高通量技术在重症肺炎患者呼吸道病原体快速检测中的应用

Rapid detection of high-throughput techniques for respiratory pathogens in patients with severve pneumonia

目的探讨不依赖序列的单引物扩增技术结合Ion Torrent高通量测序平台对重症肺炎患儿呼吸道肺泡灌洗液进行病毒病原体宏基因组检测的效果。方法取临床重症肺炎患者肺泡灌洗液标本,进行病毒基因组核酸DNA/RNA的提取,随机引物扩增建库后,进行Ion Torrent高通量测序分析,测序结果进行本地blast分析筛选病毒序列,运用MetaVir数据库进行病毒归类分析。结果一共获得591 593条序列读长,病毒序列3 545条,占0.6%,其中主要病毒及序列有腺病毒1 760条(49.6%),淋巴囊肿疾病病毒1 422条(40.1%),Taterapox病毒65条(1.8%),智利巨型病毒37条(1.0%),云杉卷蛾雕背姬蜂病毒33条(0.9%),猫白血病病毒32条(0.9%),网状内皮组织增生证病毒30条(0.8%),RD114逆转录病毒18条(0.5%);呼吸道病原体荧光定量聚合酶链反应结果为腺病毒7型,提示患者腺病毒7型感染。结论该研究初步建立的病毒宏基因组分析技术平台可用于呼吸道相关已知和未知病毒病原体鉴定。

Objective To investigate the effect of sequence independent single-primer amplification technol-ogy combined with Ion Torrent high throughput sequencing platform on viral pathogen macro genomic detec-tion in respiratory bronchoalveolar lavage fluid（BALF） in children with severe pneumonia. Methods Total vi rus DNA/RNA was extracted from BALF specimen of a child with severe pneumonia,a library contained DNA/RNA virus was constructed with the SISPA method and subjected to sequecing with the Ion Torrent PGM. A stand alone blast was analyzed to filter the reads belong to different species. Using the MetaVir database for virus classification analysis. Results A total of 591 593 reads was obtained,of which 3 545 （0. 6%） reads belong to virus. Adenovirus, lymphocystis disease virus, Taterapox virus, Chiliensis megavirus, Glypta fumiferanae ichnovirus, feline leukemia virus, reticuloendotheliosis virus and RDl14 retrovirus account for 49.6% ,40.1% ,1.8% ,1.0% ,0.9% ,0.9%,0.8% and 0.5% of the total virus reads,respectively. Futher anal ysis BALF with real time PCR demonstrated that adenovirus type 7 was the pathogen of this severe pneumonia,suggesting adenovirus type 7 infection. Conclusion A virus macro genomic analysis technology based on high throughput sequencing platform is initially established which can be used for identification of known and unknown viral s pathogens in the respiratory tract.