TaqMan荧光定量PCR检测新型隐球菌方法的建立  

Establishment of TaqMan probe real-time PCR for detection of Cryptococcus neoformans

作　　者：韩慧[1] 黄福达[1] 张秀明[1] 吴炳义[2] 

机构地区：[1]中山大学附属中山医院/中山市人民医院检验医学中心,广东中山528403 [2]南方医科大学附属南方医院临床医学实验研究中心,广州510515

出　　处：《国际检验医学杂志》2016年第19期2697-2698,2701,共3页International Journal of Laboratory Medicine

摘　　要：目的建立TaqMan荧光定量PCR方法定量检测新型隐球菌基因组DNA,为检测隐球菌性脑膜炎提供重要方法。方法在国家生物技术信息中心(NCBI)查找新型隐球菌各亚型的ITS-rDNA序列,序列比对后设计特异性引物和探针,扩增片段为114bp,构建质粒标准品,调整质粒浓度为1.42×10~8 copy/μL^1.42×10copy/μL共8个浓度梯度,分别取2μL作为模板,优化反应条件,建立标准曲线,进行敏感性、特异性、重复性评价,并检测临床确诊的15例隐球菌脑膜炎感染菌株。结果建立的荧光定量PCR可以检测2.84×10~2拷贝的质粒DNA,对临床分离的各10例其他真菌、细菌、乙型肝炎病毒DNA和人类基因组DNA均无扩增曲线,重复性良好,3个浓度的批间变异系数分别为2.86%、1.48%、1.36%,可准确检测15例新型隐球菌。结论成功建立检测新型隐球菌的荧光定量PCR方法,敏感性和特异性较高,结果稳定可靠,可早期、快速诊断隐球菌性脑膜炎。Objective To establish TaqMan probe real-time Fluorescent Quantitative PCR in detecting Cryptococcus neoformans genomic DNA,and to provide important method for detection of cryptococcal meningitis.Methods According to the Cryptococcus neoformans ITS-rDNA sequences obtained from NCBI,specific primers and probe were designed based on the conserved sequences,a specific 114 bp fragment was amplified by primers and probe,then recombinant plasmid was constructed.Eight different concentrations from 1.42×10copy/μL to 1.42×10copy/μL were used as templates by 2μL.In the optimum reaction condition,the sensitivity,specificity and repeatability were evaluated and standard curve was established.15 clinical cryptococcal meningitis strains isolated from clinical diagnosis patients were detected.Results The real-time PCR showed high sensitivity and specificity and was able to detect 2.84×10^2 copies of plasmid DNA.The detection sensitivity was 2.84×10^2 copies plasmid DNA by real-time PCR,no amplification curve was detected with human genomic DNA,other fungus,bacterias and viruses.The CV of inter-assay were2.86%,1.48% and 1.36% respectively with excellent reproducibility.Fifteen clinical isolated strains could be detected accurately.Conclusion A method of detection of Cryptococcus neoformans DNA by real-time PCR is established successfully with high sensitivity and specificity,and the results are stable,could diagnose cryptococcal meningitis rapidly and early.

关 键 词：荧光定量PCR 新型隐球菌 基因组DNA 

分 类 号：R519[医药卫生—内科学] R440[医药卫生—临床医学]