目的 建立TaqMan探针法荧光定量PCR检测方法,用于脑脊液标本细菌的快速检测.方法 针对细菌的16S rRNA基因,设计合成细菌的通用引物和革兰阳性菌、革兰阴性菌分型探针,通过构建质粒和制作脑脊液模拟标本来研究引物和探针的检测特异度和敏感度.结果 两种革兰分型探针只对相应的细菌可以检测到荧光信号,乙肝病毒DNA、白色念珠菌及人基因组DNA检测结果均为阴性,此方法最低可以检测到10个拷贝的质粒DNA以及102CFU/ml的金黄色葡萄球菌和大肠埃希菌.结论 TaqMan探针法荧光定量PCR检测细菌的特异度和敏感度高,检测快速,对脑脊液细菌的早期诊断有重要意义.%Objective To establish TaqMan Probe real-time PCR method for rapid detection of bacteria in cerebrospinal flu id specimens. Methods A pair of universial primers and a set of probe including Gram-positive probe and Gram-negative probe were designed and synthesized based on the 16S rRNA gene of bacteria. The sensitivity and specificity of primers and probes were assessed by constructing plasmid and making cerebrospinal fluid simulated samples. Results Different bacte ria could be detected correctly by Gram-specific probes. The results were allnegative for hepatitis B virus,candida albican and human genomic DNA. The lowest detection limited was 10 copies/Ul of plasmid DNA,Staphylococcus aurens and Es cherichia coli could be detected at concentrations of ≥1O2CFU/ml. Conclusion TaqMan Probe real-time PCR method with high sensitivity and specificity,with rapid detection,has great value in early diagnosis of bacteria in cerebrospinal fluid.
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