荧光定量PCR和环介导等温扩增方法检测隐球菌CAP10基.pdfVIP

2012;32(6) 南方医科大学学报（J South Med Univ ） ·817 · 基础研究 荧光定量PCR 和环介导等温扩增方法检测隐球菌CAP10基因的 比较研究 韩 慧，胡子有，吴炳义 南方医科大学南方医院临床医学实验研究中心，广东 广州 510515 摘要：目的建立荧光定量PCR 和环介导等温扩增技术(LAMP)检测隐球菌荚膜相关蛋白基因(CAP10)的方法，并比较和评价两 种方法的优缺点。方法针对新型隐球菌CAP10基因序列，使用在线软件设计引物，并构建质粒标准品，优化反应条件后用荧光 定量PCR 和LAMP 两种方法检测定量的质粒，分析其敏感性和特异性，并检测临床标本。结果荧光定量PCR 检测CAP10质粒 1 3 的敏感性为6.8×10 拷贝，LAMP 方法的敏感性为6.8×10 拷贝，PCR 检测阳性率比LAMP 方法高。两种方法特异性好，对脑膜炎 奈瑟菌、白色念珠菌、热带念珠菌、黄曲霉、黑曲霉和大肠埃希菌的检测结果均为阴性。结论成功建立了检测CAP10基因的荧 光定量PCR 方法敏感性和特异性高，但是需要特殊仪器；LAMP 方法敏感性较低，但其操作简单，无需特殊仪器和设备，加入核 酸染料即能观察结果。两者均适用于新型隐球菌感染的检测。 关键词：荧光定量PCR ；环介导等温扩增技术；新型隐球菌；CAP10 中图分类号：R446.5; R379.5 文献标志码：A 文章编号：1673-4254（2012）06-0817-04 doi: 10.3969/j.issn. 1673-4254.2012.06.013 /kcms/detail/44.1627.R1518.013.html Establishment of real-time PCR and loop-mediated isothermal amplification for detecting Cryptococcus neoformans CAP10 gene HAN Hui, HU Ziyou, WU Bingyi Research Center of Clinical Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China Abstract: Objective To establish real-time PCR and loop-mediated isothermal amplification (LAMP) systems for detecting Cryptococcus neoformans CAP10 gene. Methods Specific primers were designed targeting CAP10 gene of Cryptococcus neoformans, and the plasmid was constructed. After optimization of the reaction condition, the plasmid was quantitatively detected using real-time PCR and LAMP, and the detection sensitivity and specificity were evaluated. Clinical samples were also detected using the two methods. Results The detection sensitivity of real-time PCR and LAMP was 6.8 × 101 and 6.8 × 103 copies, respectively. Real-time PCR yielded a higher positivity rate than LAMP. Both of the two