大肠杆菌β-内酰胺酶耐药基因bla_(TEM),bla_(SHV),bla_(CTX-M)三重PCR检测方法建立  

Development of multiplex PCR for detection of β-Lactamases bla_(TEM), bla_(SHV),bla_(CTX-M) in Escherichia coli

作　　者：田国宝[1] 王红宁[1] 张安云[1] 张毅[1] 杨鑫[1] 徐昌文[1] 

机构地区：[1]四川大学生命科学学院动物疫病防控与食品安全四川省重点实验室生物资源与生态环境教育部重点实验室,四川成都610064

出　　处：《中国兽医杂志》2013年第1期3-5,共3页Chinese Journal of Veterinary Medicine

基　　金：国家"十一五"科技支撑计划"动物产品兽药残留安全控制技术研究"(2006BAK02A19)

摘　　要：本研究以我国大肠杆菌中常见的3种β-内酰胺酶耐药基因(bla,TEMblaSHV和blaCTX-M)作为目的基因,设计3对特异性引物,建立了blaTEM,blaSHV和blaCTX-M耐药基因三重PCR检测方法。三重PCR反应体系为:10×PCR缓冲液2.5μL,MgCl2(25mmol/L)2.5μL,dNTP(2.5mmol/L)4μL,blaTEM,blaSHV和blaCTX-M上下游引物(25μmol/L)分别为0.25μL、0.5μL、1.0μL,Taq DNA聚合酶(2.5U/μL)0.4μL,模板2.5μL,反应总体积为25μL。反应参数为:94℃5min,94℃50s,55℃55s,72℃1min,32个循环,72℃延伸5min。本方法的建立为大肠杆菌中β-内酰胺酶耐药基因的快速检测及分子流行病学调查提供了新的手段。Three β-Lactamase resistance genes （blaTEM, blaSHV and blaCTX-M） were the most common β-lactamase genotypes in China. We chose them as the targets to build a multiplex PCR system in this study. Three pairs of specific primers were designed according to the correlative blaTEM, blaSHV and blaCTX-M gene sequences.from the GenBank. Firstly, a preliminary multiplex PCR system was established base on single gene PCR systems. We then optimized the multiplex PCR system to make it more stable and accurate. The optimized the multiplex PCR system. 10X PCR buffer 2.5 μL, MgC12 （25 mmol/L） 2.5 μL, dNTP（2.5 mmol/L） 4μL, blaTEM, blaSHV and blaCTX-M primers 0. 25 μL,0. 5 μL and 1.0 μL, Taq DNA （2.5 U/μL） 0.4 μL, PCR template 2.5 μL, total Volume 25 μL. To our best knowledge, this is the first time to use a multiplex PCR to detect β-1actamase genes blaTEM , blaSHV and blaCTX-M of Escherichia coli.

关 键 词：大肠杆菌 三重PCR Β-内酰胺酶 耐药基因 

分 类 号：S852.612[农业科学—基础兽医学]