应用序列非依赖扩增技术检测儿童呼吸道标本中潜在病毒病原体

摘要

目的 应用序列非依赖扩增技术(sequence-independent amplification,SIA)检测常见病毒筛查阴性的5岁以下急性呼吸道感染患儿的呼吸道标本中可能存在的潜在病毒病原体,了解SIA扩增文库中各种背景核酸的组成.方法 随机选择45份常见病毒筛查阴性的5岁以下急性呼吸道感染患儿的鼻咽吸出物,0.45μm过滤和DNase/RNase处理去除病毒颗粒外的各种外源性核酸,再通过序列非依赖扩增技术对处理后的标本提取的核酸进行扩增,继而对扩增产物进行克隆、测序和BLAST比对.结果 测序403个克隆,获得有效序列368个,检出16个(16/368,4.3％)真核病毒同源序列,分别与Torque teno mini virus,Torque teno midi virus和Human bocavirus同源.此外,还检出1个真菌病毒( sclerotinia sclerotiorum hypovirulence associated DNA virus 1)同源序列和5个细菌病毒(噬菌体)同源序列.其余检出序列包含206个( 206/368,56.0％)与人基因组DNA同源的序列,11个(11/368,3.0％) rRNA同源序列,72个(72/368,19.6％)细菌同源序列,4个(4/368,1.1％)真菌同源序列,5个(5/368,1.4％)寄生虫同源序列,6个(6/368,1.6％)食源性序列,以及36个(36/368,9.8％)未能确定分类的序列.结论 核酸消化结合SIA方法可以检出常规检测方法所无法检出的潜在病毒病原体,本研究为后续系统性的查找和监测未知病毒提供了基础.%Objective Application of sequence-independent amplification (SIA) to identify the potentially viral pathogens in the clinical respiratory samples of children with acute respiratory tract infection of unknown etiology and characterize the composition of various non-viral sequences in the library of SIA amplicons.Method 45 randomly selected pediatric nasopharyngeal aspirate(NPA) samples for which no causal agent could be identified by common viruses screening were subjected to filtration & DNase/RNase treatments to remove the non-viral nucleic acid and then followed by SIA,balanced mixture of the each SIA amplicons was then cloned and sequenced,target sequences were then subjected to the BLAST search.Result A total of 403 clones were selected for sequencing and 368 sequences were validated,among them 16( 16/368,4.3％ ) sequences of eukaryotic virus from 4 specimens were identified:12 Torque teno mini virus,1Torque teno midi virus and 3 Human bocavirus homologous sequences.In addition,1mycovirus ( Sclerotinia sclerotiorum hypovirulence associated DNA virus 1) and 5 phage homologous sequences were also detected.The remaining non-viral sequences included 206 ( 206/368,56.0％ ) Homo sapiens,11( 11/368,3.0％ ) rRNA,72 (72/368,19.6％ ) bacterial,4 (4/368,1.1％ ) fungal,5 ( 5/368,1.4％ )parasitic,6(6/368,1.6％ ) foodborne and 36 ( 36/368,9.8％ ) unmatched sequences.Conclusion As for NPA samples,host nucleic acid depletion combined with SIA can be used to screen for the undetectable viruses that had not been covered by the general diagnostic techniques.Present study provided baseline information for the subsequent researches on the identification of unknown viruses.