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

用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，并需要专门知识来解释结果。为了检测和区分主要的致病分枝杆菌种，我们开发了从广泛的分枝杆菌种中扩增dna J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、非洲分枝杆菌、微小分枝杆菌、甘肃分枝杆菌、海洋分枝杆菌、胃肠分枝杆菌、猿猴分枝杆菌、华支杆菌、苏云金分枝杆菌、高登分枝杆菌、禽类分枝杆菌、胞内分枝杆菌、异种分枝杆菌、福氏分枝杆菌、合胞分枝杆菌、嗜血分枝杆菌和微小分枝杆菌)的dna J基因的核苷酸序列。我们确定了物种特异性的限制性内切酶切点，这使得我们能够通过聚合酶链式反应和限制性片段长度多态性分析相结合的方法来区分大多数分枝杆菌DNA。DNAJ基因的聚合酶链式反应方法灵敏、特异，可直接检测痰、支气管肺泡灌洗液或脑脊液等临床标本中的分枝杆菌DNA。此外，我们开发了针对结核分枝杆菌复合体、M.A vium、M.inellulare和M.kansasii的物种特异性寡核苷酸探针的斑点杂交分析，允许在对dna J基因进行聚合酶链式反应后快速识别这些物种。我们的结论是，用扩增dna J基因的属特异性引物进行聚合酶链式反应，以及随后用限制性内切酶进行RFLP分析和用物种特异性寡核苷酸探针进行斑点杂交分析，对于结核病和非结核分枝杆菌感染的鉴别诊断是最有用的。

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.