论文部分内容阅读
分别以感染百合无症病毒、黄瓜花叶病毒和百合斑驳病毒的百合及感染烟草花叶病毒和马铃薯病毒Y的烟草为试材,根据5种植物病毒外壳蛋白基因保守序列设计引物和寡核苷酸探针,并制备基因芯片。用Trizol试剂盒提取感染病毒的植物总RNA,荧光RT-PCR产物与芯片杂交,研究PCR产物是否进行变性处理、杂交时间、杂交温度、杂交液组分SSC和SDS浓度及PCR体系中非荧光引物和荧光引物比例对芯片杂交的影响。结果表明:杂交适宜条件为6×SSC、0.2%SDS的杂交液、42℃杂交60min,PCR体系中非荧光与荧光引物比例为1∶10,PCR产物要进行变性处理。经过整体条件优化后的基因芯片在杂交检测上具有较高的特异性,适于检测百合病毒病。
The primers and oligonucleotides were designed based on the conserved sequences of five plant virus coat protein genes, respectively, using lily of the lily infective virus, cucumber mosaic virus and lily mottle virus and tobacco infected with tobacco mosaic virus and potato virus Y as materials, Acid probe and prepare gene chip. Trizol kit was used to extract the total RNA of infected plant virus. Fluorescent RT-PCR products were hybridized with the chip to study whether the PCR products were denatured, hybridization time, hybridization temperature, SSC and SDS concentrations of hybridization solution and non-fluorescent primers And fluorescent primer ratio on the chip hybridization. The results showed that the optimum conditions for hybridization were 6 × SSC and 0.2% SDS, and hybridized at 42 ℃ for 60 min. The ratio of non-fluorescent and fluorescent primers in the PCR system was 1:10, and the PCR products were denatured. After the overall optimization of the gene chip hybridization test has a high specificity, suitable for detecting lily virus disease.