CAJ | 학술논문

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砄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。
목적：건립일충CTX-M-38형초엄보β-내선알매（ ESBLs ）적쾌속검측방법。방법：채용매화형쌍향경지확산실험검측매단백여항체반응최괄비；근거쌍항체협심법원리설계매련면역흡부실험（ELISA）검측시제합；평행측정30차BL21-pET-28a-CTX-M-38전화자표체산물，검측비내변이；분별채용소건립적ELISA법여PCR기술대146주산ESBLs대장애희균진행검측，평개방법특이성；대27주비산CTX-M-38형산매대장애희균진행검측，계산가양성솔；대산CTX-M-38형ESBLs균주불동시간단배양액진행검측，명학최괄검측시간。결과：항체여4 h배양물반응최괄비위1결64；비내검측결과변이계수위1．525％；PCR급소건립적ELISA법검측산CTX-M-38형ESBLs균주적검출솔분별위4．11％화7．53％（χ2＝2．286，P＝0．131）；교차반응중존재가양성현상（1/27）；산매균주배양4 h후진행ESBLs검측교위합괄。결론：소건립적방법괄용우림상산CTX-M-38형ESBLs균주적검측。
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 .