多重PCR和反向线点杂交技术快速检测鲍曼不动杆菌21个主动外排基因及临床应用  

Rapid detection of 21 active efflux genes of Acinetobacter baumannii using multiplex PCR and reverse line blot hybridization and its clinical application

作　　者：金萍[1] 肖克林[2] 熊礼宽[2] 卢月梅[3] 吴劲松[3] 吴丽娟[4] 招悦[1] 刘纯义[1] 

机构地区：[1]深圳市宝安区妇幼保健院儿科重症监护病房,广东深圳518133 [2]深圳市宝安区妇幼保健院中心实验室,广东深圳518133 [3]深圳市人民医院检验科,广东深圳518000 [4]深圳市宝安区妇幼保健院检验科,广东深圳518133

出　　处：《国际检验医学杂志》2013年第12期1489-1493,1496,共6页International Journal of Laboratory Medicine

基　　金：广东省医学科学技术研究基金资助项目(A2010561)

摘　　要：目的建立一种新的多重PCR和反向线点杂交技术(mPCR-RLB)快速同时检测鲍曼不动杆菌21个主动外排基因的方法并评价其临床应用价值。方法根据GenBank中收录的多重耐药鲍曼不动杆菌AYE株5个外排泵蛋白基因家族,选取其中21个基因(4个来自MFS家族,13个来自RND家族,2个来自MATE家族,1个来自SMR家族,1个来自ABC家族)。设计22对引物及寡核苷酸探针(1对鲍曼不动杆菌通用引物及探针),mPCR同时扩增22个目的基因,PCR产物再与固定在尼龙膜上的特异性寡核苷酸探针反向线点杂交,同时检测上述22个基因。用该法对40株经过鉴定的鲍曼不动杆菌进行检测。结果除cmlA5和cmlA基因未检出阳性外,其余19个外排泵基因均呈阳性。除第16号菌株外排泵基因检测阴性外,其余39株鲍曼不动杆菌临床株携带外排泵基因数量从5～18个不等。外排系统在多重耐药菌株及相对敏感菌株中均有分布。11个外排泵基因在多重耐药鲍曼不动杆菌菌株中阳性率明显高于敏感株,差异具有统计学意义(P<0.05)。结论建立的mPCR-RLB技术可快速同时检测鲍曼不动杆菌多个外排泵基因,短时间内明确菌株携带外排泵基因情况,对明确其多重耐药性的发生机制、避免药物成为外排泵对象、找到更有效的特异外排系统抑制剂均有重要意义。Objective To establish a new method for simultaneous rapid detection of 21 active efflux genes of Acinetobacter baumannii by using multiplex PCR and reverse line blot hybridization（mPCR-RLB） and evaluate its clinical application value.Methods 21 genes（4 from MFS family,13 from RND family,2 from MATE family,one from SMR family and one from ABC family） were selected based on GenBank records of 5 efflux pump gene families of multi-drug resistant Acinetobacter baumannii AYE.22 pairs of primers and oligonucleotide probes including a pair of universal Acinetobacter baumannii primers and probes were designed.mPCR was employed to amplify the 22 target genes simultaneously,and then the PCR products were subjected to reverse line blot hybridization with specific oligonucleotide probes which were fixed on nylon membrane.22 genes mentioned above were detected simultaneously.The method was utilized to detect 40 strains of identified Acinetobacter baumannii.Results Except cmlA5 and cmlA gene,19 efflux pump genes were found to be positive.The numbers of efflux pump genes carried by 39 clinical strains of Acinetobacter baumannii were varied from 5 to 18 except the efflux pump gene in the No.16 strain being negative.Efflux system was found both in multi-drug resistant bacterial strains and relatively sensitive bacterial strains.The positive rate of 11 efflux pump genes in multi-drug resistant Acinetobacter baumannii strains was markedly higher than that in sensitive strains with statistically significant difference（P0.05）.Conclusion Established mPCR-RLB technology can be employed to detect numbers of efflux pump genes of Acinetobacter baumannii rapidly and simultaneously,and identify the status of bacterial strain carried efflux pump gene in a short time,which is important for clarifying its multi-drug resistant mechanism,avoiding drug being the target of efflux pump and seeking out more effective inhibitors of specific efflux system.

关 键 词：聚合酶链反应 核酸杂交 鲍氏不动杆菌 多重药物流出泵基因 

分 类 号：R446.5[医药卫生—诊断学]