基于宏基因组学的鸭源样本中坦布苏病毒检测方法的建立

利用二代高通量测序技术建立检测鸭源样品中坦布苏病毒的方法。首先将样品过滤和经核酸酶处理,再利用等温链位移扩增(SPIA)技术快速制备样品总cDNA,最后经磁珠纯化和文库构建后,通过Ion Torrent进行高通量测序获得基因组信息。结果显示,通过SPIA扩增和核酸文库制备的优化,可从微量病原样本中获得327pmol/μL的核酸文库样本。Ion Torrent测序共获得1 725 436条reads,约6.2M数据,平均109bp。数据拼接并Blast发现,病原样品的核酸序列可注释鸭坦布苏病毒全部基因组数据,与首次发生在我国的鸭坦布苏病毒BYD-1株参考序列的同源性为100%。将所测病毒序列与其他5种黄病毒科成员进行同源性比对,发现与近3年发生在我国的鸭坦布苏病毒同源性最高,在98%以上。且NS基因进化树分析与鸭坦布苏病毒处于同一分支上,符合鸭坦布苏病毒特征。本研究建立的基于未知病原SPIA扩增结合高通量测序检测方法,可同步获知坦布苏病毒基因的核酸信息。

Establishment of Detecting Tembusu Virus Method from Duck Source Samples Based on Metagenomics

To explore the methods of high-throughput sequencing for Tembusu virus from duck source sam-ples based on metagenomics,first of all,interference was removed through filtering and nucleic acid enzyme treatment.Then,total cDNA was prepared by using isothermal strand-displacement amplification (SPIA) technology.The purification was made by using magnetic beads and the nucleotid library building,the cD-NA amplicons were sequenced by Ion Torrent,and the genome information was acquired.The results showed that 327 pmol/μL nucleic acid library was prepared from pathogenic samples through SPIA and op-timizing preparation of DNA library.1 725 436 reads,about 6.2 M data,were got by Ion Torrent sequen-cing,and average read was 109 bp.The nucleic acid sequence of pathogen samples can note entire genome data by Blast on NCBI with the pathogen analysis software,and the similarity was 100% compared to the BYD-1 strain reference sequence occurred first time in our country.In this study,the genome of QD strain has high similarity to duck Tembusu virus occurred during nearly three years compared with the other five flaviviruses,and the genetic relationship was up to 98%.Evolutionary tree of NS5 gene analysis showed that this detected strain and duck Tembusu virus were at the same branch,conforming to the Tembusu vi-rus's characteristics.The SPIA amplification combined with high-throughput sequencing method based on the unknown pathogens,genomic information can be synchronized to learn effectively.