DEVELOPMENT OF PCR TECHNIQUES FOR MYCOBACTERIAL DETECTION

分枝杆菌检测 PCR 技术的发展

• 批准号: 5201176
• 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/5201176
• 关键词: Mycobacterium; Mycobacterium tuberculosis; bacterial DNA; colorimetry; communicable disease diagnosis; diagnosis design /evaluation; diagnosis quality /standard; human tissue; method development; microorganism classification; polymerase chain reaction; sputum

The principal focus of this work has been to develop a reliable method for detection of mycobacteria in clinical specimens by PCR amplification of DNA. Initial attempts were made to use both species and genus specific primer sets, with the goal of enabling detection, and subsequent identification, of all clinically significant mycobacteria. The results with the genus primers were somewhat disappointing, however, with amplification of non-mycobacterial genera (including corynebacteria) presenting a problem in terms of detection format and amplification efficiency. The results with M. tuberculosis-specific amplification have been more encouraging, and we have been able to demonstrate consistent amplification of DNA from sputum specimens containing this organism. We are currently working on developing a colorimetric microtiter plate assay for detection of PCR product, and hope that with this procedure we will be able to determine if acid fast organisms detected in direct smears are M. tuberculosis or not. We have also begun using a liquid-nitrogen-based extraction procedure, which has improved the efficacy of the DNA extraction process from mycobacteria.

这项工作的主要重点一直是开发一种可靠的方法来 聚合酶链式反应检测临床标本中的分枝杆菌 DNA最初的尝试是同时使用物种和属的特异性 引物集，目标是启用检测，以及后续 所有临床上有意义的分枝杆菌的鉴定。结果是 然而，与该属的引物有些失望，与 非分枝杆菌属(包括棒状杆菌)的扩增 提出检测格式和放大方面的问题 效率。结核分枝杆菌特异性扩增的结果 更加鼓舞人心，我们已经能够证明一贯的 从含有这种微生物的痰标本中扩增DNA。我们 目前正致力于开发一种比色微滴定板分析 用于检测聚合酶链式反应产物，并希望通过这个程序我们将 能够确定在直接涂片中检测到的抗酸微生物是否为M。 不管是不是肺结核。我们还开始使用以液氮为基础的 提取程序，这提高了DNA的效率 分枝杆菌的提取工艺。