一种基于多重PCR的基孔肯雅病毒全基因组高通量测序方法的建立  

Establishment of a high-throughput sequencing platform for the whole genome of Chikungunya virus based on a multiplex-PCR method

作　　者：苏文哲[1] 李岩[2] 陆维智 谢华萍[1] 李魁彪[1] 狄飚[1] 聂凯 王环宇[3] 张周斌[4] 许松涛 Su Wenzhe;Li Yan;Lu Weizhi;Xie Huaping;Li Kuibiao;Di Biao;Nie Kai;Wang Huanyu;Zhang Zhoubin;Xu Songtao(Department of Virology,Guangzhou Center for Disease Control and Prevention,Guangzhou 510440,China;Institute of Infectious Disease Control and Prevention,Shandong Center for Disease Control and Prevention,Jinan 250000,China;National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention/State Key Laboratory for Infectious Disease Control and Prevention,Beijing 102206,China;Director′s Office,Guangzhou Center for Disease Control and Prevention,Guangzhou 510440,China)

机构地区：[1]广州市疾病预防控制中心病毒免疫部,广州510440 [2]山东省疾病预防控制中心传染病防制所,济南250000 [3]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室,北京102206 [4]广州市疾病预防控制中心主任室,广州510440

出　　处：《中华预防医学杂志》2024年第4期489-496,共8页Chinese Journal of Preventive Medicine

基　　金：广州市医学重点学科(2021-2023-11);广州市科技计划项目(202002030117、2023A03J0447)。

摘　　要：目的:建立基于多重PCR的基孔肯雅病毒全基因组高通量测序方法。方法:通过NCBI网站检索和下载CHIKV的各基因型序列,经序列比对筛选出备选参考序列,使用Primalscheme和Geneious Prime等软件进行引物方案的设计、筛选和人工优化,并使用7份CHIKV阳性样品对实验流程进行评估。结果:本研究所设计的引物方案可对各阳性样品进行成功扩增;在样品Ct值小于33时,可获得完整编码区序列;样品Ct值达到35时,可获得99.38%的编码区序列;使用多次迭代,逐步校正的拼接策略可将拼接成功率提高5.70%~25.43%。结论:建立的基于多重PCR的基孔肯雅病毒全基因组高通量测序方法操作简单,对多个浓度的基孔肯雅病毒阳性临床样品均表现良好。ObjectiveTo establish a rapid pipeline for whole genome sequencing of Chikungunya virus(CHIKV)by combining imbricated multiplex-PCR amplification and Illumina high-throughput sequencing platform.MethodsThe primary reference sequences of CHIKV were downloaded from the National Center for Biotechnology Information(NCBI)database,covering all genotypes of CHIKV.After multiple alignments using the Mafft software and phylogenetic analysis,the 20 CHIKV references were selected for primer design.The Primal Scheme tool and Geneious Prime software were used to design,evaluate and optimize the primer panel.Finally,seven CHIKV-positive samples were involved in the validation of the primer panel.ResultsAll the amplicons of the designed panel were generated successfully.The consensuses generated from the mapping results could cover 100.00% of the coding region of the CHIKV genome when the Ct-value of the sample was less than 33,as the percentage would decrease to 99.38%when the Ct-value reached 35.The mapping percentage could be increased by 5.70%-25.43%when using the stepwise correction mapping strategy.ConclusionThe multiplex-PCR amplification method for CHIKV whole genome sequencing is relatively simple and convenient,which only requires two tubes of PCR amplification and performs well on CHIKV-positive clinical samples with different concentration levels of virus.

关 键 词：基孔肯雅病毒 全基因组测序 多重PCR 高通量测序 

分 类 号：R446.5[医药卫生—诊断学]