黄瓜霜霉菌ITS区序列分析反应体系的构建及优化

Establishment and optimization of PCR reaction system for rDNA ITS of Pseudoperonospora cubensis

文章以黄瓜霜霉菌为材料,采用改良的CTAB方法提取基因组DNA,研究了黄瓜霜霉菌ITS区序列分析中PCR反应体系的各主要成分对检测结果的影响,确立了适宜的退火温度和反应体系。优化后的反应体系(25μL)为:采用引物ITS1/ITS4各40 ng,60 ng DNA模板,2.5 mmol·L-1 Mg2+2.0μL,2.5 mmol·L-1 dNTP 1.5μL,Taq酶1 U。优化的PCR扩增程序为:94℃预变性3 min,94℃50 s,52.2℃40 s,72℃1.5 min。在这一条件下运行35个循环;72℃延伸7 min。采用此反应体系和扩增程序,对采自黑龙江省不同地区的31份黄瓜霜霉菌基因组DNA进行PCR扩增,均可得到一条约为900 bp的条带。

Genomic DNA of Pseudoperonospora cubensis was extracted by the improved CTAB method, effects of major components of PCR amplification on ITS detection results were studied and the optimal reaction system was established. The optimal PCR amplification system was： 40 ng each ITS universal primers ITS1 and ITS4, 60 ng DNA template, 2.0 μL 2.5 mmol·L^-1 Mg^2＋, 1.5 μL 2.5 mmol·L^-1 each dNTP, 1 U Taq polymerase. The optimized PCR cycles started at 94 ℃ 3 min, followed by 35 cycles of 25 s at 94 ℃, 40 s at 52.2 ℃ and 1.5 min at 72 ℃, and then extended 7 min at 72 ℃. Using the established system, all the 31 genomic DNA samples could obtain an approximate 900 bp band, which collected from different regions of Heilongjiang Province.