正交设计优化南方根结线虫RAPD-PCR体系

Establishment and optimization of RAPD-PCR reaction system for Meloidogyne incognita using orthogonal design

以南方根结线虫(Meloidogyne incognita)DNA为模板,对影响南方根结线虫RAPD-PCR扩增的重要参数进行了优化试验,以期建立南方根结线虫RAPD-PCR反应最佳体系。应用L16(45)正交设计研究了Taq酶、10×PCR buffer、dNTP、primer、模板DNA对扩增反应的影响。结果表明,南方根结线虫RAPD-PCR优化体系为20μL体系中含模板3μL、10μmol/L引物1.5μL、10×反应缓冲液2.5μL、2.5 mmol dNTP mixture 2.4μL、Taq DNA聚合酶0.4μL。在此基础上筛选出扩增稳定、多态性丰富的RAPD引物,并通过梯度PCR试验,确定了引物最佳退火温度。该优化RAPD-PCR反应体系具有良好的稳定性和重现性,可应用于南方根结线虫不同居群间亲缘关系和遗传多样性的分析。

The orthogonal design was used to optimize RAPD amplification system of Meloidogyne incognita with five factors （Taq polymerase, PCR buffer, dNTP, primer and DNA template） at four levels, respectively. Through the deep analysis, a suitable RAPD-PCR reaction system was established, namely 20 p,L reaction system containing DNA template 3 p,L, 10xPCR buffer 2.5 p,L, 2.5 mmol dNTP mixture 2.4 p.L, Taq polymerase 0.4 μL, 10 μmol/L primers 1.5μL with stable amplification and rich polymorphism for RAPD were screened. The optimal annealing temperature of some primers for RAPD-PCR reaction was proposed by gradient PCR. The results provided a standardizing RAPD-PCR program for analysis of genetic diversity of Meloidogyne incognita.