目的 探索实验室高通量检测前临床样本中病毒核酸特异性纯化富集的方法.方法 以负链RNA病毒发热伴血小板减少综合征病毒(SFTSV)为模拟样本,针对病毒基因组S、M和L三个片段设计杂交探针,并进行5'端生物素修饰,与链霉亲和素修饰磁珠共价结合,纯化样本中SFTSV核酸,比较研究特异性纯化富集对样本二代测序效果的影响.结果 cDNA文库制备时全转录组扩增前或后进行特异性富集纯化均较不开展富集纯化有显著改善,有效数据比例分别为5.57％、6.39％、2.71％,差别具有显著性(x2=9 799.8,P＜0.001).对全转录组扩增前或后进行特异性富集纯化对测序结果进行比较分析时发现,扩增后进行纯化优于扩增前(P＜0.001).结论 基于核酸杂交的病毒基因组特异性富集纯化方法可有效降低二代测序中背景值,提高测序效率,具有高通量检测前特异性纯化富集样本的作用.%Objective To explore the specific purification and enrichment method for the viral genome in clinical samples before high-throughput detection.Methods The negative chain RNA virus fever with thrombocytopenia syndrome (SFTSV) as was taken the simulate samples and the hybridization probes of the segment S,M and L were desiqned respectively,modified the probes with biotin in 5 ' ends to covalent bonded with streptavidin magnetic beads to purify the viral genome and compared the influence of specific purification on the effect of high-throughput detection.Results Specific enrichment and purification for viral genome before sequencing can significantly increase the proportion of valid data and whole genome coverage.The valid data proportions of the groups purified before or after whole transcriptome amplification (WTA) were improved significantly compared to group without purifying.The valid data proportion respectively were 5.57％,6.39％,2.71％,and the difference was significant((x2 =9799.8,P ＜0.001)).And the effect of the group purified after whole transcriptome amplification was better than group purified before whole transcriptome amplification(P ＜ 0.001).Conclusions Viral genome specific enrichment and purification method based on nucleic acid hybridization can effectively reduce the background values in the second generation sequencing,improve the sequencing efficiency and play the role of purification and enriching the nucleic acid in samples specifically before high-throughput detection.
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