荧光实时定量PCR检测铜绿假单胞菌外膜蛋白oprI基因的标准品的构建  

Construction of Reference Standards for Detecting oprI Gene of Pseudomonas Aeruginosa With Fluorescence Real-time Quantitative PCR

作　　者：钟礼立[1] 张兵[1] 贺蓉[1] 林小娟[1] 张爱民[1] 蔡瑞云[1] 段贞[1] 

机构地区：[1]湖南省人民医院,湖南长沙410005

出　　处：《实用预防医学》2006年第2期265-268,共4页Practical Preventive Medicine

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。Objective To construct the reference standards for detecting the oprl gene of Pseudomonas aeruginosa used in fluorescence real - time quantitative PCR and application in quantification of bacterial burden. Methods Primers and probe were designed based on the major outer rnembranelipoprotein 1 （oprl） gene specific for Pseudomonas aeruginosa.Bacterial genomic DNA was isolated from typical Pseudomonas aeruginosa strain, linked with pMD 18 - T Vector to construct recombined plasmids and transfected into Escherichia coli. Target plasmids in white colonies selected by ampicillin screening were liuearrizated by HindⅢ restrictive enzyme and had their specificity identified by direct PCR, OD value and DNA sequencing, and the concentration was identified by OD value. The copy numbers were calculated by value of OD in A260 and then diluted into serial gradient concentrations as standard to plot a standard curve for FQ-PCR used to calculate the cultivated copy numbers Pseudomonas aeruginosa and E. coli. Results Satisfactorily prepared the target fragment of oprl gene of Pseudomonas aeruginosa and constructed the recombinant plasmid containing this target fragment which was stable and kept its sequential integrity and specificity. The correlation coefficient of the standard curve reached 0. 994. The copy numbers of type and culture strains of pseudomonas aeruginosa were calculated, and neither E. coli nor negative control copies were misinterpreted as false positive, Conclusion Reference standards for FQ PCR are constructed satisfactorily and FQ - PCR can be applied to accelerate the detection of Pseudomonas aeruginosa bacterial burden.

关 键 词：铜绿假单胞菌 oprI基因 荧光实时定量PCR 标准品 

分 类 号：R378.991[医药卫生—病原生物学]