黄瓜花叶病毒NASBA检测技术的建立

 以香蕉花叶病病样为材料,初步建立了黄瓜花叶病毒核酸序列依赖性扩增(Nucleic acid sequence based amplifica-tion,NASBA)的检测技术。通过以香蕉叶片总RNA为模板,在黄瓜花叶病毒(Cucumber mosaic virus,CMV)亚组ⅠRNA 2高保守区设计特异引物,进行NASBA反应,经5%琼脂糖凝胶电泳检测,阳性样品中出现了预期大小为310 bp的条带,而阴性和空白对照中均未出现。并对11份香蕉样品分别进行NASBA反应,并经过斑点杂交验证与RT-PCR检测比较,两者的检测结果一致,灵敏度相当,检出限量可达100 pg。

Establishment of nucleic acid sequence based amplification system for detection of Cucumber mosaic virus

The diseased samples of banana infected with CMV(Cucumber mosaic virus) were collected and a nucleic acid sequence based amplification(NASBA) detection system was developed.The specific primers were designed according to the high conserved region of RNA 2 sequence of CMV subgroupⅠ.The 310 bp specific amplification product was obtained in positive sample determined with 5% agarose gel electrophoresis.The 11 different banana samples were analyzed with NASBA and RT-PCR to find out reliability of the me-thod,the former was demonstrated by dot blot with digoxigenin-labeled probe and had the similar results with RT-PCR.Further,the sensitivities were also identical and was equal to 100 pg.