虾桃拉病毒、黄头病毒和白斑病毒液相芯片快速检测方法的建立
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摘要
本研究通过设计、合成并修饰基因特异性引物和寡核苷酸探针并标记生物素,利用该探针与荧光编码微球偶联后与虾黄头病毒(Yellow head virus,YHV)、虾桃拉综合征病毒(Taura syndrome virus,TSV)和白斑综合征病毒(White spot syndrome virus,WSSV)的PCR产物杂交反应,用液相芯片检测仪(Luminex200)检测荧光信号,初步建立了虾黄头病毒、桃拉病毒和白斑病毒的快速同步检测方法。结果显示,建立的该方法具有较好的特异性,偶联特异性探针的微球只与相应的病毒基因的PCR产物反应,而不与其他虾病病毒基因反应,YHV、TSV和WSSV检测灵敏度高分别为401.5、251.5和75.2 copies。
A liquichip technique for detection of Yellow head virus(YHV), Taura syndrome virus(TSV) and White spot syndrome(WSSV) was developed in the present study by using Luminex 200 to catch fluorescence signal. In this method, specific primers and their corresponding oligonucleotide probes were designed, synthesized and modified. Then individual YHV, TSV and WSSV probes were labeled with biotin and coupled with fluorescence-coded microspheres. The results showed that this method displayed good specificity to YHV, TSV and WSSV and no reaction to other shrimp viruses. The sensitivity test indicated the detection limit for YHV, TSV and WSSV reached to 401.5、251.5 and 75.2 copies. A liquichip technique was preliminarily developed for detection of YHV, TSV and WSSV.
A liquichip technique for detection of Yellow head virus(YHV), Taura syndrome virus(TSV) and White spot syndrome(WSSV) was developed in the present study by using Luminex 200 to catch fluorescence signal. In this method, specific primers and their corresponding oligonucleotide probes were designed, synthesized and modified. Then individual YHV, TSV and WSSV probes were labeled with biotin and coupled with fluorescence-coded microspheres. The results showed that this method displayed good specificity to YHV, TSV and WSSV and no reaction to other shrimp viruses. The sensitivity test indicated the detection limit for YHV, TSV and WSSV reached to 401.5、251.5 and 75.2 copies. A liquichip technique was preliminarily developed for detection of YHV, TSV and WSSV.
引文
[1]OIE-Listed diseases,infections and infestations in force in 2017,World Organisation For Animal Health.
[2]Manual of Diagnositc Tests for Aquatic Animals,2017,World Organisation For Animal Health.
[3]Rejan P R,Ramesamy P.A white spot disease-like syndrome in the pacific blue shrimp as a form of bacterial shell disease[J].Aquaculture,2000,183:25-30.
[4]谢芝勋,庞耀珊,刘加波,等.二温式多重PCR检测鉴别对虾白斑综合征病毒(WSSV)和传染性皮下及造血器官坏死病毒(IHHNV)的研究与应用[J].海洋科学,2005,29(12):9-12.
[5]熊炜,邱璐,李健,等.上海检验检疫局从泰国进境草虾中检出虾黄头病毒[J].检验检疫科学,2006,6(16):61-62.
[6]Bay J T,Garred P.Rapid bead-based immunoassay for measurement of mannose-binding lectin[J].Scand J Immunol,2009,69(6):570-575.
[7]Arellano-Garcia M E,Hu S,Wang J,et al.Multiplexed immunobead-based assay for detection of oral cancer protein biomarkers in saliva[J].Oral Dis,2008,14(8):705-712.
[8]Livingston A D,Campbell C J,Wagner E K,et al.Biochip sensors for the rapid and sensitive detection viral disease[J].Genome Biol,2005,6(6):112-115.
[9]Linkov F,Yurkovetsky Z,Lokshin A.Hormones as biomarkers:practical guide to utilizing Luminex technologies for biomarker research[J].Methods Mol Biol,2009,520:129-141.
[10]Yeh H Y,Shoemaker C A,Klesius P H.Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri[J].J Microbiol Methods,2005,63(1):36-44.
[2]Manual of Diagnositc Tests for Aquatic Animals,2017,World Organisation For Animal Health.
[3]Rejan P R,Ramesamy P.A white spot disease-like syndrome in the pacific blue shrimp as a form of bacterial shell disease[J].Aquaculture,2000,183:25-30.
[4]谢芝勋,庞耀珊,刘加波,等.二温式多重PCR检测鉴别对虾白斑综合征病毒(WSSV)和传染性皮下及造血器官坏死病毒(IHHNV)的研究与应用[J].海洋科学,2005,29(12):9-12.
[5]熊炜,邱璐,李健,等.上海检验检疫局从泰国进境草虾中检出虾黄头病毒[J].检验检疫科学,2006,6(16):61-62.
[6]Bay J T,Garred P.Rapid bead-based immunoassay for measurement of mannose-binding lectin[J].Scand J Immunol,2009,69(6):570-575.
[7]Arellano-Garcia M E,Hu S,Wang J,et al.Multiplexed immunobead-based assay for detection of oral cancer protein biomarkers in saliva[J].Oral Dis,2008,14(8):705-712.
[8]Livingston A D,Campbell C J,Wagner E K,et al.Biochip sensors for the rapid and sensitive detection viral disease[J].Genome Biol,2005,6(6):112-115.
[9]Linkov F,Yurkovetsky Z,Lokshin A.Hormones as biomarkers:practical guide to utilizing Luminex technologies for biomarker research[J].Methods Mol Biol,2009,520:129-141.
[10]Yeh H Y,Shoemaker C A,Klesius P H.Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri[J].J Microbiol Methods,2005,63(1):36-44.