【摘要】 目的建立FQ-PCR(real-time Fluorescent Quantitative PCR)系统定量检测新型隐球菌CAP10基因,为将其应用于新型隐球菌感染的诊断和疗效判断奠定基础。方法根据新型隐球菌5种血清型(A、B、C、D、AD)中同源序列设计引物和探针,PCR扩增CAP10基因片段,并将其克隆到pGEM-T Easy载体上,构建质粒标准品;建立FQ-PCR体系,优化反应条件,建立标准曲线,进行特异性、敏感性、重复性实验。结果FQ-PCR系统用于检测CAP10基因片段,敏感性为1拷贝数/μl,重复性好,批内变异系数(CV)为1.36%,批间变异系数(CV)为1.86%,特异性好,对临床其他常见脑膜炎病原体不出现特异性扩增曲线。结论成功建立了新型隐球菌CAP10基因FQ-PCR检测方法,结果稳定、准确,有望为判断新型隐球菌性脑膜炎疗效、预后提供新的依据。更多还原

【Abstract】 Objective Cryptococcus neoformans CAP10 gene detection was developed using real-time Fluorescent Quantitative PCR technique with the objective of its preliminary application in the clinical laboratory diagnosis and therapeutic effect judgment.Methods According to the constant sequence of five serological types(A,B,C,D,AD)of cryptococcus neoformans,specific primers and probes were designed.CAP10 gene was amplified with PCR and ligated into a pGEM-T Easy vector.A method for detection of CAP10 gene using FQ-PCR was established after determination of the sensitivity and specificity of the test as well as the reproducibility of the assay.Results The sensitivity of this method was 1 copy/μl and the CV of inter-assay and intra-assay were 1.36% and 1.86% respectively with excellent specificity and reproducibility.Conclusion A method of detection of cryptococcus neoformans CAP10 gene by real-time Fluorescent Quantitative PCR was established successfully and the results were stable and accurate.It might become a new method for evaluating the therapeutic effect and the prognosis of cryptococcus neoformans meningitis.更多还原