建立检测产气肠杆菌的TaqMan荧光定量PCR和普通PCR方法

目的 建立敏感、特异的普通聚合酶链反应(PCR)方法和TaqMan 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TaqMan FAM探针,建立对产气肠杆菌进行检测的普通PCR方法和TaqMan 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TaqMan 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/l和1.0105 cfu/g,TaqMan 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33 copies/l和1.0104 cfu/g;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;在稳定性评价试验中,普通PCR方法的重复性良好,TaqMan 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TaqMan 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。

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

Objective To establish a sensitive and specific conventional PCR assay and a TaqMan 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 TaqMan real time PCR assay. Results The specificities of conventional PCR assay and TaqMan PCR assay were high in detecting E. aerogenes. The sensitivity of conventional PCR assay for standard plasmid and stimulated feces specimens was 100 copies/l and 1.0105 cfu/g respectively. The sensitivity of TaqMan real time PCR assay for standard plasmid and feces simulated specimens were 33 copies/l and 1.0104 cfu/g respectively. Conventional PCR assay had good repeatability. The inter-andintra-CVs of TaqMan real time PCR assay for standard plasmid were 0.15%-0.98% and 0.55~1.63% respectively. Conclusion Conventional PCR assay and TaqMan real time PCR assay in our study were sensitive and specific for the detection of E. aerogenes.