兰花5种病毒可视化基因芯片检测方法建立

Visual DNA microarrays for detection of five orchid virus

为建立运用可视基因芯片技术快速、准确检测兰花病毒的方法,选择黄瓜花叶病毒、齿舌兰环斑病毒、建兰花叶病毒编码外壳蛋白(coat protein,CP)基因、辣椒褪绿病毒编码核衣壳蛋白N基因、落葵皱纹花叶病毒编码CI蛋白基因为目标基因,设计引物和探针(5'标记一段poly T)。利用多重RT-PCR方法进行病毒核酸扩增,将扩增产物与固定于芯片的特异性探针杂交,经清洗、可视化显色后进行结果分析。在优化的检测条件下,本研究筛选出2组多重引物组合Cm(F2-R2a)、Ba(F1-R1);Or(F1-R1)、Cy(F2-R2)和Ca(F1-R1),5条特异性探针。所建立的可视基因芯片具有较好的特异性和重复性,可检测出病毒阳性质粒的量为不低于10~3拷贝·μL^(-1)。

In order to develop a rapid and accurate method based on the thin-film biosensor chips for the detection of orchid viruses, genes （coding coat protein of CMV, ORSV, CymMV; nucleocapsid protein N of CaCV; CI protein of BaRMV） were used as the target genes to design primers and probes. The olignucleotide probes were labelled with 5′polyT group. The nucleic acids were amplified by multiplex RT-PCR method, and then hybridized with specific probes fixed on the microarray followed by washing and visual color development. Results indicated that, under the optimum conditions, two groups of multiple primers, Cm（F2-R2a）, Ba（F1-R1） and Or （F1-R1）, Cy （F2-R2）, Ca （F1-R1） and five specific probes were screened out. The microarray method showed high sensitivity and specificity, which could detect no less than 10^3 copies·μL^-1 for virus positive plasmids.