FTA滤膜与环介导等温扩增技术结合快速检测消毒乳中的蜡样芽孢杆菌  

FTA Filter Combined with Loop-Mediated Isothermal Amplification Technique for Rapid Detection of Bacillus Cereus in City Milk

作　　者：齐哲[1] 张伟[1] 刘卫华[1] 亢春雨[1] 张先舟[1] 陈珊珊[1] 魏昭[1] 张亚爽[1] 

机构地区：[1]河北农业大学食品科技学院,河北保定071000

出　　处：《中国食品学报》2009年第3期156-161,共6页Journal of Chinese Institute Of Food Science and Technology

基　　金：河北省自然科学基金资助项目(C2008000216)

摘　　要：目的:建立环介导等温扩增技术(LAMP)快速检测蜡样芽孢杆菌的方法。方法:根据公布的蜡样芽孢杆菌溶血素基因的hblA基因的保守序列设计引物,将FTA滤膜提取DNA与LAMP法相结合,检测蜡样芽孢杆菌和人工污染蜡样芽孢杆菌的消毒乳。结果:LAMP方法检测灵敏度高,蜡样芽孢杆菌的检出限为4.4cfu/mL,比PCR检测灵敏度高10倍;人工污染消毒乳中蜡样芽孢杆菌的检出限为57cfu/mL。LAMP扩增可在20min内完成,对消毒乳中蜡样芽孢杆菌的检测(包括DNA提取、LAMP和电泳)可在2h内完成。结论:初步建立LAMP检测蜡样芽孢杆菌的快速检测方法,该方法灵敏度高、特异性好、耗时短,为食品中蜡样芽孢杆菌快速检测构建了一个技术平台。Objective： An assay using Loop-mediated isothermal amplification technology （LAMP） was developed for directly, rapidly detection of bacillus cereus. Method： The specific LAMP primers of Bacillus cereus were designed on the basis of the published sequence of strain hblA and the gene was purified by FTA filter, also FTA-LAMP was used both in bacillus cereus and city milk contaminated with bacillus cereus. Results： Under the optimum reaction conditions, a rapid technique for detection of bacillus cereus was initially established by LAMP. The LAMP assay was found to be 10-fold more sensitive than the traditional PCR assay, detection limit 4.4 cfu/mL for bacillus cereus. The LAMP detection limit was 57 cfu/mL for contaminated city milk. The improved FTA-LAMP assay enables detection of bacillus cereus in less than 2h（contain DNA purify, LAMP and electrophoretic analysis）. Conclusion： LAMP amplification using FTA filters provides a faster and more sensitive method for bacillus cereus detection than the standard cultivation and PCR method and it has higher specificity. A technical platform was established for the rapid detection of bacillus cereus in food.

关 键 词：FTA滤膜 环介导等温扩增 LAMP 链置换酶 消毒乳 蜡样芽孢杆菌 

分 类 号：R155.5[医药卫生—营养与食品卫生学]