布鲁菌环介导等温扩增检测试剂盒的研制  

Development of Loop-mediated Isothermal Amplification Kit for Detection of Brucella

作　　者：李秀梅[1] 郭恋[1] 梁智选[1] 李颖[1] 郭立力[1] 石瑜[1] 朱雅宁[1] 王红军 

机构地区：[1]天津市动物疫病预防控制中心,天津300402 [2]天津市畜牧业发展服务中心,天津300384

出　　处：《动物医学进展》2014年第7期6-10,共5页Progress In Veterinary Medicine

基　　金：国家高技术研究发展计划(863计划)项目(2012AA101605);天津市科技支撑计划重点项目(10ZCZDNC02800)

摘　　要：利用环介导等温扩增(LAMP)技术建立布鲁菌的检测方法。通过对反应条件的优化,组装布鲁菌LAMP检测试剂盒,并对试剂盒的各项性能进行评价。结果显示,该试剂盒适用于布鲁菌的快速检测,在63℃条件下1h内可检出布鲁菌,其检测限达5copies/μL,特异性为100%,无假阳性和假阴性出现。反复冻融20次或-20℃下贮存1年,均不影响试剂盒的使用效果。对牛奶样品的检测结果显示,阳性检出率为3.33%,与奶牛布鲁菌病PCR诊断技术(NY/T1467-2007)的检测结果相符。该试剂盒具有灵敏度高、特异性强和稳定性好等特点,可满足大批量样品快速筛选的要求。A loop-mediated isothermal amplification（LAMP）method for detection of Brucella was established.By optimization of the reaction conditions of LAMP,LAMP kit was assembled,meanwhile various performance were evaluated in this paper.The results showed that the kit was available to rapidly detect Brucella.The Brucellacould be detected by the kit within 1hour at constant temperature of 63℃.Limit of detection was at the level of 5copies/μL and the specificity of the approach was 100% without any false negative or positive action.Besides,the kit worked well after being frozen-thawed for 20 times or stored at-20℃for one year.The milk samples of the cows with brucellosis were detected with the developed method and PCR method,and the results showed that the coincidence rate of these two methods was100%,postive detection rate was 3.33%.The detection kit developed in this study has good performances in specificity,sensiticity and stability etc and is suitable for the rapid screening of mass samples.

关 键 词：布鲁菌 环介导等温扩增 检测试剂盒 Omp25基因 

分 类 号：S852.614[农业科学—基础兽医学]