碳青霉烯类和喹诺酮类双重耐药核酸胶体金试纸条快速检测方法的建立及试剂盒研制  

Establishment of a rapid detection method for carbapenem and quinolone resistant nucleic acid colloidal gold test strips and development of a reagent kit

作　　者：潘蓓珍 杨继飞 王岳峰 刘岩 周钰娇 马玉贺 孙丽媛 PAN Beizhen;YANG Jifei;WANG Yuefeng;LIU Yan;ZHOU Yujiao;MA Yuhe;SUN Liyuan(Department of Clinical Pathogen Laboratory,School of Medical Technology,Beihua University,Jilin 132013,China)

机构地区：[1]北华大学医学技术学院临床病原学检验教研室,吉林132013

出　　处：《中国免疫学杂志》2024年第11期2386-2390,2398,共6页Chinese Journal of Immunology

基　　金：吉林省科技发展计划项目(20230204085YY);北华大学研究生创新计划项目([2022]027)。

摘　　要：目的:建立双重核酸胶体金试纸条快速检测鲍曼不动杆菌(Ab)OXA和par C耐药基因的方法并研制试剂盒。方法:采用加热煮沸法提取Ab的DNA,根据NCBI选择Ab的OXA和par C基因序列作为靶基因片段,设计引物并分别用6-FAM、地高辛和生物素进行标记,研发耐药基因检测试剂,采用双重核酸胶体金试纸条实现快速、可视化检测。采用分子克隆、测序技术克隆阳性对照品评价试剂盒的特异性、灵敏度和稳定性。结果:水煮法提取的Ab DNA浓度和纯度较好。克隆测序后的质粒DNA与GenBank数据库中基因序列的同源性均为100%;试剂盒的特异性良好,仅Ab出现阳性,其他菌属等均为阴性;双重核酸胶体金试纸条中Ab的DNA浓度降到10-3ng/μl时仍出现红色线,与电泳的最低检测限10-2ng/μl相比,灵敏度高出10倍;试剂盒分别在第3、6和9个月进行检测,稳定性较好。结论:本研究建立的Ab双重耐药检测试剂盒可同时检测Ab的OXA和par C耐药基因,具有高灵敏度、强特异性、快速简便等优点,为临床Ab碳青霉烯类和喹诺酮类抗生素耐药检测提供了一种新型快速的检测方法。Objective:To establish a method for rapid detection of OXA and par C resistance genes of Acinetobacter baumannii(Ab)by double nucleic acid colloidal gold strip and to develop kit.Methods:DNA of Ab was extracted by heating and boiling method.OXA and par C genes sequences of Ab were selected as target gene fragments based on NCBI.Primers were designed and labeled with 6-FAM,digoxin and biotin,respectively.Drug resistance gene detection reagents were developed,and nucleic acid gold test strips were used for rapid and visual detection.Molecular cloning and sequencing techniques were used to clone positive control samples and evaluate specificity,sensitivity and stability of kit.Results:DNA concentration and purity of Ab extracted by boiling method were good.Homology between cloned and sequenced plasmid DNA and gene sequence in GenBank database was 100%,respectively.Speci-ficity of kit was good,with only Ab showing positive results and other bacterial genera showing negative results;DNA concentration of Ab in double nucleic acid colloidal gold test strip decreased to 10-3 ng/μl,a red line still appeared,whose sensitivity was 10 times higher consistent with minimum detection limit of electrophoresis 10-2 ng/μl;test kits were tested at 3rd,6th and 9th months,and showed good stability.Conclusion:Double resistance detection kit established in this study can simultaneously detect OXA and par C resis-tance of Ab,who has advantages of high sensitivity,strong specificity,rapid and simple,and provides a new method for detection of carbapenem and quinolone antibiotic resistance of Ab.

关 键 词：鲍曼不动杆菌 OXA par C 分子克隆 胶体金 核酸试纸条 

分 类 号：R378[医药卫生—病原生物学]