应用纳米磁珠荧光PCR检测棉花曲叶病毒

摘要

为了提供快速灵敏检测棉花曲叶病毒(Cotton leaf curl virus,CLCuV)的技术以防止其传播扩散,本研究根据CLCuV外壳蛋白基因(Coat protein,CP)保守序列设计了引物和TaqMan探针,并结合纳米磁珠(Magnetic nanoparticles,MNPs)建立了该病毒的MNP实时荧光PCR (Real-time PCR)检测方法.该方法检测阈值约为525 fg·μL-1 DNA.通过对CLCuV、非洲木薯花叶病毒、烟草线条病毒、黄瓜花叶病毒及番茄斑萎病毒的检测表明,该方法具有良好的特异性；同时该方法无需任何PCR后处理,交叉污染风险小.本方法可用于CLCuV的快速检测.%For developing a rapid and sensitive detection technique to detect cotton leaf curl virus (CLCuV) in order to prevent its introduction and spread, we have developed a Real-time TaqMan PCR combining with magnetic nanoparticles (MNPs) method. By using TaqMan probe combining with MNPs technique and coat protein gene as the target gene, the MNP fluorescent PCR detection method was established after specificity and sensitivity experiments. The sensitivity of the method was 525 fg ?u.L' of DNA, which was same that of the conventional PCR gel electrophoresis method. The good specificity of this method was evaluated by applying the proposed method to detect five viruses, CLCuV, African cassava mosaic virus (ACMV), Tobacco streak virus (TSV), Cucumber mosaic virus (CMV) and Tomato spotted wilt virus (TSWV). The MNP fluorescent PCR method was rapid and accurate for CLCuV detection of field leaf samples and could reduce the risk of cross-contamination without any PCR post-processing, which is a feasible technique to routine testing assays.