摘要
为了建立一种快速、准确地检测布氏杆菌S2疫苗株的环介导等温扩增(LAMP)方法,将S2基因组与牛种、羊种野毒株基因组比较,筛选出S2疫苗特有基因trbJ作为检测靶标序列,设计LAMP特异性引物,在对该反应组分、反应条件进行单因素优化的同时,还对该引物特异性、灵敏度、重复性进行测试。结果显示,该方法在65℃等温条件下扩增,反应结束后加入SYBR Green I荧光染料,即可通过肉眼直接观察结果。该方法灵敏性、特异性、重复性良好,能准确鉴别出S2疫苗,与对照菌株无交叉反应。与普通PCR技术相比,LAMP检测灵敏度高100倍,最低可检测出6.89 pg/μL的DNA模板,并且不同批次试剂间的重复性良好。本试验成功建立了一种快速、准确、可视化地检测S2疫苗的LAMP方法。
In order to establish a rapid and accurate loop mediated isothermal amplification method(LAMP) for detection of the Brucella S2 vaccine strain, the S2 genome was compared with that of B.abortus and B.melitensis. The specific Trbj gene of the S2 vaccine was selected as the detection target sequence. LAMP specific primers were designed, and the reaction components and reaction conditions were optimized by single factors. At the same time, the specificity, sensitivity and repeatability of the primers were tested. The results of the method were observed directly by naked eye after adding SYBR Green I fluorescent dye after the reaction was finished. The results showed that the method had good sensitivity, specificity and repeatability and could identify the S2 vaccine accurately, and had no cross reaction with the control strain. Compared with the conventional PCR, the sensitivity of LAMP was 100 times higher, and the minimum DNA template of 6.89 pg/μL could be detected, and the repeatability of different batches of reagents was good. To conclude, a rapid, accurate and visual lamp detection method for the S2 vaccine was successfully established here.
作者
穆嘉明
毛晓伟
石雅琴
秦云
王文龙
MU Jiaming;MAO Xiaowei;SHI Yaqin;QIN Yun;WANG Wenlong(College of Veterinary Medicine,Inner Mongolia Agricultural University/Key Laboratory of Clinical Diagnosis and Treatment Technology for Animal Diseases,Ministry of Agriculture and Rural Affairs,Hohhot 010018,China)
出处
《畜牧与兽医》
北大核心
2021年第11期80-84,共5页
Animal Husbandry & Veterinary Medicine
基金
内蒙古自治区高等学校科学研究项目(NJZZ19041)
内蒙古自治区科技计划项目(20140174,20120244)。
