DEVELOPMENT OF PCR TECHNIQUES FOR MYCOBACTERIAL DETECTION

分枝杆菌检测 PCR 技术的发展

• 批准号: 2571417
• 负责人: E O'SHAUGHNESSY
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2571417
• 关键词: Mycobacterium tuberculosis; bacterial DNA; bacterial genetics; fluorescent dye /probe; human tissue; method development; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; southern blotting; transposon /insertion element

This work's principal focus has been to develop a reliable method for detecting Mycobacterium tuberculosis from clinical specimens through the amplification of mycobacterial DNA using the polymerase chain reaction (PCR). DNA is extracted from clinical samples using a liquid nitrogen-based procedure. Primers specific to the insertion sequence IS6110 are used. This insertion sequence is specific to M. tuberculosis complex and is present in 1 to 20 copies in the M. tuberculosis genome. Internal controls have been developed and are used to detect PCR inhibitors in the samples. Amplification products are visualized using gel electrophoresis. To confirm the presence of the specific amplicons, FCR products are also analyzed by Southern blot and hybridization with chemiluminescent probes. We have been able to demonstrate amplification of M. tuberculosis DNA from sputum specimens. Amplification of M. tuberculosis DNA from buffy coat and tissue specimens is being optimized; we have been able to amplify M. tuberculosis DNA from buffy coats spiked with organism and from a small number of tissue specimens.

这项工作的主要重点是开发一种可靠的方法 用于从临床标本中检测结核分枝杆菌 通过聚合酶扩增分枝杆菌DNA 链式反应(PCR)。从临床样本中提取DNA使用 一种以液氮为基础的程序。插入片段的特异性引物 使用序列IS6110。该插入序列是M所特有的。 结核复合体，在M。 结核病基因组。已经制定了内部控制，并正在 用于检测样品中的聚合酶链式反应抑制物。扩增产物 用凝胶电泳法进行可视化。确认…的存在 对特异扩增产物、FCR产物也进行了Southern分析 用化学发光探针进行杂交和杂交。我们一直在 能够证明痰中结核分枝杆菌DNA的扩增 标本。结核分枝杆菌棕黄色外壳DNA的扩增及鉴定 组织标本正在优化；我们已经能够扩增出M。 棕黄色大衣中添加了有机体的结核DNA和一种 少量的组织标本。