摘要
利用DNA环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP),以rfbE基因为靶序列,设计LAMP引物,建立LAMP反应体系与条件,进行灵敏度和特异性试验。结果显示,整个检测过程仅需1h即可通过肉眼直接目测试验结果。在灵敏度试验中,rfbE-LAMP对O157菌液和模拟阳性样品的最低检测限分别为1CFU/mL和1CFU/g。在特异性试验中,以其他血清型大肠杆菌以及沙门杆菌基因组DNA为模板对rfbE基因进行检测,均没有发生非特异性扩增反应。试验建立的LAMP检测方法具有操作简单安全、检测灵敏度高、特异性高的特点,能够满足基层实验室、应急检测或现场监测等方面的使用需求。
We established a rapid detection method for E. coli O157 based on loop-mediated iso- thermal amplification （LAMP）. A set of LAMP primers was designed, using rfbE gene as the tar- get sequence. Meanwhile, we established optimal reaction systems and conditions to carry out the sensitivity and specificity experiments. The experiment results showed that the whole detection process took only one hour and could be observed visually. In the experiment of sensitivity,rfbE- LAMP had a detection limit of 1 CFU/mL and 1 CFU/g to bacterial culture and prepared sam- ples. In the experiment of specificity, we detected rfbE gene in other E. coli and Salmonella and did not amplify the bands. The method established in this study could rapidly detected rfbE gene and visualize the result. The rfbE-LAMP is easy to operate and has high sensitivity and specificity and thus has great application value in basic research laboratories, emergent detection and spot detection.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2013年第7期1027-1031,共5页
Chinese Journal of Veterinary Science
基金
国家科技支撑计划资助项目(2012BAK08B07)
江苏省农业支撑计划-社会发展项目(BE2011771)
江苏省自然科学基金资助项目(BK2010068)
关键词
环介导恒温扩增技术
rfbE基因
快速检测
loop-mediated isothermal amplification （LAMP）
rfbE gene
rapid detection
