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建立非洲木薯花叶病毒(African cassava mosaic virus,ACMV)灵敏快速的纳米磁珠实时荧光PCR检测技术,防止其传入中国。采用TaqMan探针结合纳米磁珠(magnetic nanoparticles,MNPs)技术,以外壳蛋白基因为目标基因,通过特异性及灵敏度试验建立MNP荧光PCR检测方法。成功建立了非洲木薯花叶病毒(ACMV)的MNP荧光PCR检测方法;该方法检测阈值约为13pg DNA,比普通PCR及ELISA方法灵敏度高25倍;该方法具有良好的特异性,能够有效区分双生病毒属的ACMV、棉花曲叶病毒(CLCuV)、番茄黄化曲叶病毒(TYLCV)及番茄严重曲叶病毒(ToSLCV)。MNP荧光PCR方法能够快速灵敏的检测茎叶样品中的ACMV,无需任何PCR后处理,交叉污染风险小,适于口岸检验检疫。
To establish a sensitive and rapid nano-magnetic bead real-time fluorescence PCR detection technology for African cassava mosaic virus (ACMV) to prevent its introduction into China. Using TaqMan probe combined with magnetic nanoparticles (MNPs) technology, the coat protein gene was used as target gene to establish MNP fluorescent PCR detection method by specificity and sensitivity test. MNP fluorescent PCR detection method of African cassava mosaic virus (ACMV) was successfully established. The method has a threshold of about 13 pg DNA and is 25 times more sensitive than the common PCR and ELISA methods. The method has good specificity and can effectively distinguish twins ACMV, Corynebacterium curviruses (CLCuV), TYLCV and ToSLCV. MNP fluorescence PCR method can quickly and sensitively detect ACMV in stem and leaf samples without any post-PCR treatment, and has the advantage of low cross-contamination risk and is suitable for port inspection and quarantine.