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目的 :筛选出反应体系的最佳优化方案及最佳扩增条件 ,用于铜绿假单胞菌随机扩增多态性DNA分析 (RAPD)。方法 :应用单因素设计和反应曲面设计进行RAPD反应体系的优化 ,同时比较不同的退火温度、延伸温度及热循环仪类型对RAPD指纹图的影响。结果 :单因素设计明显受到多因素间交互作用的影响 ,从而使优化的反应体系具有多样性 ;反应曲面设计得出相对稳定的优化反应体系 (关键成分浓度 )为 :Mg2 + 2 .0mmol/L、引物 0 .5 μmol/L和模板 0 .5ng/μl。退火温度以38～ 4 3℃、延伸温度以 62～ 72℃为最佳。结论 :反应曲面设计是优化RAPD反应体系的最佳方案。扩增条件中退火和延伸温度分别以 38～ 4 3℃和 62～ 72℃为最佳
OBJECTIVE: To screen out the best optimization scheme and optimal amplification conditions of the reaction system for the random amplified polymorphic DNA analysis (RAPD) of Pseudomonas aeruginosa. Methods: The single factor design and reaction surface design were used to optimize the RAPD reaction system. The effects of annealing temperature, extension temperature and thermocycler type on RAPD fingerprinting were compared. Results: The single-factor design was obviously influenced by the interaction of many factors, which resulted in the diversity of the optimized reaction system. The optimized reaction system (key component concentration) of the reaction surface design was: Mg2 +2.0 mmol / L , Primer 0.5 μmol / L and template 0.5 ng / μl. Annealing temperature of 38 ~ 43 ℃, the extension temperature of 62 ~ 72 ℃ for the best. Conclusion: Reactive surface design is the best way to optimize RAPD reaction system. Annealing and extension temperature in the amplification conditions were 38 ~ 43 ℃ and 62 ~ 72 ℃ for the best