【摘要】 目的探讨有利于制备多种病毒长探针平行检测芯片的最佳制备及杂交条件。方法通过引物直接标记方法,对70mer探针寡核苷酸芯片的点样浓度、点样后固定方案、杂交温度、杂交时间进行优化。结果70mer寡核苷酸探针点样浓度在10μmol/L时就可获得较高的的荧光强度,探针点样干燥后在3600mJ条件下紫外交联固定效果最好,在65℃条件下杂交8h能获得最优杂交结果,所用杂交液是5×SSC(0.5%SDS、50%甲酰胺、1%鲑鱼精DNA)。结论本实验优化了70mer探针寡核苷酸芯片制备及杂交条件;此为进一步研制多种海洋环境人致病性病毒检测芯片奠定了基础。更多还原

【Abstract】 Objective To optimize the preparation and hybridization conditions of long oligonucleotide microarray for Astrovirus detection.Methods To optimize the probe concentration spotted on the slide,UV cross-linking energy,the hybridization time and temperature by dye primer.Results The stable hybridization signal was obtained at the probe spotting concentration of 10 μmol/L.On the condition cross-linking at 3 600 mJ we got best immobilization results.The optimized hybridization buffer is composed of 50 % formamide,5×SSC,0.5 %SDS and 1% salmon sperm DNA.The optimal hybridization time and temperature was 8 hours and 65 ℃ respectively.Conclusion The preparation and hybridization conditions of 70 mer oligonucleotide microarray was eptimized.This study may lay the foundation for the parallel detection of different species of viruses in marine environment.更多还原