植物病毒检测芯片的杂交条件优化

利用芯片点样仪将5种侵染马铃薯的病毒/类病毒(苜蓿花叶病毒、黄瓜花叶病毒、黄瓜花叶病毒-卫星病毒、马铃薯病毒Y、马铃薯块茎纺锤状类病毒)的保守区寡核苷酸(Oligonucleotide,oligo)探针和PCR探针点样于玻片，并以植物18S rRNA作为内参照制成基因芯片。研究探针浓度、杂交时间、杂交温度以及点样液对芯片杂交的影响，并验证优化后病毒检测芯片的特异性。结果表明，寡核苷酸探针浓度介于5-20 ?mol/L之间对杂交信号强度影响不大，PCR探针浓度与杂交信号强度间呈线性关系；在45℃杂交4 h时，芯片的杂交信号最强，且该条件下进行杂交对两种探针芯片的影响趋势一致；点样液中以DMSO的杂交效果最好。经过整体条件优化后的两种探针芯片在杂交检测上具有较高的特异性，适于检测植物病毒。

Optimization of Hybridization Condition for Plant Virus Detection Array

Based on the conserved region nucleotide sequences of four viruses (Alfalfa Mosaic Virus, ALMV; Cucumber mosaic virus, CMV; Cucumber mosaic virus-satellite, CMV-sat; Potato virus Y, PVY) and one viroid (Potato spindle tuber viroid, PSTVd) which could be pathogenic on potato world and one inner control(18S rRNA), specific oligonucleotide probes and primers were designed and spotting for hybridization detection.There is examine the influence of probe concentration、hybridization time、hybridization temperature and spotting solutions on microarray signal.Finally there is validate the specificity of optimized plant virus detection array.The experiment indication: There is not significant influence on hybridization signals with the concentration of the oligo probes between 5-20 µM,PCR probe show the linear relationship in influence of hybridization signals. Microarray hybridization at 45℃ for 4 h, the intensity of hybridization signals was the most significant, and under this condition the influence tendency on oligonucleotide probes and PCR probes was nearly same. With the comparison of different spotting solutions, DMSO had the best spotting reproducibility. Through the whole condition optimized, the oligonucleotide probe microarray and PCR probe microarray were provided with better specificity in the hybridized detection.