液相芯片技术同时检测对虾桃拉病毒和虾黄头病毒的研究
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摘要
为建立一种同时检测对虾桃拉病毒、虾黄头病毒的液相芯片快速检测技术,用DNAStar软件对GenBank中对虾桃拉病毒的CP2基因和虾黄头病毒的N基因进行序列分析,设计对虾桃拉病毒、虾黄头病毒特异性引物并标记生物素。探针氨基化修饰,与荧光编码微球偶联后与对虾桃拉病毒、虾黄头病毒病毒RT-PCR产物杂交反应,用液相芯片仪器检测荧光信号。该检测体系对对虾桃拉病毒、虾黄头病毒病毒核酸检测灵敏度高,其最低检测限为100pg,且与白斑病毒、鲤春病毒血症病毒、传染性造血器官坏死病毒核酸等无交叉反应。该方法灵敏度及特异性高,为同时快速检测对虾桃拉病毒、虾黄头病毒提供了简捷快速的技术手段。
The CP2gene of Taura syndrome virus(TSV)and N gene of yellow head disease virus(YHDV) in the GenBank were analysed by the software DNAStar 7.0,and specific TSV and YHDV primers labeled with biotin were prepared and coupled with fluorescence-coded microspheres to develop liquichip technique to detect TSV and YHDV simultaneously.The aminationed probe was used for hybridization reaction to PCR products of TSV and YHDV,and then the liquichip detection technique for detection of TSV and YHDV was established using BD FACSArray to detect fluorescence signal in the reaction system.This assay was highly sensitive to TSV,and YHDV with a lower detection limit of 100pg,and was not susceptible to cross reaction with other viruses,including white spot syndrome virus(WSSV),spring viremia of carp virus(SVCV),and infectious haematopoietic necrosis virus(IHNV).In conclusion,the liquichip technique assay is sensitive,and specific for TSV and YHDV detection,and provides a novel, convenient,and rapid approach for the detection of TSV and YHDV.
The CP2gene of Taura syndrome virus(TSV)and N gene of yellow head disease virus(YHDV) in the GenBank were analysed by the software DNAStar 7.0,and specific TSV and YHDV primers labeled with biotin were prepared and coupled with fluorescence-coded microspheres to develop liquichip technique to detect TSV and YHDV simultaneously.The aminationed probe was used for hybridization reaction to PCR products of TSV and YHDV,and then the liquichip detection technique for detection of TSV and YHDV was established using BD FACSArray to detect fluorescence signal in the reaction system.This assay was highly sensitive to TSV,and YHDV with a lower detection limit of 100pg,and was not susceptible to cross reaction with other viruses,including white spot syndrome virus(WSSV),spring viremia of carp virus(SVCV),and infectious haematopoietic necrosis virus(IHNV).In conclusion,the liquichip technique assay is sensitive,and specific for TSV and YHDV detection,and provides a novel, convenient,and rapid approach for the detection of TSV and YHDV.
引文
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[8]Mengelle C,Mansuy J M,Prere M F,et al.Simultaneous detection of gastrointestinal pathogens with a multiplex Luminex-based molecular assay in stool samples from diarrhoeic patients[J].Clin Microbiol Infect,2013,29(10):1469-1475.
[9]Ryan J T,Rose T M.Development of whole-virus multiplex luminex-based serological assays for diagnosis of infections with kaposi’s sarcomaassociated herpesvirus/human herpesvirus 8homologs in macaques.[J].Clin Vaccine Immunol,2013,20(3):409-419.
[10]Wang L,Zhang H X,Zhang J H,et al.In vitro effects of recombinant zebrafish IFN on spring viremia of carp virus and infectious hematopoietic necrosis virus[J].J Interferon Cytokine Res,2006,26(4):256-259.
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[14]Peterson A W,Wolf L K,Georgiadis R M.Hybridization of mismatched or partially matched DNA at surfaces[J].J Am Chem Soc,2002,124(49):14601-14607.
[15]Gotoh M,Hasegawa Y,Shinohara Y,et al.A new approach to determine the effect of mismatches on kinetic parameters in DNA hybridization using an optical biosensor[J].DNA Res,1995,2(6):285-293.
