摘要
目的构建基于靶向纳米孔测序的诺如病毒、腺病毒、肠道病毒等儿童易感病毒的多重快速检测和分型方法,并对其进行方法学验证。方法提取待测样本的核酸,对诺如病毒、肠道病毒先进行逆转录,然后利用上述病毒特定基因片段的混合引物对扩增目标核酸;再构建文库并进行纳米孔测序,通过生物信息学分析鉴定病毒种类和基因型;最后,以临床样本和人工污染牡蛎样本进行方法学验证。结果该方法能在6小时内现场完成快速检测,可精准鉴定临床腹样本中的病毒类型和基因型,最高检测限Ct值达40。在人工污染诺如病毒的牡蛎样本检测中,本方法的检测灵敏度与实时荧光PCR相当,并可精准鉴定诺如病毒基因型。结论成功建立了一种基于靶向纳米孔测序的诺如病毒、腺病毒、肠道病毒等病毒的多重快速检测和分型方法,可满足相关监管部门对未知病毒样本的高通量快速检测需求。
Objective To develop a multiplex rapid detection and typing method for viruses susceptible in children,such as norovirus,adenovirus,and enterovirus based on targeted nanopore sequencing technology,and perform methodological verification.MethodsThe nucleic acids extracted from test specimens were applied to reverse transcription for norovirus and enterovirus.The mixed primers of the specific gene fragments of the above viruses were used to amplify the target nucleic acid for library preparation and nanopore sequencing.The species and genotypes of the viruses were identified by bioinformatics method.Finally,clinical samples and artificially contaminated oyster samples were used for methodological verification.ResultsThe method completed multiplex rapid detection within 6 hours and precisely identified the species and genotypes of the virus in clinical samples.The maximum detection limit by Ct value reached 40.For the oyster samples that were artificially contaminated by norovirus,the detection sensitivity of this method was equivalent to that of real time PCR,and the genotypes of norovirus were precisely identified.ConclusionsA multiplex rapid detection and typing method for viruses such as norovirus,adenovirus,and enterovirus based on targeted nanopore sequencing technology was constructed.The method can meet the needs of high-throughput and rapid analysis of samples with unknown virus in relevant regulatory authorities.
作者
陈晓峰
聂旋
徐兴宇
唐旖
孙梦鸽
王大鹏
王鹏
毛凌峰
吕陈昂
Chen Xiaofeng;Nie Xuan;Xu Xingyu;Tang Yi;Sun Mengge;Wang Dapeng;Wang Peng;Mao Lingfeng;Lyu Chenang(Hangzhou Baiyi Technology Co.,Ltd.,Hangzhou 310051,China;School of Agriculture and Biology,Shanghai Jiao Tong University,Shanghai 200240,China;Microbiological Laboratory,Anhui Provincial Center for Disease Control and Prevention,Hefei 230601,China)
出处
《国际病毒学杂志》
北大核心
2025年第3期194-198,共5页
International Journal of Virology
基金
上海市科学技术委员会科技创新行动计划(22N31900100)。
