从混合线虫样品和植物组织中直接检测香蕉穿孔线虫的ITS-PCR方法

摘要

[目的]建立直接从多种线虫混合样品以及香蕉和红掌根组织中检测鉴定香蕉穿孔线虫的PCR方法.[方法]使用Primer Premier 5.0在香蕉穿孔线虫的ITS区设计1对特异性引物,运用PCR技术对目的线虫DNA进行特异性检测.[结果]通过对17种24个种群线虫的检测表明,所设计的引物只能从香蕉穿孔线虫种群中特异扩增出rDNA-ITS片段,产物大小为518 bp.利用该特异引物以及建立的DNA提取方法和PCR体系,可以直接从香蕉穿孔线虫与短体线虫、螺旋线虫、肾状线虫、根结线虫、茎线虫、矮化线虫、纽带线虫、丝尾垫刃线虫、滑刃线虫和小杆线虫混合样品中特异扩增出目的线虫的rDNA-ITS片段,并可以分别从混合有不少于3条香蕉穿孔线虫的2 cm香蕉或红掌根组织(约0.1g)中特异检测出目的线虫的DNA片段. [结论]本研究设计的特异性引物以及建立的DNA提取方法和PCR体系可直接从多种线虫混合的样品以及香蕉和红掌根组织中快速检测鉴定出香蕉穿孔线虫.%[ Objective ] The objective of this study is to build an ITS-PCR (internal transcribed spacer-polymerase chain reaction) method for detecting Radopholus similis (Cobb) Thome from mixtures of different nematodes, or tissues of Musa spp. And Anthurium andraeanum. [Method] A specific primer pair was designed according to ITS region of Rdna from R. Similis using Primer Premier 5.0. A single-step PCR method was used to specially detect the target ITS-Rdna of R. Similis. [Result] The specificity of the designed primers was confirmed that a 518 bp PCR product was amplified from 7 populations of R. Similis, not from the other 16 species including 17 populations. Using the established DNA extraction method and PCR system by the specific primers, the target ITS-Rdna fragment was specially amplified from DNA templates of the mixture of individuals of/?, similis with species of Pratylenchus, Helicotylenchus, Rotylenchulus, Meloidogyne, Ditylenchus, Tylenchorhynchus, Hoplolaimus, Filenchus, Aphelenchoides, and Rhabditida, and also from the DNA mixtures of at least 3 individuals of R. Similis with root tissues (2 cm long, about 0.1 g) of banana and Anthurium andraeanum. I Conclusion ] The ITS-PCR method established in this study was proven to be a reliable and useful technique for fast and accurate detecting of R, similis from mixed nematode samples and mixtures of root tissues of banana or A.andraeanum.