利用长距离PCR扩增国内肠道病毒流行株基因组方法的建立和评价
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摘要
目的:通过调查近年来我国肠道病毒EV-71型和柯萨奇病毒A16型流行株的全基因组序列,建立一种能够获得我国肠道病毒序列的通用扩增方法,为今后的手足口病流行病学分析、致病机理研究等打下基础。方法:收集我国近5年各地报道的肠道病毒流行株全基因组序列作为参考序列进行比对分析,在保守区设计通用引物,利用3'RACE、长距离PCR扩增及简并引物扩增肠道病毒全基因组序列,采用Ion Torrent PGM二代测序仪对扩增产物进行深度测序,以对扩增方法进行验证和评价。结果:通过比对肠道病毒流行株序列设计了通用扩增引物,经二代测序实验获得了肠道病毒全基因组序列;以系列比例模拟混合病毒感染,该扩增方法能够同时获得2株肠道病毒的全基因组序列;能够完整地揭示肠道病毒重组情况。结论:建立了针对我国近年来肠道病毒流行株的通用全基因组扩增方法,在病毒培养液中肠道病毒的提取与扩增中显示了较高的灵敏度,能够反映混合病毒感染、重组病毒的情况。
Objective: To collect the genome-wide sequences of enterovirus EV-71 and coxsackievirus A16 strains in recent years in China, and establish a universal amplification method for obtaining Chinese enterovirus sequences, laying the foundation for the epidemiological analysis and pathogenesis of hand, foot and mouth disease in the future. Methods: The whole genome sequences of enterovirus epidemic strains reported in China in the past five years were collected as reference sequences to be compared and analyzed, and then, the universal primers were designed in the conserved regions. The enterovirus genome-wide sequence was amplified using 3'RACE,long-distance PCR amplification, and degenerate primers. The amplified products were subjected to deep sequencing using an Ion Torrent PGM second generation sequencer to verify and evaluate the amplification method. Results: Universal amplification primers were designed by comparing the sequences of enterovirus epidemic strains,and the whole genome sequence of enterovirus was obtained by second-generation sequencing. The amplification method was capable of simultaneously obtaining the whole genome sequences of two enteroviruses from a mixed virus infection by a series of ratios, therefore, it can reveal the combination of enterovirus. Conclusion: A universal genome-wide amplification method for enterovirus epidemic strains in China has been established. It shows high sensitivity in the extraction and amplification of enteroviruses in virus culture fluids, and can reflect mixed virus infection and combined virus.
Objective: To collect the genome-wide sequences of enterovirus EV-71 and coxsackievirus A16 strains in recent years in China, and establish a universal amplification method for obtaining Chinese enterovirus sequences, laying the foundation for the epidemiological analysis and pathogenesis of hand, foot and mouth disease in the future. Methods: The whole genome sequences of enterovirus epidemic strains reported in China in the past five years were collected as reference sequences to be compared and analyzed, and then, the universal primers were designed in the conserved regions. The enterovirus genome-wide sequence was amplified using 3'RACE,long-distance PCR amplification, and degenerate primers. The amplified products were subjected to deep sequencing using an Ion Torrent PGM second generation sequencer to verify and evaluate the amplification method. Results: Universal amplification primers were designed by comparing the sequences of enterovirus epidemic strains,and the whole genome sequence of enterovirus was obtained by second-generation sequencing. The amplification method was capable of simultaneously obtaining the whole genome sequences of two enteroviruses from a mixed virus infection by a series of ratios, therefore, it can reveal the combination of enterovirus. Conclusion: A universal genome-wide amplification method for enterovirus epidemic strains in China has been established. It shows high sensitivity in the extraction and amplification of enteroviruses in virus culture fluids, and can reflect mixed virus infection and combined virus.
引文
[1]Liu W,Wu S,Xiong Y,et al.Co-circulation and genomic recombination of coxsackievirus A16 and enterovirus 71 during a large outbreak of hand,foot,and mouth disease in Central China[J].PLoS One,2014,9(4):e96051.
[2]Yip C C,Lau S K,Zhou B,et al.Emergence of enterovirus 71"double-recombinant"strains belonging to a novel genotype D originating from southern China:first evidence for combination of intratypic and intertypic recombination events in EV71[J].Arch Virol,2010,155(9):1413-1424.
[3]Tan X,Huang X,Zhu S,et al.The persistent circulation of enterovirus 71 in People's Republic of China:causing emerging nationwide epidemics since 2008[J].PLoS One,2011,6(9):e25662.
[4]Zhu F,Xu W,Xia J,et al.Efficacy,safety,and immunogenicity of an enterovirus 71 vaccine in China[J].N Engl J Med,2014,370(9):818-828.
[5]Zhu F C,Meng F Y,Li J X,et al.Efficacy,safety,and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China:a multicentre,randomised,double-blind,placebo-controlled,phase 3 trial[J].Lancet,2013,381(9882):2024-2032.
