环介导恒温扩增法快速检测短小芽孢杆菌  

Establishment of Loop-mediated Isothermal Amplification Method for Detection of Bacillus pumilus

作　　者：应琪[1] 李太武[1,2] 苏秀榕[1] 李晔[1] 周君[1] 崔静[1] 王中华[1] 李松伟[1] 

机构地区：[1]宁波大学生命科学与生物工程学院,宁波315211 [2]宁波城市职业技术学院,宁波315110

出　　处：《生物技术通报》2011年第2期179-183,共5页Biotechnology Bulletin

基　　金：海洋公益性行业科研专项(2011418007);宁波大学研究生科研创新基金项目(NG09JLA024)

摘　　要：短小芽孢杆菌(Bacillus pumilus)是一种能引起食源性疾病的腐败菌,对其进行快速检测具有重要意义。针对短小芽孢杆菌木聚糖(xynA)基因,设计了4条特异性引物(两条内引物和两条外引物),通过条件优化,首次将一种新颖的核酸扩增技术——环介导恒温扩增技术应用于短小芽孢杆菌的快速检测。采用该技术,63℃温育1h的条件下扩增短小芽孢杆菌DNA,琼脂糖凝胶电泳得到特异性梯度条带。PCR和LAMP的检测灵敏度分别约为162和16.2拷贝每反应。结果表明,该方法检测短小芽孢杆菌特异性强、灵敏度高、操作简便、检测成本低,1h即可完成,有望发展成为快速检测短小芽孢杆菌的有效手段。Bacillus pumilus has been considered as one of the most important spoilage bacterial which also causes foodborne illness.A set of four primers,two outer and two inner primers,was designed specifically to recognize the xylanase（xynA） gene of B.pumilus.After the optimization of loop-mediated isothermal amplification（LAMP） reaction mix,the LAMP method was applied for rapid detection of B.pumilus for the first time by incubation at 63℃ for only 1 h.LAMP amplification products had a ladder-like appearance when electrophoresed on an agarose gel.The detection limit of the LAMP assay was 16.2 copies,which was found to be higher than the commonly used PCR method.These results suggested that detection of B.pumilus by LAMP is an effective and low-cost procedure with high specificity and sensitivity that requires no specialized equipment.This assay is expected to become a valuable tool for rapid detection and identification of B.pumilus.

关 键 词：短小芽孢杆菌 环介导恒温扩增技术(LAMP) 木聚糖(xynA)基因 快速检测 

分 类 号：TS254.7[轻工技术与工程—水产品加工及贮藏工程]