16S rDNA用作荧光定量PCR靶基因快速检测铜绿假单胞菌  

Rapid Detection of Pseudomonas aeruginosa by the Fluorescence Quantitative PCR Assay Targetting 16S rDNA

作　　者：薛利军[1] 王永智[1] 任浩[1] 童一民[1] 赵平[1] 朱诗应[1] 戚中田[1] 

机构地区：[1]第二军医大学微生物学教研室,全军医学微生物学重点实验室,上海200433

出　　处：《生物工程学报》2006年第5期789-794,共6页Chinese Journal of Biotechnology

基　　金：军队"十一五"医学专项课题资助(No.06Z026)。~~

摘　　要：对20余种细菌16SrDNAs进行多序列比对与进化树分析,设计铜绿假单胞菌(Pseudomonasaeruginosa,PA)荧光定量PCR(fluorescencequantitativePCR,FQ-PCR)特异性引物。提取PA基因组DNA,以特异性引物扩增16SrDNA靶片段,并构建重组质粒pMDT-Pfr。将梯度稀释的pMDT-Pfr质粒作为模板,用于建立定量标准曲线。以SYBRGreenI荧光染料建立20μL反应体系,对不同浓度的PADNA样品进行FQ-PCR检测。同时,以金黄色葡萄球菌、伤寒杆菌、福氏志贺菌、变形杆菌、表皮葡萄球菌、大肠杆菌和结核杆菌的基因组DNA作阴性对照,验证FQ-PCR方法检测PA的特异性。结果显示,设计的FQ-PCR引物的靶向序列,仅对PA16SrDNA有高度同源性;FQ-PCR方法检测PA,其灵敏度达3.6pg/μL的基因组DNA或(2.1×103±3.1×102)拷贝/μL的16SrDNA基因,并且具有很强的特异性;从细菌DNA提取到FQ-PCR检测,可在2h左右完成PA鉴定。较传统的培养鉴定法而言,以16SrDNA作为FQ-PCR靶基因快速检测PA,具有很好的研究价值与应用前景。The 16S rDNA specific primers were designed for rapid detection of Pseudomonus aeruginosa （PA） by the fluorescence quantitative PCR （FQ-PCR） assay, based upon multiple sequence alignment and phylogenetic tree analysis of the 16S rDNAs of over 20 bacteria. After extraction of PA genomic DNA, the target 16S rDNA fragment was amplified by PCR with specific primers, and used to construct recombinant pMDT-Pfr plasmid, the dilution gradients of which were subjected to the standard quantitation curve in FQ-PCR assay. Different concentrations of PA genomic DNA were detected by FQ-PCR in a 20μL of reaction system with SYBR Green Ⅰ. At the same time, various genomic DNAs of Staphylococcus aureus, Salmonella typhi, ShigeUa flexneri, Proteus vulgaris, Staphylococcus epidermidis, Escherichia coli, and Mycobacterium tuberculosis were used as negative controls to confirm specificity of the FQ-PCR detection assay. Results demonstrated that the predicted amplified product of designed primers was of high homology only with PA 16S rDNA, and that sensitivity of the FQ-PCR assay was of 3.6pg/μL of bacterial DNA or （2.1 × 10^3 ± 3.1 × 10^2） copies/μL of 16S rDNA, accompanied with high specificity, and that the whole detection process including DNA extraction could be completed in about two hours. In contrast to traditional culture method, the FQ-PCR assay targeting 16S rDNA gene can be used to detect PA rapidly, which exhibits perfect application prospect in future.

关 键 词：铜绿假单胞菌 16S RDNA 荧光定量PCR 检测 

分 类 号：R450[医药卫生—治疗学] Q789[医药卫生—临床医学]