弗氏枸橼酸杆菌TaqMan实时荧光定量-聚合酶链反应检测方法的建立

Establishment of novel real-time TaqMan PCR assay for detection of Citrobacter freundii

目的建立针对弗氏枸橼酸杆菌的TaqMan实时荧光定量-聚合酶链反应(real time-PCR)检测方法。方法针对弗氏枸橼酸杆菌的特有序列设计引物和TaqMan探针,扩增目的基因建立标准曲线,确定检测方法的灵敏度;对20种其他肠道致病菌及院内感染中常见的致病菌进行检测,评价该检测方法的特异性;使用牛奶模拟标本评价方法在实际检测工作中应用性。结果 TaqMan real time-PCR检测方法对弗氏枸橼酸杆菌重组质粒的检测灵敏度为1.0×101拷贝/反应体系;该检测方法在检测30种其他肠道致病菌及院内感染中常见的致病菌时未出现特异性扩增。该检测方法对牛奶模拟样本中弗氏枸橼酸杆菌检测下限为1.0×102cfu/ml的菌量;通过对1.0×107、1.0×105和1.0×103三个浓度质粒标准品的重复检测,确定本方法的组内变异系数为1.90%～3.91%;组间变异系数为1.52%～1.69%。结论本研究建立的TaqMan real time-PCR检测方法可作为检测弗氏枸橼酸杆菌灵敏、特异、快速的方法。

Objective To establish a real-time TaqMan polymerase chain reaction（PCR） assay for the detection of Citrobacter freundii.Methods Primers and probe were designed based on the sequences of tricarboxylic transport（tct） gene.The target gene was cloned to pMD20-T vector to build the standard curve of this assay and evaluate the sensitivity of the assay.The specificity was evaluated by using 20 other enteropathogenic bacteria and isolates causing nosocomial infection.Results Sensitivity test of recombinant plasmids showed that the sensitivity could reach 1×10^1copies /reaction.Specificity test showed that no specific amplifications were presented for the 20 other enteropathogenic bacteria and the isolates causing nosocomial infection.The detection limit of this assay for artificially contaminated milk was 1.0×10^2cfu/ml.Conclusion This real-time TaqMan PCR assay is sensitive and specific for the rapid detection of Citrobacter freundii.