荧光实时定量PCR检测铜绿假单胞菌方法建立及价值  

Development and evaluation of fluorescence real-time quantitative PCR for detecting pseudomonas aeruginosa

作　　者：徐璇[1] 钟礼立[2] 张兵[2] 蔡瑞云[3] 段贞[3] 林小娟[2] 张爱民[2] 

机构地区：[1]中南大学湘雅三医院,湖南长沙410013 [2]湖南省人民医院儿科 [3]湖南省人民医院检验科

出　　处：《中国医师杂志》2006年第12期1614-1617,共4页Journal of Chinese Physician

摘　　要：目的建立荧光实时定量PCR(FQ-PCR)定量测定铜绿假单胞菌的方法,检测该方法的灵敏度并与传统细菌培养比较其特异性吻合率。方法选择铜绿假单胞菌的oprI基因为目的基因设计探针引物,建立Taqm an探针FQ-PCR体系定量检测5种标准菌株、3种病毒株、白色念珠菌、肺炎支原体、人基因组DNA及临床培养96株菌株。结果31株铜绿假单胞菌、铜绿假单胞菌与其它菌种或人全血的混合液均产生特异扩增信号,标准曲线的相关系数为0.997;其他菌种(76株)无扩增;乙肝病毒、巨细胞病毒及肺炎支原体及健康人全血均无扩增,与传统培养相比特异性吻合率达100%,铜绿假单胞菌标准株按等级浓度稀释后其拷贝数也呈等级梯度递减,实际可检测到的最小菌落数为100个/m l,全程检测<4 h。结论荧光实时定量法检测oprI基因可以运用于铜绿假单胞菌的快速检测。Objective To establish a fluorescent real-time quantitative PCR（FQ-PCR） assay to rapidly detect pseudomonas aeruginosa and to estimate the sensitivity and specificity of this method. Methods Primers and probe were designed based on the major outer membrane lipoprotein I （oprI） gene which is specific for pseudomonas aeruginosa. 5 standard bacteria strains, 3 different viruses, mycoplasma pneumoniae, fungus, human genomic DNA, and 96 strains of cultured bacteria were tested by FQ-PCR assay. Results 31 strains of pseudomonas aeruginosa and pseudomonas aeruginosa mixed with other strains or human blood were all amplified specifically by FQ-PCR, and the correlation coefficient of the standard curve reached as high as 0. 997. No cross-reaction was found with human genomic DNA, other bacteria, viruses, mycoplasma pneumoniae and fungus. Compared with traditional bacterial culture, the specificity of FQ-PCR was 100%. In addition, the FQ-PCR showed very high sensitivity and was able to detect at least 10 copies of oprI gene. The clinical minimum concentration which can be detected wasl00cfu/ml. The number of copies descended along with the dilution of the pseudomonas aeruginosa. The whole detection time was less than 4 hours. Conclusion FQ-PCR characterized by its rapidity, sensitivity and specificity might have great value in detecting bacterial burden of pseudomonas aeruginosa.

关 键 词：聚合酶链反应/方法 假单胞菌 铜绿/分离和提纯 

分 类 号：R450[医药卫生—治疗学]