医院临床标本中阴沟肠杆菌遗传多样性分析及应用TaqMan荧光定量PCR方法快速检测阴沟肠杆菌的研究（英文）

Genetic diversityof Enterobacter cloacae strains isolated from hospital in China and TaqMan probe real-time polymerase chain reaction for rapid detection of it

目的调查医院临床标本中阴沟肠杆菌基因群的构成比例,并建立TaqMan荧光定量PCR方法对阴沟肠杆菌进行特异、灵敏、快速检测。方法通过hsp60基因分型分析临床标本中基因群的构成,并以外膜蛋白基因(ompX)为靶基因设计引物及FAM探针,建立TaqMan荧光定量PCR方法对阴沟肠杆菌基因群检测,并评价该方法的特异性、灵敏性和稳定性。结果 237株临床标本共归为10个基因群。其中群III,VI和VIII菌株数量最多,占总菌株数的71%;群I占11%;其他6个群总共占18%。无基因群VII,X和XII。TaqMan荧光定量PCR方法能对阴沟肠杆菌不同基因群进行特异检测:对十个基因群和群I的质粒标准品的检测下限分别为36拷贝/μL和21拷贝/μL,对粪便模拟标本的检测下限为104个菌落形成单位/μL;TaqMan荧光定量PCR方法对质粒标准品和粪便模拟标本检测的扩增曲线良好;结论医院临床标本中可以检测到十个基因群,具有遗传多样性的特征。本研究建立的TaqMan荧光PCR方法特异性好、灵敏度高,能够用于阴沟肠杆菌的快速检测。

Enterobacter cloacae are clinically important as nosocomial pathogens. In order to investigate the genetic diversity of the clinical E. cloacae, 237 isolates obtained from routine diagnostic laboratory were examined with analysis of heat shock protein 60 gene （hsp60） sequence. Based on the neighbor-joining tree of the hsp60 gene sequence, ten genetic clusters of E. cloacae could be isolated from the clinical samples. Three genetic clusters （III, VI and VIII） represent almost 71% of the iso- lates; cluster I accounts for 11%; cluster VII, X and XII were absent. The remaining six clusters are minority in our study, which totally accounted for 18% of all strains. Based on out membrane protein X （ompX） gene sequence analysis of 237 strains, two sets of primers and probes were designed which were specific for ten clusters and cluster I respectively. The limit of detections of the assay were 3.6× 10^1 copiesμL for ten clusters and 2.1 × 10^1 copies/μL for cluster I strains within 40 cy- cles. This method was also successfully applied to detect ten clusters and cluster I strains from swab samples, the limit detec- tion for swab samples with inoculated bacteria were 10^4 CFU/mL. In the study, we analyzed the genetic clusters of E. cloacae isolated from hospital setting, and developed a novel real-time polymerase chain reaction method for rapid detection of ten clus- ters and cluster I.