屎肠球菌TaqMan荧光定量PCR检测方法的研究  

TaqMan real-time PCR detection of Enterococcus faecium

作　　者：金东[1] 于波[1] 叶长芸[1] 刘凯[1] 侯雪新[1] 孙渭歌[1] 徐建国[1] 李振军[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所,传染病预防控制国家重点实验室,北京102206

出　　处：《中国人兽共患病学报》2013年第6期551-555,共5页Chinese Journal of Zoonoses

基　　金：国家科技重大专项(No.2011ZX10004-001,No.2012ZX10004-501);卫生科研行业专项(No.201302006)联合资助~~

摘　　要：目的建立屎肠球菌的TaqMan荧光定量PCR检测方法。方法根据屎肠球菌的ddl基因设计TaqMan荧光定量PCR特异的引物及探针,在多种常见致病菌及条件致病菌中检测其特异性;将目的基因克隆到pMD18-T载体中建立标准曲线,检测方法的灵敏度和稳定性;使用腹水模拟标本验证方法的应用性。结果在特异性评价中,除了阳性对照外其余菌株均未见特异性扩增曲线。建立屎肠球菌TaqMan荧光定量PCR方法的标准曲线,确定本方法对质粒标准品的检测下限为20copy/管。通过对1.0×107、1.0×105和1.0×1033个浓度质粒标准品的重复检测,确定屎肠球菌TaqMan荧光定量PCR方法的组内变异系数为1.90%～3.91%;组间变异系数为1.52%～1.69%。应用屎肠球菌TaqMan荧光定量PCR方法对含菌量为1.6×100～1.6×108cfu/mL浓度梯度的腹水模拟标本进行检测,其检测灵敏度为1.6×102cfu/mL。结论本研究建立了屎肠球菌TaqMan荧光定量PCR检测方法。To establish TaqMan real-time PCR assay of Enterococcus faecium and provide reliable technical basis for clinical application, we designed the primers and probe of Enterococcus faecium according to ddl gene and detected the specific ity in common pathogens and conditioned pathogens. The target gene was cloned to pMD18-T vector to build the standard curve of this method. We made the ascites simulation specimens with the concentrations from 1.6 X 100 1.6 X 108 cfu/mL by 10 se-ries dilutions of Enterococcus faecium strain and mixing well with sterile ascites to evaluate the application in clinical lab. The target gene was positive in Enterococcus faecium standard strain and clinical isolates but negative in other pathogens and condi- tioned pathogens by TaqMan real time PCR assay. According to the standard curve, we acquired the sensitivity of this method was 20 copy/reaction. The stability evaluation indicated that the inter and intra-assay coefficients of variability were1. 52^- 1.69~/oo and 1. 90%--3.91%, respectively. The sensitivity of TaqMan real-time PCR assay of the ascites simulation specimens was 1.6 X 10^2cfu/mL. In conclusion, the TaqMan real time PCR assay developed in our study could be used in the detection of Enterococcus faecium in ascites specimens.

关 键 词：屎肠球菌 TaqMan荧光定量PCR 标准曲线 

分 类 号：R378.1[医药卫生—病原生物学]