[6]Viboud C,Nelson M I,Tan Y,et al.Contrasting the epidemiological and evolutionary dynamics of influenza spatial transmission[J].Philos Trans R Soc Lond B Biol Sci,2013,368(1614):20120199.
[7]侯俊.肠道病毒71型基因分析、病毒拯救、毒力及免疫原性的比较研究[D].北京:解放军医学院,2014.
[8]Hu M,Li Z,Wang J,et al.Determinants of the incidence of hand,foot and mouth disease in China using geographically weighted regression models[J].PLoSOne,2012,7(6):e38978.
[9]Li R,Liu L,Mo Z,et al.An inactivated enterovirus71 vaccine in healthy children[J].N Engl J Med,2014,370(9):829-837.
[10]He S Z,Chen M Y,Xu X R,et al.Epidemics and aetiology of hand,foot and mouth disease in Xiamen,China,from 2008 to 2015[J].Epidemiol Infect,2017,145(9):1865-1874.
[11]Yi E J,Shin Y J,Kim J H,et al.Enterovirus 71 infection and vaccines[J].Clin Exp Vaccine Res,2017,6(1):4-14.
[12]Oberste M S,Pallansch M A.Enterovirus molecular detection and typing[J].Rev Med Microbiol,2005,16(4):163-171.
[13]Oshiki M,Miura T,Kazama S,et al.Microfluidic PCRamplification and Mi Seq amplicon sequencing techniques for high-throughput detection and genotyping of human pathogenic RNA viruses in human feces,sewage,and oysters[J].Front Microbiol,2018,9:830.
[14]Li J,Pan H,Wang X,et al.Epidemiological surveillance of hand,foot and mouth disease in Shanghai in 2014-2016,prior to the introduction of the enterovirus 71vaccine[J].Emerg Microbes Infect,2018,7(1):1-7.
[15]武建民,袁义平.东台市2012-2015年手足口病病原学特征[J].海峡预防医学杂志,2017,23(5):42.
[2]Yip C C,Lau S K,Zhou B,et al.Emergence of enterovirus 71"double-recombinant"strains belonging to a novel genotype D originating from southern China:first evidence for combination of intratypic and intertypic recombination events in EV71[J].Arch Virol,2010,155(9):1413-1424.
[3]Tan X,Huang X,Zhu S,et al.The persistent circulation of enterovirus 71 in People's Republic of China:causing emerging nationwide epidemics since 2008[J].PLoS One,2011,6(9):e25662.
[4]Zhu F,Xu W,Xia J,et al.Efficacy,safety,and immunogenicity of an enterovirus 71 vaccine in China[J].N Engl J Med,2014,370(9):818-828.
[5]Zhu F C,Meng F Y,Li J X,et al.Efficacy,safety,and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China:a multicentre,randomised,double-blind,placebo-controlled,phase 3 trial[J].Lancet,2013,381(9882):2024-2032.
[6]Viboud C,Nelson M I,Tan Y,et al.Contrasting the epidemiological and evolutionary dynamics of influenza spatial transmission[J].Philos Trans R Soc Lond B Biol Sci,2013,368(1614):20120199.
[7]侯俊.肠道病毒71型基因分析、病毒拯救、毒力及免疫原性的比较研究[D].北京:解放军医学院,2014.
[8]Hu M,Li Z,Wang J,et al.Determinants of the incidence of hand,foot and mouth disease in China using geographically weighted regression models[J].PLoSOne,2012,7(6):e38978.
[9]Li R,Liu L,Mo Z,et al.An inactivated enterovirus71 vaccine in healthy children[J].N Engl J Med,2014,370(9):829-837.
[10]He S Z,Chen M Y,Xu X R,et al.Epidemics and aetiology of hand,foot and mouth disease in Xiamen,China,from 2008 to 2015[J].Epidemiol Infect,2017,145(9):1865-1874.
[11]Yi E J,Shin Y J,Kim J H,et al.Enterovirus 71 infection and vaccines[J].Clin Exp Vaccine Res,2017,6(1):4-14.
[12]Oberste M S,Pallansch M A.Enterovirus molecular detection and typing[J].Rev Med Microbiol,2005,16(4):163-171.
[13]Oshiki M,Miura T,Kazama S,et al.Microfluidic PCRamplification and Mi Seq amplicon sequencing techniques for high-throughput detection and genotyping of human pathogenic RNA viruses in human feces,sewage,and oysters[J].Front Microbiol,2018,9:830.
[14]Li J,Pan H,Wang X,et al.Epidemiological surveillance of hand,foot and mouth disease in Shanghai in 2014-2016,prior to the introduction of the enterovirus 71vaccine[J].Emerg Microbes Infect,2018,7(1):1-7.
[15]武建民,袁义平.东台市2012-2015年手足口病病原学特征[J].海峡预防医学杂志,2017,23(5):42.