PCR直接测序法在脓肿分枝杆菌鉴定中的应用

目的探讨聚合酶链式反应（Polymerase chain reaction,PCR）与DNA测序技术鉴定脓肿分枝杆菌的效果。方法利用细菌16S r RNA基因通用引物PCR扩增疑似分枝杆菌的16S r RNA基因的DNA片段并进行DNA测序分析。DNA测序获得序列经生物信息学比对分析获得相关分枝杆菌的信息;针对相关分枝杆菌分别设计非同源区域特异性引物对1 500 bp的克隆DNA片段扩增验证。结果细菌16S r RNA基因通用引物扩增获得约1 500 bp的DNA片段,但DNA测序仅获得其中部分DNA序列约296 bp;部分DNA序列经生物信息学比对分析后获得四种同源性高达98%的分枝杆菌信息,并且四种分枝杆菌的特异性引物对1 500 bp的克隆产物进行扩增后仅在脓肿分枝杆菌中获得特异性PCR产物。结论疑似结核患者体内的抗酸染色阳性菌为非结核分枝杆菌中的脓肿结核分枝杆菌,并且PCR直接测序法可快速鉴定细菌的种属。

Application of PCR and direct sequencing technologies in the identification of Mycobacterium abscessus

Objective To explore the identification of Mycobacterium abscessus with PCR, direct sequencing approaches and bioinformatics. Methods DNA fragments of 16 S r RNA gene were acquired using PCR and DNA sequence obtained by DNA sequencing. DNA about Mycobacterium was analyzed by bioinformatics. Specific primers of non-homologous region for relevant branches Bacillus were designed respectively to clone DNA fragment of 1 500 bp product. Results DNA fragment of 1 500 bp was acquired with PCR, but only a part of the DNA sequence of approximately 296 bp was obtained. After DNA sequence bioinformatics analysis, four kind of Mycobacterium with homology of up to 98% were obtained. Finally, we only obtained PCR special product in M. abscessus when four kinds of specific primers for Mycobacterium were used to clone the DNA fragment of 1 500 bp. Conclusion The Mycobacterium in the suspected TB patients is M. abscessus, and species of bacteria can be identified rapidly by PCR and direct sequencing approaches.