四种分枝杆菌快速检测方法的研究

Methods for Rapid Detection of Four Mycobacterial Species

目的建立敏感、特异的快速检测4种分枝杆菌的方法。方法用特异性引物对结核分枝杆菌、鸟分枝杆菌、胞内分枝杆菌和堪萨斯分枝杆菌菌悬液DNA进行PCR扩增,验证其敏感性和特异性。将上述4种分枝杆菌的特异性引物同时放入PCR扩增反应体系中,以此反应体系分别对该4种分枝杆菌菌悬液DNA、及其两两组合或特定的三重组合进行扩增,同时验证其敏感性。结果4种分枝杆菌的特异性引物分别放入各自的PCR扩增反应体系中,可分别特异性地扩增出该4种分枝杆菌对应的DNA片段,敏感性达1×101～1×102个菌细胞/mL。4种分枝杆菌的特异性引物同时放入同一PCR扩增反应体系中可分别特异性地扩增出对应的4种分枝杆菌单菌及其两两组合或三种分枝杆菌组合的DNA片段,敏感性达1×102～1×103个菌细胞/mL;4种分枝杆菌的特异性引物对其他分枝杆菌进行扩增,结果均为阴性。结论多重PCR方法能敏感、特异地快速检测4种分枝杆菌。

Objective To develop a rapid method with high sensitivity and specificity to detect 4 mycobacterial species(M. tuberculosis, M. avium, M. intracellulare and M. kansasii) which are the most common opportunistic Mycobacteria in AIDS patients. Methods The sensitivities and specificities of PCR were determined with different primer pairs targeting various mycobacterial genes. Multiplex PCR with combination of 4 primer pairs was used to detect the template mixtures of either 1, 2 or 3 mycobacterial DNA. Sensitivities of multiplex PCR were measured. Results Specific DNA fragments of 4 mycobacterial species mentioned above could be detected by PCR and sensitivities ranged from 1 × 101 ~ 1 × 102 cells/mL, while the other 17 mycobacterial strains were all PCR-negative. Multiplex PCR could amplify the corresponding 1, 2 or 3 DNA fragments, depending on the number of template DNA added, and sensitivities of multiplex PCR ranged from 1 × 102 ~ 1 × 103 cells/mL. Conclusions Multiplex PCR is a rapid, sensitive and specific method for differentiation and detection of Mycobacteria.