建立FQ-PCR定量体系检测新型隐球菌CAP10 mRNA  

Developing Fluorescent-quantitative PCR System to Detect the mRNA of Capsule Associated Protein 10 Gene of Cryptococcus Neoformans

作　　者：戴琳孙[1] 林旎[2] 陈勇[2] 江凌[2] 程祖建[2] 欧启水[2] 

机构地区：[1]福建医科大学附属第一医院神经外科,福州350005 [2]福建医科大学附属第一医院检验科福建医科大学医学检验系,福州350005

出　　处：《福建医科大学学报》2009年第1期45-48,共4页Journal of Fujian Medical University

基　　金：福建省自然科学基金(2008J0081);福建省自然科学基金自由申请项目(X0650045);福建省教育厅科技计划项目(JA07086)

摘　　要：目的建立荧光定量PCR(FQ-PCR)技术定量检测新型隐球菌(CN)的荚膜相关蛋白10(CAP10)基因mRNA,为新型隐球菌的诊断、预后及疗效判断提供依据。方法用逆转录PCR的方法从CN标准株(ATCC34874)中扩增得到CAP10,构建重组质粒标准品pGEMT-Easy-CAP10,以倍比稀释的标准品制备标准曲线。设计引物和探针,建立FQ-PCR反应体系并对其重复性、特异性和线性范围进行评价。结果成功构建了重组质粒标准品pGEMT-Easy-CAP10,并用倍比稀释的标准品制备了标准曲线Y=-3.11X+38.84。批内重复性试验CV值为0.31%,批间重复性试验CV值为2.73%。FQ-PCR体系能特异扩增新型隐球菌,特异性好,该体系的线性范围为101copies/μL～108copies/μL。结论成功建立CN的CAP10 mRNA的定量检测方法;该方法重复性好,特异性强。Objective To develop the fluorescent-quantitative PCR system for detecting the mRNA expression of the capsule associated protein 10 of cryptococcus neoformans （CN）. The study provides basis for the diagnosis, prognosis, and evaluation of therapeutic efficacy of CN. Methods The CAP10 gene was amplified from standard strain of cryptococcus neoformans by reverse-transcription PCR tO construct the standard substance named pGEMT-Easy-CAP10. A standard curve was set up by using the serially diluted standard substance. The fluorescent-quantitative PCR system was developed, and its stability, specificity, and linear range were appraised. Results We successfully constructed the plasmid standard substance and set up a standard curve. The intra-assay and inter-assay variation were 0.31% and 2.73% respectively. The FQ-PCR system would specifically amplify the CAP10 gene of CN. The linear range was from 10lcopies/μL to 10^8 copies/μL. Conclusion The FQ-PCR technique for the detection of the capsule associated protein 10 gene of cryptococcus neoformans has been successfully developed in that it is specific and stable, and its linear range is wide, It would be useful for detecting the infection of CN.

关 键 词：细胞荚膜 隐球菌 新型 真菌蛋白质类 逆转录聚合酶链反应 mRNA 信使 

分 类 号：R450[医药卫生—治疗学]