实时荧光定量PCR检测新生隐球菌DNA方法的建立

实时荧光定量PCR检测新生隐球菌DNA方法的建立 Establishment of Real-Time Fluorescence Quantitative PCR for Detection of Cryptococcus neoformans DNA 程鹏飞 CHENG Pengfei1 1 first-author 蒋晓迪 JIANG Xiaodi1 1 曹轩 CAO Xuan1 1 赵友云 ZHAO Youyun1 1 王业富 WANG Yefu1 1 江小明 JIANG Xiaoming2 2 杨永 YANG Yong2 2 武汉大学生命科学学院/病毒学国家重点实验室 武汉大学生命科学学院/病毒学国家重点实验室 College of Life Sciences,Wuhan University/State Key Laboratory of Virology College of Life Sciences,Wuhan University/State Key Laboratory of Virology 武汉产品质量监督检验所 武汉产品质量监督检验所 Wuhan Institute of Industrial Product Quality Supervision and Inspection Wuhan Institute of Industrial Product Quality Supervision and Inspection 以新生隐球菌DNA中一段高度保守序列为目的序列,结合PCR和DNA探针技术优点,设计出特异引物和探针,并优化新生隐球菌荧光定量PCR反应体系,研究出实时荧光定量PCR检测技术检测新生隐球菌DNA的方法.结果显示:标准株及临床样本株新生隐球菌保守序列为124 bp;重组质粒pUCM-T中目的基因片段序列碱基符合率100%;当质粒标准品浓度1.0×103 1 .0×107Copies/mL时,相关系数为0.997,有很好的线性关系;各浓度标准品的Ct值变异系数均小于5%,表明此技术的重复性好,用特异性引物扩增可检测出5.0×102Copies/mL的新生隐球菌DNA,说明该方法敏感性很好. Based on a conserved DNA sequence of Cryptococcus neoformans as the target fragment,we optimized the fluorescence quantitative PCR reaction system and developed the real-time fluorescence quantitative PCR detecting technology by combining the advantages of PCR and DNA probe.The results showsed that the conserved DNA sequence of Cryptococcus neoformans of standard strains and clinical isolates aws 124 bp.The target gene fragment in recombinant plasmid pUCM-T was found to have a 100% sequence similarity to the corresponding GenBank sequence.When plasmid DNA was diluted from 1.0×103 1 .0×107 Copies/mL,the correlation coefficient of standard curve was 0.997,which showed a nice linear relationship.The variation coefficient of Ct values of all standard DNA was less than 5%,which indicated a nice repetition.Specific primers were used to detect 5.0×102 copies/mL of C.neoformans DNA,and the result showed that the sensitivity of the method was very good. 新生隐球菌 DNA 实时荧光定量PCR Cryptococcus neoformans DNA real-time FQ-PCR 10.14188/j.1671-8836.2010.03.022 R440 2010-03-01 2021-04-01 3 56