An Improved Barcoded Oligonucleotide Primers-based Next-generation Sequencing Approach for Direct Identification of Viral Pathogens in Clinical Specimens

查看全文 作　　者：WANG Churl [1]Hua;NIE [1]Kai;ZHANG [1]Yi;WANG [1]Ji;ZHOU Shuai [1,2]Feng;LI Xin [1]Na;ZHOU Hang [1]Yu;QI Shun [3]Xiang;MA Xue [1]Jun 机构地区：[1]Key Laboratory for Medical Virology, National Health and Family Planning Commission, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China;[2]Center for Disease Prevention and Control of Hunan Province, Changsha 410005, Hunan, China;[3]Institute for Viral Disease Control and Prevention, Center for Disease Control and Prevention of Hebei, Shijiazhuang 050000,Hebei, China 出　　处：《Biomedical and Environmental Sciences》索引2017年第30卷第1期,共13页 基　　金：supported by the China Mega-Project for Infectious Disease(2016ZX10004-101,2016ZX10004-215);Beijing Municipal Science&Technology Commission Project(D151100002115003);Guangzhou Municipal Science&Technology Commission Project(2015B2150820) 摘　　要：Objective To provide a feasible and cost-effective next-generation sequencing(NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples. Methods Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide(random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline. Results NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease(HFMD) and identified other potential viruses or differentiated misdiagnosis events. Conclusion The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice. 关 键 词：肠道病毒 临床应用 寡核苷酸 直接识别 测序方法 病原体 条形码 引物