【摘要】 目的构建荧光实时定量PCR(FQ-PCR)标准品测定铜绿假单胞菌oprI基因,以此测定铜绿假单胞菌菌量。方法以铜绿假单胞菌的oprI基因为目的基因设计探针引物,提取细菌基因组DNA,与pMD 18-T Vector连接并转化到大肠杆菌中。用氨卞青霉素筛选出白色菌落,提取含目的基因质粒,并用HindⅢ限制酶进行线性化处理,通过直接PCR、OD值测定及DNA片段测序鉴定其特异性。根据OD值确定浓度,制备FQ-PCR梯度浓度参考标准品,做出标准曲线,检测各样品菌菌量。结果铜绿假单胞菌oprI基因的目的片段成功制备,获得稳定的重组质粒,保持了目的片段的特异性和序列完整性,并获得很好的标准曲线(相关系数0.994),成功检测了铜绿假单胞菌标准菌株、培养株的菌量,而大肠杆菌及阴性对照无扩增。结论成功构建FQ-PCR检测铜绿假单胞菌oprI基因的定量参考标准,荧光实时定量法可以快速检测铜绿假单胞菌。更多还原

【Abstract】 Objective To construct the reference standards for detecting the oprI gene of Pseudomonas aeruginosausedin fluorescence real-ti me quantitative PCR and applicationin quantification of bacterial burden.Methods Pri mers andprobe were designed based on the major outer membranelipoprotein I(oprI) gene specific for Pseudomonas aeruginosa.Bacterial genomic DNA was isolated fromtypical Pseudomonas aeruginosa strain,linked with pMD18-T Vector to constructrecombined plasmids and transfected into Escherichia coli.Target plasmids in white colonies selected by ampicillin screeningwere linearrizated by HindⅢrestrictive enzyme and hadtheir specificityidentified by direct PCR,OD value and DNA sequenc-ing,andthe concentration was identified by OD value.The copy numbers were calculated by value of ODin A260andthen di-luted into serial gradient concentrations as standardto plot a standard curvefor FQ-PCR usedto calculatethe cultivated copynumbers Pseudomonas aeruginosa and E.coli.Results Satisfactorily prepared the target fragment of oprI gene of Pseu-domonas aeruginosa and constructedthe recombinant plasmid containingthis target fragment which was stable and kept its se-quential integrity and specificity.The correlation coefficient of the standard curvereached0.994.The copy numbers of type andculture strains of pseudomonas aeruginosa were calculated,and neither E.coli nor negative control copies were misinterpretedas false positive.Conclusion Reference standards for FQ-PCR are constructed satisfactorily and FQ-PCR can be ap-plied to accelerate the detection of Pseudomonas aeruginosa bacterial burden.更多还原