Kox L F, van Leeuwen J, Knijper S, Jansen H M, Kolk A H
Department of Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands.
J Clin Microbiol. 1995 Dec;33(12):3225-33. doi: 10.1128/jcm.33.12.3225-3233.1995.
A PCR and a reverse cross blot hybridization assay were developed for the detection and identification of mycobacteria in clinical samples. The PCR amplifies a part of the DNA coding for 16S rRNA with a set of primers that is specific for the genus Mycobacterium and that flanks species-specific sequences within the genes coding for 16S rRNA. The PCR product is analyzed in a reverse cross blot hybridization assay with probes specific for M. tuberculosis complex (pTub1), M. avium (pAvi3), M. intracellulare (pInt5 and pInt7), M. kansasii complex-M. scrofulaceum complex (pKan1), M. xenopi (pXen1), M. fortuitum (pFor1), M. smegmatis (pSme1), and Mycobacterium spp. (pMyc5a). The PCR assay can detect 10 fg of DNA, the equivalent of two mycobacteria. The specificities of the probes were tested with 108 mycobacterial strains (33 species) and 31 nonmycobacterial strains (of 17 genera). The probes pAvi3, pInt5, pInt7, pKan1, pXen1, and pMyc5a were specific. With probes pTub1, pFor1, and pSme1, slight cross hybridization occurred. However, the mycobacterial strains from which the cross-hybridizing PCR products were derived belonged to nonpathogenic or nonopportunistic species which do not occur in clinical samples. The test was used on 31 different clinical specimens obtained from patients suspected of having mycobacterial disease, including a patient with a double mycobacterial infection. The samples included sputum, bronchoalveolar lavage, tissue biopsy samples, cerebrospinal fluid, pus, peritoneal fluid, pleural fluid, and blood. The results of the PCR assay agreed with those of conventional identification methods or with clinical data, showing that the test can be used for the direct and rapid detection and identification of mycobacteria in clinical samples.
开发了一种聚合酶链反应（PCR）和反向杂交印迹分析方法，用于检测和鉴定临床样本中的分枝杆菌。PCR使用一组对分枝杆菌属特异且位于编码16S核糖体RNA（rRNA）基因内物种特异序列侧翼的引物，扩增编码16S rRNA的部分DNA。PCR产物在反向杂交印迹分析中用针对结核分枝杆菌复合群（pTub1）、鸟分枝杆菌（pAvi3）、胞内分枝杆菌（pInt5和pInt7）、堪萨斯分枝杆菌复合群 - 瘰疬分枝杆菌复合群（pKan1）、偶发分枝杆菌（pXen1）、龟分枝杆菌（pFor1）、耻垢分枝杆菌（pSme1）以及分枝杆菌属（pMyc5a）的探针进行分析。PCR检测可检测到10 fg的DNA，相当于两个分枝杆菌。用108株分枝杆菌菌株（33个种）和31株非分枝杆菌菌株（17个属）测试了探针的特异性。探针pAvi3、pInt5、pInt7、pKan1、pXen1和pMyc5a具有特异性。使用探针pTub1、pFor1和pSme1时，出现了轻微的交叉杂交。然而，产生交叉杂交PCR产物的分枝杆菌菌株属于临床样本中不会出现的非致病性或非机会性物种。该检测方法应用于从疑似患有分枝杆菌病的患者获取的31份不同临床标本，包括一名患有双重分枝杆菌感染的患者。样本包括痰液、支气管肺泡灌洗、组织活检样本、脑脊液、脓液、腹水、胸水和血液。PCR检测结果与传统鉴定方法或临床数据一致，表明该检测可用于临床样本中分枝杆菌的直接快速检测和鉴定。
