多重聚合酶链反应检测结核菌耐异烟肼相关基因

目的：建立耐异烟肼(isoniazid,INH)结核分枝杆菌(M.tuberclasis,MTB)多重聚合酶链反应(multiple PCR,multi-PCR)检测系统，在一次扩增中快速，特异地同时检出aphC启动子，inhA和kagG基因，用于快速初步诊断结核分枝杆菌对INH的耐药性．方法：根据结核分枝杆菌的aphC启动子，inhA和kagG序列，分别设计出3对特异性寡聚核苷酸引物，采用multi—PCR技术，同时检出对结核分枝杆菌耐INH起作用的3个基因．结果：应用multi—PCR反应体系，对引物的相关性实验结果表明引物之间不会因相互干扰而出现假阳性结果；multi—PCR扩增的预期结果为：单基因引物出现一条特异性扩增区带，多重基因引物出现2或3条特异性扩增区带，经实验达到预期的扩增结果；对H37Rv标准株、INH敏感株及INH耐药株分别采用常规PCR和multi—PCR进行同时扩增，结果两种扩增方法均能扩增出预期的目的片段，符合率达100％．结论：multi—PCR能有效地为多基因耐药的临床病原体的诊断提供快速、准确的诊断手段，更能提高检验效率。

Study on M. tuberculosis isoniazid-resistant isolates by multi-PCR

AIM: To develop a new multi PCR system to detect the aphC promoter, inhA and katG genes of the M. tuberculosis isoniazid resistance isolates in a single reaction and to diagnose the M. tuberculosis isoniazid resistance isolates quickly. METHOD: According to the sequence of aphC promoter, inhA and katG genes of the M. tuberculosis , three pairs of oligonucleotide primers were designed to examine the isoniazid resistance by multi PCR. RESULTS: The results of the pertinence test indicated that there was no interference among the three pair of primers. One pair of primers were found to amplify one specific section while three pairs were found to amplify 2 or 3 specific sections. To analyze the isoniazid sensitivity isolates and resistance isolates, both conventional PCR and multi PCR were and H37Rv was used as control. The two methods amplified the anticipated sections and the rate of consistency was 100%. CONCLUSION: The multi PCR not only diagnoses quickly and accurately the multi gene drug resistance clinical isolates but also improves the analysis efficiency.