白色念珠菌菌丝相培养条件及RAPD反应体系优化的研究

Optimization of Culture Condition and RAPD Reaction System for Candidia Albicans Hyphal-Form

目的优化门色念珠菌菌丝相培养条件，为白色念珠菌菌丝相的分子生物学研究提供必要条件；建立白色念珠菌菌丝相RAPD的最佳反应体系，应用于其基因组DNA扩增。方法在RPM11640培养基的基础上，通过单因素试验研究了小牛血清用量、培养液pH值、培养温度和转种次数等对白色念珠菌菌丝相形成的影响。采用单因素试验，分别研究了Mg^2＋浓度、dNTPs浓度、Taq酶的浓度、引物浓度和模板DNA浓度对白色念珠菌菌丝相RAPD反应的影响；应用L16（4^5）正交试验对RAPD反应条件进行了优化。结果白色念珠菌菌丝相诱导的最佳条件为：每100ml培养液中的小牛血清用量为10ml，培养液pH值为7．5，培养温度为36℃，转种次数为12次。白色念珠菌菌丝相的最适RAPD反应体系为：Mg^2＋ 1．25mmol／L、dNTPs 0．4mmol／L、随机引物0．1μmol／L、TaqDNA聚合酶5U／50μl、模板DNA495ng／50μl。结论通过单因素和正交试验，获得了较适白色念珠菌菌丝相培养条件和其基因组RAPD扩增条件。

Objective To optimized the culture condition of Candidia Albieans hyphal-form for its molecular biological research, establish the optimal RAPD reaction system of Candidia Albicans hyphal-form for genomie DNA amplification. Methods On the basis of RPMI1640 culture medium, the effects of fetal bovine serum amount, pH degree, culture temperature, and sub-culture frequency on Candidia Albicans hyphal-form formation were studied in a single-factor experimentation. Meanwhile, the effects of concentrations of Mg2＋, dNTPs, DNA, primer, and Taq polymerase on Candidia Albicans hyphal-form RAPD amplification were also studied. And the L16 （45） orthogonal experiment was applied to optimize the reaction condition. Results The opti- mal culture condition of Candidia Albieans hyphal-form for the amount of fetal bovine serum was 10ml per 100ml culture medium, pH was 7.5, culture temperature was 36℃, and sub-culture frequency was 12. The optimal RAPD reaction system for Candidia Albicans hyphal-form was 36℃ annealing temperature, 1.25mmol/L Mg2＋, 0. 4mmol/L dNTPs, 0. 1 μmol/L primer, 495ng/50μl template DNA, and 5U/50μl Taq DNA ploymerase, respectively. Conclusion The optimal culture condition and RAPD reaction system of Candidia Albieans hyphal-form were established by single-factor and orthogonal experimentations.