百合病毒检测芯片的杂交条件优化

Optimization of Hybridization Condition for Lily Virus Detection Microarray

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。

In the present study the lily infected by Lily symptomless virus（LSV）,Cucumber mosaic virus（CMV）,Lily mottle virus（LMoV）and tobacco infected by Tobacco mosaic virus（TMV）,Potato virus Y（PVY）were chosen as test material.Based on conserved coat protein region nucleotide sequences of five kinds of viruses,primers and probes were designed and the microarray were designed and fabricated.Total RNA of infected plant was extracted by the Trizol regent,and products that Cy3 labeled RT-PCR amplification hydrid with the microarray.The effects of whether the PCR products were denatured,hybridization time,hybridization temperature,hybridization buffer and the ratio of non-market primer to market primer with fluorescence in PCR system on microarray hybridization were evaluated.The result showed that the optimal hybridization time was 60minutes;temperature was 42℃;SSC concentration was 6×SSC,SDS was0.2%;The ratio of non-market primer to market primer with fluorescence was 1∶10in the PCR system,and the PCR products must to be degenerated before hybridization.After the overall optimization,the gene chips had a high specificity in the hybridization,and could be used for detection of lily viruses fast and accurately.