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

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.