreaks of Vibrio parahaemolyticus.[Method]In this study,a real-time loop-mediated isothermal amplified(Rti-LAMP)DNA assay system targeting the specific tlh gene was developed for the rapid detection of viable Vibrio parahaemolyticus(VP)in marine fish without enrichment.The Rti-LAMP assay was combined with bentonite coated activated carbon(BCAC)and ethidium bromide monoazide(EMA)to treat fish samples.[Result]The results showed that the limitation of detection for VP was 1 CFU/reaction by Rti-LAMP assay in 1 h.The BCAC-EMA-Rti-LAMP assay involved seeding 1 g portions of fish with various numbers of VP cells.Four grams of BCAC was used to treat fish samples to remove DNA amplification inhibitors,then 6μg/mL EMA was chosen as the optimum final concentration for discrimination of viable VP from dead VP cells in Rti-LAMP assay.The minimum detection limit of viable VP was 6 CFU/g within 5 h.Thirtythree fresh marine fish samples collected from local markets were detected by the BCAC-EMA-Rti-LAMP and BCAC-Rti-LAMP,using VP culture as a control.The results showed that there were 3 positive samples detected by BCAC-EMA-Rti-LAMP,two positive samples by VP culture,and 6 positive samples by BCAC-Rti-LAMP.[Conclusion]The results indicated that the BCAC-EMA-Rti-LAMP could effectively distinguish viable VP,and was more sensitive and rapid than VP culture,which is expected to be used in the rapid detection of fish contaminated VP without enrichment.

关 键 词：副溶血弧菌 鱼肉 实时荧光环介导等温扩增 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]