RAPD扩增分枝杆菌DNA优化条件及菌型分型的诊断应用  

The optimized condition for differentiation of Mycobactreium strains by using RAPD and its appliation in strain typing and diagnosis

作　　者：许丽[1] 杨应周[1] 谭卫国[1] 林世平[1] 胡志上[2] 吴清芳[1] 张玉华[1] 

机构地区：[1]深圳市慢性病防治院,广东深圳258020 [2]中国医学科学院基础医学研究所,北京100005

出　　处：《中国热带医学》2006年第5期752-754,共3页China Tropical Medicine

基　　金：深圳市科委资助项目(200204213)

摘　　要：目的探讨随机扩增多态性DNA(RandomlyamplifiedpolymorphicDNA,RAPD)技术在分枝杆菌分型诊断中优化条件及其应用价值。方法优化RAPD技术扩增分枝杆菌DNA的条件,并分别扩增深圳市临床病人分离株DNA,对比RAPD电泳条带特征。结果以条带稳定、丰富、清晰为标准,确定引物1RAPD最佳反应条件为50μl反应体系中,含100ng模板DNA,2.5mmol/LMgCl2,2UDNA聚合酶,引物0.5μmol/L,dATP、dGTP、dCTP和dTTP各250μmol/L,反应40个循环,每个循环为94℃1min,36℃1min,72℃2min。所扩增结核杆菌临床分离株的DNA条带丰富、清晰,各条带差异较明显。结论RAPD技术能较好地鉴别MTB和NTM。Objective To explore the optimized condition for type differentiation of Mycobacteria strains and its value in applification by means of randomly amplified polymorphic DNA （RAPD） analysis. Methods RAPD condition for amplifying Mycobacteria DNA of clinically separated Mycobacteril strains from patients in Shenzhen was optimized, and then compared the features of RAPD eleetrophoresis strips. Results The best reaction condition for RAPD, taking stable, abundant and clear strips as criteria, were as the follows ： in 50μl reaction system, there was 100ng template DNA, 2.5mmol/L MgCl2, 2U DNA polymerization enzyme, 0.5μmol/L primer and 250μmol/L of dATP, dGTP ,dCTP and dTTP respectively ; 40 reaction cycles, whose cycle procedure was： 94℃ lmin,36℃ 1min,72℃2min. At the above reaction conditions for RAPD, DNA of climieally separated Mycobacteria strains were amplified, and the electrophoresis strips were abundant, clear, and evidently different from each other. Conclusion Mycobacterium tuberculosis and no- Mycobacterium tuberculosis can be differentiated by RAPD.

关 键 词：分枝杆菌 非典型性 结核杆菌 RAPD 分型 

分 类 号：R378.91[医药卫生—病原生物学]