[16]Alam M.Diagnostic limitations to accurate diagnosis of cholera[J].Clin Microbiol,2010,48(11):3918-3922.
[17]Bessede E,Delcamp A,Sifre E.New methods for detection of campylobacters in stool samples in comparison to culture[J].Clin Microbiol,2011,49(3):941-944.
[18]Baums I B.Luminex detection of fecal indicators in river samples,marine recreational water,and beach sand[J].Mar Pollut Bull,2007,54(5):521-536.
[19]Liu J,Meng C,Zhang S H,et al.Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of gastroenteritis[J].Clin(Virol),2011,50(4):308-313.
[2]黄新新,陆承平.桃拉综合征病毒中国株ZHZC3全基因测序及分子结构预测[J].中国病毒学,2006,21(3):267-272.
[3]Robles-Sikisaka R,Hasson K W,Garcia D K,et al.Genetic variation and immunohistochemical differences among geographic isolates of Taura syndrome virus of penaeid shrimp[J].J Gen Virol,2002,83(12):3123-3130.
[4]陈爱平;江育林;钱冬,等.水生动物疫病病种介绍之:黄头病[J].中国水产,2011(4):64-65.
[5]Wongteerasupaya C,Sriurairatan S,Viccker J E,et al.Yellow-head virus of Penaeus monodonis an RNA virus[J].Dis Aquat Organ,1995,22(1):45-50.
[6]Wongteerasupaya C,Tongchuea W,Boonseang V,et al.Detection of yellow-head virus(YHV)of Penaeus monodon by RT-PCR amplication[J].Dis Aquat Organ,1997,31(1):181-186.
[7]Robles-sikisaka R,Garcia D K,Klimoel K R,et al.Nucleotide sequence of 3′-end of the genome of Taura syndrome virus of shrimp suggests that it is related to insectpicornaviruses[J].Arch Viro,2001,146(5):941-952.
[8]Mengelle C,Mansuy J M,Prere M F,et al.Simultaneous detection of gastrointestinal pathogens with a multiplex Luminex-based molecular assay in stool samples from diarrhoeic patients[J].Clin Microbiol Infect,2013,29(10):1469-1475.
[9]Ryan J T,Rose T M.Development of whole-virus multiplex luminex-based serological assays for diagnosis of infections with kaposi’s sarcomaassociated herpesvirus/human herpesvirus 8homologs in macaques.[J].Clin Vaccine Immunol,2013,20(3):409-419.
[10]Wang L,Zhang H X,Zhang J H,et al.In vitro effects of recombinant zebrafish IFN on spring viremia of carp virus and infectious hematopoietic necrosis virus[J].J Interferon Cytokine Res,2006,26(4):256-259.
[11]世界动物卫生组织.桃拉综合征·水生动物疾病诊断手册[M].北京:中国农业出版社,2012:160-176,204-217.
[12]Hulse R E,Kunkler P E,Fedynyshyn J P,et al.Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue[J].Neurosci Methods,2004,136(1):87-98.
[13]Maskos U,Southern E M.A study of oligonucleotide reassociation using large arrays of oligonucleotides synthesised on a glass support[J].Nucleic Acids Res,1993,21(20):4663-4669.
[14]Peterson A W,Wolf L K,Georgiadis R M.Hybridization of mismatched or partially matched DNA at surfaces[J].J Am Chem Soc,2002,124(49):14601-14607.
[15]Gotoh M,Hasegawa Y,Shinohara Y,et al.A new approach to determine the effect of mismatches on kinetic parameters in DNA hybridization using an optical biosensor[J].DNA Res,1995,2(6):285-293.
[16]Alam M.Diagnostic limitations to accurate diagnosis of cholera[J].Clin Microbiol,2010,48(11):3918-3922.
[17]Bessede E,Delcamp A,Sifre E.New methods for detection of campylobacters in stool samples in comparison to culture[J].Clin Microbiol,2011,49(3):941-944.
[18]Baums I B.Luminex detection of fecal indicators in river samples,marine recreational water,and beach sand[J].Mar Pollut Bull,2007,54(5):521-536.
[19]Liu J,Meng C,Zhang S H,et al.Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of gastroenteritis[J].Clin(Virol),2011,50(4):308-313.