检测耐利福平结核分枝杆菌寡核苷酸芯片的研制及应用  

Development and application of oligonucleotide microarray for detecting rifampin-resistant Mycobacterium tuberculosis strains

作　　者：谢弋[1] 丁雨 黄坚 王升启[1] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850 [2]深圳益生堂生物企业有限公司,深圳518026

出　　处：《军事医学科学院院刊》2003年第5期353-355,共3页Bulletin of the Academy of Military Medical Sciences

基　　金：国家自然科学基金资助项目 (398890 0 1) ;军队杰出人才基金资助项目

摘　　要：目的 :探讨用寡核苷酸芯片检测结核分枝杆菌耐利福平株的rpoB基因突变的可行性。方法 :制备了检测耐利福平结核分枝杆菌寡核苷酸芯片 ,并且通过优化引物比例和杂交温度以获得最佳结果。应用该寡核苷酸芯片检测 6例结核分枝杆菌的rpoB基因突变 ,并与测序结果比较。结果 :不同引物比例PCR产物经热变性后杂交 ,杂交结果无明显差异。杂交温度可以影响杂交结果的特异性和杂交信号的强度。引物比例 1∶1,杂交温度 5 0℃为最佳条件。 6例标本的芯片检测结果显示其中有 1例野生型标本 ,5例突变标本 :5 16位GAC→AAC ,GTC ,TAC ,GAG ,5 31位TCG→TTG。其结果与测序结果完全相符。结论 :寡核苷酸芯片可以推广应用到临床作为耐药结核病快速诊断的一种有效方法。Objective:To explore the feasibility of detecting the mutations of rpoB gene in rifampin-resistant Mycobacterium tuberculosis strains by oligonucleotide microarray.Methods:Oligonucleotide microarray with 22 probes were prepared. The ratio of the forward primer to the reverse one for PCR and the hybridization temperature were optimized in order to get the best result. Then 6 strains of clinical isolates of M.tuberculosis were examined by the oligonucleotide microarray for the presence of mutations in rpoB gene,which was compared with sequencing results.Results:The results showed that the ratio of primers for PCR did not affect the hybridization results if the PCR products were heat denatured before hybridization, while the hybridization temperature could affect the specificity and the intensity of hybridization signals, and the best ratio of primers was 1∶1 and the best hybridization temperature was 50℃. Of the 6 samples, 1 strain was wild type,while the other 5 strains were mutation type, including GAC →AAC, GAC→GTC, GAC→TAC, GAC→GAG at the 516th codon for 4 strains,respectively, and TCG→TTG at the 531st codon for one strain,which were consistent with those of DNA sequencing.Conclusion:This research implies that oligonucleotide microarray can be applied to quick diagnosis of rifampin-resistant M.tuberculosis.

关 键 词：检测 耐利福平结核分枝杆菌 寡核苷酸芯片 研制 应用 

分 类 号：R346[医药卫生—基础医学]