CTX-M-38型超广谱β-内酰胺酶快速检测方法的建立  

Establishment of a quick detection method of CTX-M-38 type ESBLs

作　　者：郭小兵[1] 代志峰[1] 宋海容[1] 张傅山[1] 叶亚菲[1] 明亮[1] 

机构地区：[1]郑州大学第一附属医院检验科,郑州450052

出　　处：《郑州大学学报（医学版）》2014年第1期66-69,共4页Journal of Zhengzhou University(Medical Sciences)

基　　金：河南省医学科技攻关项目2011020014

摘　　要：目的:建立一种CTX-M-38型超广谱β-内酰胺酶(ESBLs)的快速检测方法。方法:采用梅花形双向琼脂扩散实验检测酶蛋白与抗体反应最适比;根据双抗体夹心法原理设计酶联免疫吸附实验(ELISA)检测试剂盒;平行测定30次BL21-pET-28a-CTX-M-38转化子表达产物,检测批内变异;分别采用所建立的ELISA法与PCR技术对146株产ESBLs大肠埃希菌进行检测,评价方法特异性;对27株非产CTX-M-38型产酶大肠埃希菌进行检测,计算假阳性率;对产CTX-M-38型ESBLs菌株不同时间段培养液进行检测,明确最适检测时间。结果:抗体与4 h培养物反应最适比为1∶64;批内检测结果变异系数为1.525%;PCR及所建立的ELISA法检测产CTX-M-38型ESBLs菌株的检出率分别为4.11%和7.53%(χ2=2.286,P=0.131);交叉反应中存在假阳性现象(1/27);产酶菌株培养4 h后进行ESBLs检测较为合适。结论:所建立的方法适用于临床产CTX-M-38型ESBLs菌株的检测。Aim：To establish a quick detection method of CTX-M-38 type ESBLs .Methods： The reaction optimal ratio between CTX-M-38 type ESBLs and polyclonal antibody was confirmed by double agar diffusion test .The ELISA kit was designed according to double-antibody sandwich principle .The products of CTX-M-38 transformant were detected 30 times to obtain the information of experimental repeatability and intraassay variation ;146 ESBLs-producing E.coli strains were detected by PCR and the designed method respectively to evaluate the experimental specificity ;27 isolates of ESBLs-producing E.coli strains（not carrying CTX-M-38 type ESBLs） were detected to evaluate the experimental cross-reactivity;the culture medium of CTX-M-38 type ESBLs-producing E.coli was detected at various time to confirm the optimal detective time of ESBLs.Results：The reaction optimal ratio between CTX-M-38 type ESBLs and polyclonal antibody was 1：64;the results of the parallel testing indicated that the CV was 1.525%;the detection rate of PCR and ELISA was 4.11%and 7.53%（χ2 =2.286,P=0.131）;the designed ELISA might have the phenomenon of false-positivity（1/27）; the optimal detection time for ESBLs was being cultured for 4 h.Conclusion：The established ELISA can meet the need for detecting CTX-M-38-type ESBLs-producing strains .

关 键 词：CTX-M-38 超广谱Β-内酰胺酶 酶联免疫吸附实验 CTX-M-38 

分 类 号：R392[医药卫生—免疫学]