尿液中大肠埃希菌实时荧光定量聚合酶链反应检测方法的建立

目的建立实时荧光定量聚合酶链反应（RQ—PCR）检测大肠埃希菌的方法，检测泌尿道感染患者尿液样本，评估应用RQ-PCR法在检测大肠埃希菌尿路感染的意义。方法选择大肠埃希菌β-右旋半乳糖苷酶基因作为检测靶基因设计引物，建立SYBRGREENIRQ—PCR检测体系。结果引物特异性好，标准曲线相关系数在0．990～0．996之间。熔解曲线显示产物特异性较强，无非目的条带和二聚体产生。在最低检测限（10^2拷贝／止）以上，能对大肠埃希菌样本进行检测，且重复性好。结论RQ—PCR是一种快速、敏感、特异、重复性好的定量检测大肠埃希菌质粒DNA方法，可用于定量检测临床患者尿液样本中大肠埃希菌含量。

The establishment of real-time fluorescent quantitation polymerase chain reaction for detection of Escherichia coli in urine

Objective To establish a method for detecting Escherichia coli in urine of patients with urinary tract infection by real-time fluorescent quantitation polymerase chain reaction （RQ-PCR） and evaluate the significance of this method. Methods Escherichia coli β-D-galactosidase gene was used to design primers for RQ-PCR. SYBR GREEN I was used in RQ-PCR, Results The specifcity of primer was good, and the correlation coefficient of standard curve was 0. 990-0. 996. Melt-curve analysis showed that the primers were specific, and no nonspecific products such as nonspecific bands and primer-dimers were produced. Escherichia coli could be detected when the minimum detection limit of Escherichia coli was above 102 copies/μL. The repeatability was good. Conclusions RQ- PCR is a fast, sensitive and specific metbod with good repeatability to examine Escherichia coli plasmid DNA, which can be used to detect Escherichia coli level in the urine of the patients with urinary infection.