建立SYBR Green实时RT-PCR定量方法检测铜绿假单胞菌MexAB-OprM mRNA水平  

SYBR Green Ⅰ-based Real-time Fluorescent Quantitative RT-PCR Method for Detecting mRNA Level of MexAB-OprM in Pseudomonas aeruginosa

作　　者：吴春明[1] 李洪涛[1] 覃慧敏[1] 李国军[1] 宋建新[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院,湖北武汉430030

出　　处：《中华医院感染学杂志》2007年第11期1349-1352,共4页Chinese Journal of Nosocomiology

基　　金：国家自然科学基金(30571647)

摘　　要：目的建立检测铜绿假单胞菌MexAB-OprMmRNA含量的实时荧光定量逆转录（RT-）PCR方法，并用其测定铜绿假单胞菌PAE01株和MexAB-OprM过度表达株mexAB-oprM的基因表达水平。方法基于SYBRGreenI荧光探针技术，构建克隆载体pMDl8-T-mexB和pMDl8-T-rpsL作为标准品，建立实时荧光定量RT-PCR方法；在Rotor-gene3000型检测仪上测定了PAE01株和MexAB-OprM过度表达株的MexAB-OprMmRNA的表达量。结果所建立的实时RT-PCR方法的mexB、rpsL在（10^2～10^9）拷贝／ml、（10^4～10^9）拷贝／ml范围内，Ct值与起始模板浓度具有良好的线性关系，二者相关系数分别为0．99977和0．99999；MexAB-OprM过度表达株的mexB相对含量显著高于PAE01株（P〈0．01）。结论成功建立了检测铜绿假单胞菌mexAB-oprM基因表达含量的荧光定量方法，MexAB-OprM过度表达株的MexAB-OprMmRNA含量明显高于PAE01株。OBJECTIVE To establish a real-time fluorescent qugntitative reverse transcription-polymerase chain reaction （RT-PCR） method for detecting MexAB-OprM mRNA in Pseudornonas aeruginosa and measure the expression level of mexAB-oprM gene in PAE 01 strain and MexAB-OprM overexpressing strain. METHODS Based on fluorescent SYBR Green Ⅰ methodology, a real-time fluorescent quantitative RT-PCR was set up. In this method, two cloning vectors pMD18-T-mexB and pMD18-T-rpsL were constructed as standard plasmids. The expression of MexAB-OprM mRNA in PAE 01 strain and MexAB-OprM overexpressing strain were measured by using Rotor-gene 3 000 fluorescent quantitative PCR instrument. RESULTS Under the range of 102-109 copies/ml of mexB and 104-109 copies/ml of rpsL, the standard curves of pMD18-T-mexB and pMD18-T-rpsL indicated the linear relationship between cycle threshold CT and template concentration. And their correlation coefficients were 0. 99977 and 0. 99999, respectively. The relative mexB mRNA expression in MexAB OprM overexpressing strain was significantly higher than that in PAE 01 strain （P 〈 0. 01）. CONCLUSIONS A real-time fluorescent quantitative RT-PCR method for detecting the expression of MexAB-OprM mRNA in P. aeruginosa has been successfully established. The expression level of mexAB-oprM is increased in MexAB-OprM overexpressing strain.

关 键 词：铜绿假单胞菌 聚合酶链反应 mRNA 

分 类 号：R446.1[医药卫生—诊断学]