DEVELOPMENT OF PCR TECHNIQUES FOR MYCOBACTERIAL DETECTION

这项工作的主要重点是开发一种可靠的方法， PCR扩增检测临床标本中的分枝杆菌 DNA.最初的尝试是使用种属特异性 引物组，目的是能够检测，以及随后的 鉴定所有临床显著分枝杆菌。结果 与属引物有点令人失望，然而， 非分枝杆菌属（包括棒状杆菌）的扩增 在检测格式和扩增方面存在问题 效率结果与M.结核特异性扩增具有 更加令人鼓舞，我们已经能够证明， 从含有这种微生物的痰液标本中扩增DNA。我们 目前正在开发一种比色微量滴定板测定法， 用于PCR产物的检测，并希望通过这种方法，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。 肺结核或没有。我们也开始使用液氮为基础的 提取程序，这提高了DNA的功效 从分枝杆菌中提取的方法。

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.