摘要
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.
出处
《医学分子生物学杂志》
CAS
CSCD
2012年第1期21-26,共6页
Journal of Medical Molecular Biology
基金
安徽省高校省级科学研究资助项目(No.KJ20118201)