Chumakov Federal Scientific Center for Research and Development of Immune and Biological Products of Russian Academy of Sciences, Moscow, Russian Federation.
Research Institute of Occupational Health, Moscow, Russian Federation.
Sci Rep. 2017 Aug 29;7(1):9752. doi: 10.1038/s41598-017-10342-w.
Despite recent advances, our knowledge of potential and rare human pathogens is far from exhaustive. Current molecular diagnostic tools mainly rely on the specific amplification of marker sequences and may overlook infections caused by unknown and rare pathogens. Using high-throughput sequencing (HTS) can solve this problem; but, due to the extremely low fraction of pathogen genetic material in clinical samples, its application is only cost-effective in special, rather than routine, cases. In this study, we present a method for the semi-specific enrichment of viral conservative sequences in a HTS library by hybridization in solution with genus-specific degenerate biotinylated oligonucleotides. Nucleic acids of the test viruses (yellow fever virus and Japanese encephalitis virus) were enriched by solution hybrid selection using pan-flavivirus oligonucleotides. Moreover, enterovirus (family: Picornaviridae, genus: Enterovirus) sequences were successfully enriched using foot-and-mouth disease virus (family: Picornaviridae, genus: Aphthovirus) oligonucleotide. The enrichment factor relative to the background nucleic acid was about 1,000-fold. As hybridization has less stringent oligonucleotide match requirements than PCR, few oligonucleotides are sufficient to cover the potential sequence variation in the whole genus and may even enrich nucleic acids of viruses of other related genera. Efficient enrichment of viral sequences makes its use in diagnostics cost-efficient.
尽管最近取得了一些进展，但我们对潜在的和罕见的人类病原体的了解还远远不够。目前的分子诊断工具主要依赖于标记序列的特异性扩增，可能会忽略未知和罕见病原体引起的感染。使用高通量测序（HTS）可以解决这个问题；但是，由于临床样本中病原体遗传物质的比例极低，其应用仅在特殊情况下，而不是常规情况下，具有成本效益。在本研究中，我们提出了一种通过与属特异性的简并生物素化寡核苷酸在溶液中杂交，对 HTS 文库中的病毒保守序列进行半特异性富集的方法。使用泛黄病毒寡核苷酸对测试病毒（黄热病病毒和日本脑炎病毒）的核酸进行溶液杂交选择富集。此外，使用口蹄疫病毒（小 RNA 病毒科，口疮病毒属）寡核苷酸成功富集了肠道病毒（小 RNA 病毒科，肠道病毒属）序列。与背景核酸相比，富集因子约为 1000 倍。由于杂交比 PCR 具有更宽松的寡核苷酸匹配要求，因此只需少量寡核苷酸即可覆盖整个属的潜在序列变异，甚至可能富集其他相关属病毒的核酸。病毒序列的高效富集使得其在诊断中的应用具有成本效益。
