建立检测产气肠杆菌的TaqMan荧光定量PCR和普通PCR方法  

Establishing conventional PCR and TaqMan real time PCR assays for detection of Enterobacter aerogenes

作　　者：王怡倩[1] 叶长芸[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感染性疾病诊治协同创新中心,北京102206

出　　处：《疾病监测》2016年第2期153-158,共6页Disease Surveillance

基　　金：国家科技重大专项(No.2013ZX10004-101)~~

摘　　要：目的建立敏感、特异的普通聚合酶链反应（PCR）方法和Taq Man荧光定量PCR方法对产气肠杆菌进行快速检测。方法以产气肠杆菌组氨酸脱氢酶基因（hdc）为靶基因设计引物以及Taq Man FAM探针,建立对产气肠杆菌进行检测的普通PCR方法和Taq Man荧光定量PCR方法,并评价该方法的特异性、灵敏性和稳定性。结果普通PCR和Taq Man荧光定量PCR方法均能对产气肠杆菌进行特异检测;普通PCR方法对质粒标准品和粪便模拟标本的检测下限分别为100 copies/μl和1.0×105cfu/g,Taq Man荧光定量PCR方法对质粒标准品和粪便模拟标本的检测下限分别为33copies/μl和1.0×104cfu/g;Taq Man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;在稳定性评价试验中,普通PCR方法的重复性良好,Taq Man荧光定量PCR方法对质粒标准品检测Ct值的组内差异为0.15%~0.98%,组间差异为0.55%~1.63%。结论本研究建立的检测产气肠杆菌的普通PCR方法和Taq Man荧光PCR方法特异性好、灵敏度高,能够用于产气肠杆菌的快速检测。Objective To establish a sensitive and specific conventional PCR assay and a Taq M an real time PCR assay for the detection of Enterobacter aerogenes. Methods The primers and probe were designed according to the public sequences of hdc gene coding histidine decarboxylase on NCBI. The specificity was evaluated by using 30 other genus and species strains.The plasmid harboring hdc gene and feces simulated specimens were used to evaluate the sensitivity of the conventional PCR assay and Taq M an real time PCR assay. Results The specificities of conventional PCR assay and Taq M an PCR assay were high in detecting E. aerogenes. The sensitivity of conventional PCR assay for standard plasmid and stimulated feces specimens was 100 copies / μl and 1. 0 × 105 cfu / g respectively. The sensitivity of Taq M an real time PCR assay for standard plasmid and feces simulated specimens were 33 copies / μl and 1. 0 × 104 cfu / g respectively. Conventional PCR assay had good repeatability. The inter-andintra-CVs of Taq M an real time PCR assay for standard plasmid were 0. 15%- 0. 98% and 0. 55 ~1. 63% respectively. Conclusion Conventional PCR assay and Taq M an real time PCR assay in our study were sensitive and specific for the detection of E. aerogenes.

关 键 词：产气肠杆菌 普通PCR TaqMan荧光定量PCR 

分 类 号：R440[医药卫生—诊断学]