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

Development of a Liquid Chip Technique for Simultaneous Detection of Taura Syndrome Virus(TSV)and Yellow Head Disease Virus(YHDV)

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 CP2 gene 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 100 pg, 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.