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Diffcrential diagnosis of tuberculosis and nontuberculous infectious diseases by PCR-RFLP and sequence-specific oligonucleotide probes

PCR-RFLP和序列特异性寡核苷酸探针鉴别诊断结核病和非结核感染性疾病

基本信息

批准号：
02557105
负责人：
NAGAI Ryozo
金额：
$ 7.1万
依托单位：
University of Tokyo,
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

Identification of tuberculous and non-tuberculous mycobacteria by biochemical methods is a long-term process which takes up to 8 weeks for completion and requires expertise for interpretation of the results. In order to detect and differentiate the major pathogenic mycobacterial species, we developed genus-specific primers which amplify the dnaJ gene from the broad spectrum of mycobacterial species and determined the nucleotide sequences within the dnaJ gene from 19 mycobacterial species (M.tuberculosis, M.bovis BCG,M.africanum, M.microti, M.kansasii, M.marinum, M.GASTRI, M.simiae, M.scrofulaceum, M.szulgai, M.gordonae, M.avium, M.intracellulare, M.xenopi, M.fortuitum, M.chelonei, M.hemophilum and M.paratuberculosis).On the basis of the dnaJ gene sequences, we identified the species-specific restriction sites, which allows us to differentiate most of the mycobacterial DNA by a combination of the PCR with the restriction fragment length polymorphism analysis (PCR-RFLP). The PCR assay for the dnaJ gene was sensitive and specific enough to detect the mycobacterial DNA directly in clinical materials, such as sputa, bronchoalveolar lavages or cerebrospinal fluids. Furthermore, we developed dot blot hybridization analysis using species-specific oligonucleotide probes for the M.tuberculosis complex, M.a vium, M.intracellulare and M.kansasii, allowing a rapid identification of these species following PCR for the dnaJ gene. We conclude that PCR with the genus-specific primer which amplifies the dnaJ gene and subsequent both RFLP analysis with restriction enzymes and dot blot analysis with species-specific oligonucleotide probes are most useful for differential diagnosis of tuberculosis and non-tuberculous mycobacterial infections.

通过生化方法鉴定结核和非结核分枝杆菌是一个长期的过程，需要长达8周的时间才能完成，并需要专业知识来解释结果。为了检测和区分主要致病性分枝杆菌，我们开发了广谱扩增dnaJ基因的属特异性引物，并测定了19种分枝杆菌（结核分枝杆菌、牛分枝杆菌、卡介苗、卡介苗）的dnaJ基因的核苷酸序列。非洲分枝杆菌、微分枝杆菌、kansasii分枝杆菌、marinum分枝杆菌、gastri分枝杆菌、simiae分枝杆菌、scrofulaceum分枝杆菌、szulgai分枝杆菌、gordonae分枝杆菌、avium分枝杆菌、胞内分枝杆菌、xenopi分枝杆菌、fortutuum分枝杆菌、chelonei分枝杆菌、血友病分枝杆菌和副结核分枝杆菌)。在dnaJ基因序列的基础上，我们确定了物种特异性的限制性内切位点，这使得我们能够通过PCR和限制性内切片段长度多态性分析（PCR- rflp）的组合来区分大多数分枝杆菌的DNA。PCR检测dnaJ基因具有足够的敏感性和特异性，可直接检测痰液、支气管肺泡灌洗液或脑脊液等临床材料中的分枝杆菌DNA。此外，我们还利用物种特异性寡核苷酸探针对结核分枝杆菌复合体、结核分枝杆菌、胞内分枝杆菌和堪萨斯分枝杆菌进行了斑点杂交分析，从而通过PCR对这些物种的dnaJ基因进行了快速鉴定。我们得出结论，用扩增dnaJ基因的属特异性引物进行PCR，随后用限制性内切酶进行RFLP分析，用种特异性寡核苷酸探针进行点印迹分析，对结核和非结核分枝杆菌感染的鉴别诊断最有用。