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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。
In order to establish the best RAPD-PCR reaction system for Meloidogyne incognita DNA, we optimized the RAPD-PCR amplification of Meloidogyne incognita. The effect of Taq polymerase, 10 × PCR buffer, dNTP, primer and template DNA on the amplification reaction was studied by L16 (45) orthogonal design. The results showed that the optimized system of RAPD-PCR was 20μL containing 3μL of template, 1.5μL of 10μmol / L primer, 2.5μL of 10 × reaction buffer, 2.4μL of 2.5mM dNTP mixture and 0.4μL of Taq DNA polymerase. On the basis of this, the RAPD primers with stable amplification and rich polymorphism were screened and the optimal annealing temperature was determined by gradient PCR. The optimized RAPD-PCR reaction system has good stability and reproducibility and can be applied to analyze the genetic relationship and genetic diversity among different populations of Meloidogyne.