白背飞虱RAPD-PCR反应体系的正交设计优化

Optimization of RAPD- PCR reaction system with orthogonal design for Sogatella furcifera

白背飞虱Sogatella furcifera(Horváth)是水稻重要害虫之一。研究采用L16(45)正交设计优化试验,构建了白背飞虱RAPD最优反应体系,并对引物退火温度和反应循环次数进行了优化。结果表明:白背飞虱RAPDPCR最优反应体系为2μL 10×PCR Buffer(10 mmol/L Tris-HCl;50 mmol/L KCl),2.4 mmol/L Mg2+,0.15 mmol/L dNTPs,0.7μmol/L引物,0.8U Taq DNA Polymerase和30 ng模板DNA,ddH2O补充至20μL。本研究采用的10条引物的最佳退火温度在35-39℃间,以引物相应最优退火温度和40次PCR反应循环可获得较好的白背飞虱RAPD-PCR结果。

Sogatellafurcifera is one of the most important pests of rice. To establish and optimize RAPD - PCR reaction system for S. furcifera, the factors influencing RAPD system were studied with L16 （45 ） orthogonal design, and the optimal annealing temperature and cycle times were proposed. The results showed that the optimal conditions for RAPD - PCR system consists of 2 μL 10 × PCR buffer （ 10 mmol/L Tris- HC1; 50 mmol/L KC1）, 2.4 mmol/L Mg2＋, 0. 15 mmol/L dNTPs mixture, 0.7μmol/L primer, 0. 8U Taq DNA polymerase and 30 ng DNA template, and adds ddH20 to 20 μL in total. The optimal annealing temperature is 35 -39 ℃ for 10 primers used in the present study. The clear and reproducible DNA bands were obtained by the optimal annealing temperature and the 40 reaction cycles.