应用rpoB基因PCR反向斑点杂交快速鉴定分枝杆菌菌种的研究

摘要/Abstract

摘要： 目的根据分枝杆菌rpoB基因序列建立一种准确、快速的分枝杆菌菌种鉴定方法。方法以分枝杆菌rpoB基因编码序列为靶基因,用聚合酶链反应(PCR)反向斑点杂交技术检测24种分枝杆菌标准株、8种非分枝杆菌标准株、37株分枝杆菌临床分离株。结果分枝杆菌与非分枝杆菌标准株经PCR扩增后,分枝杆菌标准株均扩增出360 bpDNA片段,非分枝杆菌除甲型溶血性链球菌和假白喉棒状杆菌出现同样片段外,其它菌种均未见扩增。敏感性试验可检测出1 pg结核分枝杆菌DNA。探针特异性试验表明,21种寡核苷酸探针除海分枝杆菌与偶然分枝杆菌的探针有交叉杂交,其余均为特异性杂交。应用该方法对37株分枝杆菌临床分离株进行鉴定,结果与常规方法鉴定结果相符。结论应用rpoB基因序列和PCR反向斑点杂交技术鉴定分枝杆菌菌种快速、准确,具有较高的应有价值。

关键词: 分枝杆菌, 聚合酶链反应, 寡核苷酸探针, 核酸杂交

Abstract: Objective To establish a rapid,sensitive and specific method for identification of mycobacterium species through rpoB gene. Method Based on the sequence of rpoB gene,the standard strains of 24 Mycobacteria and 8 nonmycobacteria,37 clinical isolates of mycobacteria were detected by PCRreverse dot blot hybridization assay. Results 360 bp DNA fragments were amplified from all mycobacterial strains tested and were not found in all nonmycobacterial strains besides Hemolytic streptococcus and Corynebacterium pseudodiphtheriticum. 1 pg of M.tuberculosis DNA could be detected by PCR assay.Among 21 oligonucleotide probes,probe-M,fortuitum cross-hybridized with M.marinum,the other probes were specific.The Results of clinical isolates identified by this assay were in agreement with those obtained by conventional methods. Conclusion RpoB-based PCR-reverse dot blot hybridization assay might become a rapid,effective method for identification of mycobacterium species.

Key words: Mycobacterium, Polymerase Chain Reaction, Oligonucleotide Probes, Nucleic Acid Hybridization

樊博,王巍,李国利,李洪敏,. 应用rpoB基因PCR反向斑点杂交快速鉴定分枝杆菌菌种的研究[J]. 中国防痨杂志, 2006, 28(4): 212-215.

Fan Bo,Wang Wei,Li Guoli,et al.. Rapid identification of mycobacterium species by amplification of rpoB gene and reverse dot blot hybridization assay[J]. Chinese Journal of Antituberculosis, 2006, 28(4): 212-215.

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