液相芯片技术同时检测真鲷虹彩病毒和锦鲤疱疹病毒方法的建立

Development of a Liquid Chip Technique for Simultaneous Detection of Red Sea Bream Iridovirus(RSIV) and Koi Herpesvirus(KHV)

为建立一种同时检测真鲷虹彩病毒、锦鲤疱疹病毒的液相芯片快速检测技术,用DNAStar软件对GenBank中真鲷虹彩病毒的CP基因和锦鲤疱疹病毒的TK基因进行序列分析,设计真鲷虹彩病毒、锦鲤疱疹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真鲷虹彩病毒、锦鲤疱疹病毒PCR产物杂交反应,用液相芯片仪器检测荧光信号。试验结果表明,液相芯片检测体系能够正确的检测出真鲷虹彩病毒、锦鲤疱疹病毒。真鲷虹彩病毒、锦鲤疱疹病毒病毒核酸的最低检出量为10pg和100pg,检测特异性高,说明初步建立了同时检测真鲷虹彩病毒、锦鲤疱疹病毒的液相芯片技术,为进一步构建其他水生动物病原体快速高通量检测平台奠定基础。

The CP gene of Red Sea Bream Iridovirus （RSIV） and TK gene of Koi Herpesvirus （KHV） in the GenBank was analysed using the software DNAStar 7 .0 ,and specific RSIV and KHV primers labeled with biotin was prepared and coupled with fluorescence-coded microspheres to develop liquidchip technique to detect RSIV and KHV simultaneously .The aminationed probe was used for hybridization reaction to PCR products of RSIV and KHV ,then Liquidchip detection technique for detection of RSIV and KHV was established by BD FACSArray to detect fluorescence signal in the reaction system .The results showed that RSIV and KHV was accurately and high specifically detected by liquidchip detection technique with minimal limit of 10 pg of RSIV DNA and 100 pg of KHV DNA .The Liquidchip detection technique for detection of RSIV ,and KHV was established preliminarily ,which provided a foundation for further research on rapidly high throughout detection of other aquatic animal pathogens .