da Silva Rocha A, da Costa Leite C, Torres H M, de Miranda A B, Pires Lopes M Q, Degrave W M, Suffys P N
Department of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
J Microbiol Methods. 1999 Sep;37(3):223-9. doi: 10.1016/s0167-7012(99)00062-7.
Polymerase chain reaction amplification of part of the gene coding for the heat shock protein hsp65 followed by restriction enzyme analysis (PRA) is a recently described tool for rapid identification of mycobacteria. In this study, the speed and simplicity of PRA for identification of isolates of mycobacteria from patients with clinical symptoms of tuberculosis was evaluated and compared with identification results obtained by commercially available methods. Established PRA patterns were observed for nineteen isolates of Mycobacterium tuberculosis, eleven belonging to the complex M. avium-intracellulare, four of M. kansasii, one of M. fortuitum, one of M. abscessus, three of M. gordonae and one of the recently described species M. lentiflavum, as identified by commercially available methods. Two isolates of M. fortuitum and one of M. gordonae had unique and so far undescribed PRA patterns, suggesting geographically-related intra-species variation within the hsp65 sequence. We propose the inclusion of these new patterns in the PRA identification algorithm and have defined more accurately the molecular weight values of the restriction fragments. This is the first report on the isolation of M. lentiflavum in Brazil suggesting that identification by means of PRA could be useful for detection of mycobacterial species that are usually unnoticed. Where the use of several commercial techniques in combination was necessary for correct identification, PRA demonstrated to be a simple technique with good cost-benefit for characterization of all mycobacterial isolates in this study.
聚合酶链反应扩增热休克蛋白hsp65编码基因的部分片段，随后进行限制性酶切分析（PRA），是一种最近描述的用于快速鉴定分枝杆菌的工具。在本研究中，评估了PRA用于从有结核病临床症状患者中鉴定分枝杆菌分离株的速度和简便性，并与通过市售方法获得的鉴定结果进行了比较。通过市售方法鉴定出，19株结核分枝杆菌、11株鸟分枝杆菌-胞内分枝杆菌复合体、4株堪萨斯分枝杆菌、1株偶然分枝杆菌、1株脓肿分枝杆菌、3株戈登分枝杆菌和1株最近描述的淡黄分枝杆菌呈现出既定的PRA模式。2株偶然分枝杆菌和1株戈登分枝杆菌具有独特且迄今未描述的PRA模式，表明hsp65序列存在与地理相关的种内变异。我们建议将这些新模式纳入PRA鉴定算法，并更准确地定义了限制性片段的分子量值。这是巴西关于淡黄分枝杆菌分离的首次报告，表明通过PRA进行鉴定可能有助于检测通常未被注意到的分枝杆菌种类。在需要联合使用多种商业技术进行正确鉴定的情况下，PRA证明是一种简单的技术，在本研究中对所有分枝杆菌分离株进行鉴定时具有良好的成本效益。
