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为建立一种快速检测锦鲤疱疹病毒(CyHV-3)的环介导等温扩增方法(LAMP),本研究根据Gen Bank中登录的CyHV-3 Sphi基因编码区序列设计了LAMP特异性引物,经反应体系和反应条件优化,建立了检测CyHV-3的LAMP方法。结果表明,本研究建立的LAMP检测方法与鲤痘疱疹病毒、金鱼造血器官坏死病病毒和鳗鱼疱疹病毒均无交叉反应,特异性强;最低检出率为13.9拷贝/μL,灵敏度高。使用LAMP方法和普通PCR方法对四川地区的15个检测点采用鱼鳃拭子非致死性采集的临床样品进行检测,阳性检出率均为31.40%(27/86)。表明本实验建立的该检测方法具有高灵敏度、高特异性、快速和简便的特性,适用于临床上对锦鲤疱疹病毒病的快速诊断。
In order to establish a loop-mediated isothermal amplification method (LAMP) for rapid detection of the koi herpesvirus (CyHV-3), LAMP-specific primers were designed based on the coding sequence of CyHV-3 Sphi gene registered in GenBank. After optimization of the reaction system and reaction conditions, a LAMP method for detecting CyHV-3 was established. The results showed that the LAMP detection method established in this study had no specific cross-reaction with carp pox, goldfish hematopoietic necrosis virus and eel herpesvirus. The minimum detection rate was 13.9 copies / μL with high sensitivity. The LAMP method and the ordinary PCR method were used to detect clinical samples collected from non-lethality of 15 fish gill swabs at 15 sites in Sichuan Province. The positive detection rate was 31.40% (27/86). It is indicated that this assay established in this experiment has the characteristics of high sensitivity, high specificity, quickness and simplicity, and is suitable for the rapid diagnosis of koi herpesvirus in clinic.