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描述了一种基于双倍PCR技术 ,用种特异性引物快速鉴定大豆胞囊线虫方法。应用两组引物扩增胞囊线虫的rDNA。第一组引物含有2个通用引物D3A和D3B ,用于扩增大亚基核糖体基因(28SrDNAgene)的D3扩展区 ;第二组引物包括大豆胞囊线虫种特异性引物GlyF1和一个通用引物rDNA2,用于扩增ITS2区的片断和部分28S基因。用此双倍PCR扩增技术检测了来自中国、俄罗斯、美国和巴西的53个大豆胞囊线虫群体 ,结果表明 ,用两组引物进行双倍PCR扩增 ,所有53个大豆胞囊线虫群体都产生两个明显的片断(181bpand345bp) ,其中181bp片断是SCN特异性引物GlyF1和rDNA2扩增出大豆胞囊线虫中特异性片断。在所测试的其它胞囊线虫群体 ,仅仅扩增出345bp的片断 ,而不能扩增出大豆胞囊线虫的特异性片断。用该项技术能从大豆胞囊线虫的单个胞囊或单头二龄幼虫的微量DNA中 ,扩增出大豆胞囊线虫的特异性片断 ,该技术的敏感性达到单条二龄幼虫的水平
A method for rapid identification of soybean cyst nematode using species-specific primers based on double-PCR was described. Two groups of primers were used to amplify rDNA of cyst nematode. The first set of primers contained two universal primers, D3A and D3B, for amplifying the D3 extension of the large subunit ribosomal gene (28S rDNAgene). The second set of primers included the Glycine max and the universal primer rDNA2 , Used to amplify the ITS2 region and part of the 28S gene. A total of 53 Soybean cyst nematode populations from China, Russia, the United States and Brazil were detected by this double PCR amplification. The results showed that all 53 soybean cyst nematode populations Two distinct fragments (181bp and 345bp) were generated. The 181bp fragment was amplified by specific primers SCN specific GlyF1 and rDNA2. In other cyst nematode populations tested, only the 345 bp fragment was amplified, but no specific fragment of soybean cyst nematode was amplified. Using this technique, specific fragments of Soybean cyst nematode can be amplified from the single cyst of Soybean cyst nematode or the trace DNA of single-staged second instar larvae, and the sensitivity of this technique reaches the level of single second instar larvae