小苍兰3种病毒多重RT-PCR检测体系的建立

摘要

根据GenBank中已发表的小苍兰花叶病毒Freesia mosaic virus (FreMV)、黄瓜花叶病毒Cucumubermosaic virus (CMV)和菜豆黄花叶病毒Bean yellow mosaic virus (BYMV)外壳蛋白(CP)基因序列保守区域分别设计特异性引物,通过优化多重PCR反应条件,建立能同时检测小苍兰3种病毒的多重PCR检测体系.该体系能够一次扩增出FreMV,CMV和BYMV的特异片段,其大小分别是340、628和212 bp.测序结果表明,3种病毒序列与相应的参考序列相似性均达97％以上.灵敏度测定结果表明,从≥10-2mg的感病植物组织中能够检测到这3种病毒.%By using the specific primers designed on the basis of conserved sequences of coat protein (CP) gene, a multiplex PCR protocol for the detection of three main viruses (i. e. , Freesia mosaic virus, FreMV, Cucumber mosaic virus, CMV and Bean yellow mosaic virus, BYMV) in freesia was developed. Three specific fragments were simultaneously amplified in a single PCR reaction. Their molecular weights were determined to be 340 bp, 628 bp and 212 bp. The sequence analysis indicated that the three viruses shared at least 97 percent homology with other related viruses. The method's sensitivity was shown to be capable of positively identifying the three viruses with≥ 10-2mg of freesia tissue sample.