属级芯片筛查技术在马铃薯纺锤块茎类病毒属检测中的应用

Application of screening microarray technology in genus level for detection of Pospiviroid

摘要:

建立马铃薯纺锤块茎类病毒属 (Pospiviroid) 类病毒有效的芯片筛查技术，对该属类病毒进行筛查。分析了该属类病毒序列，得到19条具有属级鉴定特征的探针。这些探针符合GC含量在40%与60%之间、单个核苷酸含量不大于50%、无多于4个核苷酸的连续重复及不形成多于6个核苷酸的发卡结构的标准；将探针点制到玻璃片基上制备芯片。芯片探针有效性实验结果表明，菊花矮化类病毒 (Chrysanthemum stunt viroid, CSVd) 及番茄雄性株类病毒 (Tomato planta macho viroid, TPMVd) 样品杂交可获得有效信号；灵敏度实验结果表明，该芯片可检测到200 pg/μL的总RNA。该芯片可用于Pospiviroid类病毒的筛查。

Abstract:

The aim was to establish an effective screening microarray at genus level for Pospiviroid. We analyzed nucleotide sequences from Pospiviroid viroid and designed 19 probes with genus identification characteristics. The standards of these probes included the characters of (i) a GC content between 40 and 60%, (ii) less than 50% of single nucleotide, (iii) less than 4 continuous mononucleotides, and (iv) less than 6 nucleotides in the inner hairpin. We synthesized microarrays by using these probes on glass slides. The validation results of microarray probes show effective signals from chrysanthemum stunt viroid and tomato planta macho viroid standard samples hybridization. The sensitivity results show that the microarray detected 200 pg/μL of total RNA. The microarray can be used to screen Pospiviroid viroid.