基于实时荧光环介导等温扩增快速检测鸡肉中的大肠杆菌O157:H  

Rapid Detection of Escherichia coli O157:H7 from Chicken Using a Real-time Loop-mediated Amplification Isothermal Assay

作　　者：赵远洋 王瑾 林丽萍 郜彦彦 吴国平 Zhao Yuanyang;Wang Jin;Lin Liping;Gao Yanyan;Wu Guoping(School of Food Science arid Engineering, Jiangxi Agricultural University, Key Lab far Agricultural Products Processing and Quality Control of None hang City, None hang 330045)

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,南昌330045

出　　处：《中国食品学报》2019年第3期281-288,共8页Journal of Chinese Institute Of Food Science and Technology

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

摘　　要：以大肠杆菌O157:H7的VT2基因序列设计特异性引物,利用Midori Green新型核酸荧光染料,建立鸡肉中大肠杆菌O157:H7实时荧光定量环介导等温扩增(Rti-LAMP)检测方法。纯培养大肠杆菌O157:H7检测灵敏度达3.5 CFU/反应,需时45 min。模拟大肠杆菌O157:H7污染鸡肉样品,经37℃增菌4 h,用Whirl-pak无菌袋过滤离心得沉淀,提取样品DNA模板用于Rti-LAMP反应,检测大肠杆菌O157:H7灵敏度达140 CFU/g,整个检测流程约7 h。采用蒙脱石封闭的活性炭前处理污染鸡肉样品,不经增菌过程,结果表明:该Rti-LAMP检测方法灵敏度达12 CFU/g,整个检测耗时约4.5 h。市购鸡肉样品51份,以大肠杆菌国标检测法为对照,研究基于Rti-LAMP联合增菌或封闭活性炭预处理而不经增菌的两种方法,对比检测临床鸡肉样品大肠杆菌O157:H7污染率。结果:国标法和增菌的Rti-LAMP两种方法均检测到同一鸡肉样品为大肠杆菌O157:H7阳性,经封闭活性炭预处理而未经增菌的Rti-LAMP则检测到8份鸡肉样品大肠杆菌O157:H7阳性。研究表明,封闭活性炭预处理的Rti-LAMP检测方法较国标法更灵敏、快速、简便、特异地检测鸡肉中大肠杆菌O157:H7污染。In this study, a real-time loop amplified DNA assay system(Rti-LAMP) was developed for the rapid detection of Escherichia coli(E. coli)O157:H7 on the chicken. It showed that the lowest level of 3.5 CFU/reaction for pure E. coli culture could be detected and finished in 45 minutes by the Rti-LAMP targeting the VT2 gene using Midori green as nucleic acid dye. Furthermore, seeding 25 g portions of chicken with various numbers of E. coli CFU, followed enrichment culture at 37 ℃ for 4 h, then filtration through Whirl-pak bag and pelleting the bacterial cells by centrifugation at 13 000 g. The resulting pellets were suspended in saline and processed for cell lysis and DNA purification. 2 μL purified DNA samples was incorporated into 25 μL Rti-LAMP reactions at 65 ℃ for 60 min. The lowest level 140 CFU/g of E. coli consistently detected by the Rti-LAMP assay. The entire assay could be completed in 7 h. Moreover, the samples were pretreated with activated carbon coated with bentonite and without enrichment for Rti-LAMP reactions. The results showed that the sensitivity of the sample treated with coated activated carbon was 12 CFU/g and finished in 4.5 h.Fifty-one chicken samples purchased from market were detected by the Rti-LAMP assay with enrichment, the Rti-LAMP with bentonite coated activated carbon(BCAC) pretreated but without enrichment, as the control of E. coli O157:H7 culture. The same one sample was E. coli O157:H7 positive by the Rti-LAMP assay with enrichment and E. coli culture,and other 7 samples(total 8 samples) were also E. coli O157:H7 positive detected by the Rti-LAMP pretreated with BCAC. These data suggested that the Rti-LAMP combined with BCAC pretreatment was more sensitive, rapid, simple and specific than that of culture for detection of E. coli O157:H7 retrieved from Chicken.

关 键 词：大肠杆菌O157:H7 鸡肉 实时环介导等温扩增 增菌 封闭的活性炭 

分 类 号：TS251.55[轻工技术与工程—农产品加工及贮藏工程]