多重PCR联合毛细管电泳技术检测碳青霉烯酶基因  

Detection of carbapenemase genes using multiplex polymerase chain reaction with capillary electrophoresis

作　　者：孙宁 于娟 余柏增 陈勇 曹进 黄红娟 王卫萍 张立平 李晓军 SUN Ning;YU Juan;YU Baizeng;CHEN Yong;CAO Jin;HUANG Hongjuan;WANG Weiping;ZHANG Liping;LI Xiaojun(Department of Clinical Laboratory, General Hospital of Eastern Theater Command, PLA, Nanjing 210002, Jiangsu;Department of Clinical Laboratory, Lishui People′s Hospital, Nanjing 211200, Jiangsu;State Key Laboratory of Life Analytical Chemistry, Nanjing University, Nanjing 210002, Jiangsu, China)

机构地区：[1]东部战区总医院基础医学实验室,南京210002 [2]南京市溧水区人民医院检验科,南京211200 [3]南京大学生命分析化学国家重点实验室,南京210002

出　　处：《临床检验杂志》2021年第5期340-345,共6页Chinese Journal of Clinical Laboratory Science

基　　金：国家临床重点专科军队建设项目(2014ZDZK003);国家自然科学基金(81601857);南京市卫生健康委项目(YKK18214、YKK18216)。

摘　　要：目的建立并评价多重PCR联合毛细管电泳技术(mPCR-CE)同时检测碳青霉烯酶基因bla_(KPC)、bla_(NDM)、bla_(OXA-48)和bla_(VIM)的方法。方法设计特异性引物,建立多重PCR扩增体系,利用毛细管电泳检测扩增产物。以携带bla_(KPC)、bla_(NDM)、bla_(OXA-48)和bla_(VIM)的细菌作为阳性对照菌株,以携带其他β-内酰胺酶基因细菌作为阴性对照,用于mPCR-CE的灵敏度、特异性评价。收集68株碳青霉烯耐药菌株,利用mPCR-CE方法进行检测,并与PCR扩增后测序结果比较。结果mPCR-CE检测携带bla_(KPC)、bla_(NDM)和bla_(VIM)细菌最低检测限均为1.5×10^(2) CFU/mL,检测携带bla_(OXA-48)细菌为1.5×10^(3) CFU/mL,并且与携带其他β-内酰胺酶基因的细菌无交叉反应。经mPCR-CE检测,68株临床分离菌株中37株携带bla_(KPC),8株携带bla_(NDM),未检测到携带bla_(OXA-48)和bla_(VIM)的菌株,结果与PCR扩增后测序结果完全一致。结论mPCR-CE可用于临床分离菌株的主要碳青霉烯酶基因检测。Objective To establish and evaluate a multiplex PCR combined with capillary electrophoresis(mPCR-CE)in simultaneous detection for bla_(KPC),bla_(NDM),bla_(OXA-48) and bla_(VIM) of carbapenemase gene.Methods Specific primers were designed and multiplex PCR amplification system was optimized.The products of PCR were determined by capillary electrophoresis.The isolates carrying bla_(KPC),bla_(NDM),bla_(OXA-48) and bla_(VIM) as positive controls and the isolates carrying otherβ-lactamase genes were used as negative controls,and the specificity and sensitivity of mPCR-CE were evaluated.A total of 68 clinical isolates were collected and detected by mPCR-CE,and then the results were confirmed by Sanger sequencing after PCR.Results The minimum detection limit of mPCR-CE was 1.5×10^(2) CFU/mL for the isolates harboring bla_(KPC),bla_(NDM) or bla_(VIM),while 1.5×10^(3) CFU/mL for the isolates carrying bla_(OXA-48).There was no cross-reaction with other isolates carryingβ-lactamase genes.Among the 68 clinical isolates detected by mPCR-CE,37 carried bla_(KPC) and 8 carried bla_(NDM).No bla_(OXA-48) and bla_(VIM) was detected.The results were consistent with the sequencing after PCR amplification.Conclusion mPCR-CE assay can be effectively used to detect the major carbapenemase genes in clinical isolates.

关 键 词：碳青霉烯酶基因 多重PCR 毛细管电泳 

分 类 号：R446.5[医药卫生—诊断学]