天津市锦鲤疱疹病毒PCR检测与序列分析

PCR Detection of Koi Herpesvirus and Its Sequence Analysis in Tianjin City

2017年6月,天津市滨海新区、北辰区等地一些养殖场的锦鲤出现急性死亡,发病3 d后死亡率高达85%以上。病鱼呈现眼部凹陷、烂鳃、内脏出血等临床症状,初步诊断为锦鲤疱疹病毒病。针对锦鲤疱疹病毒(KHV/CyHV)的TK、SphI-5、ORF25、ORF56、ORF81基因设计特异性引物,建立PCR方法进行KHV基因检测。结果显示:死亡疑似病例的KHV检测阳性;测序获得的TK、SphI-5、ORF25、ORF56、ORF81基因片段长度分别为409、300、317、939和156bp,与CyHV-3-J2病毒株基因序列同源性为97.6%～100%。系统发育树分析显示,其与CyHV-3亲缘关系最近,自然聚为一支,最终确定为CyHV-3型。以上研究为锦鲤疱疹病毒病的分子诊断及流行病学调查奠定了基础。

In June2017,an outbreak of acute deaths appeared in the koi carps in some farms in Binhai New Area and Beichen District of Tianjin City.Three days after the outbreak,the mortality rate was as high as85%,and the diseased carps showed clinical symptoms such as endophthalmos,rotten gills and visceral hemorrhage,and the epidemic was initially diagnosed as koi herpesvirus disease(KHVD).Based on the sequences of TK、SphI-5、ORF25、ORF56and ORF81gene of KHV/CyHV,a series of specific primers were designed and used to test KHV by PCR method.As a result,KHV nucleic acids could be detected,and the sequence lengths of TK、SphI-5、ORF25、ORF56and ORF81gene fragments were409bp,300bp,317bp,939bp and156bp,respectively.The sequence homology of the gene fragments between KHV/CyHV and CyHV-3-J2strain was97.6%to100%.Phylogenetic tree analysis also showed the isolated strain was closely related to CyHV-3,which two naturally clustered into one branch,finally,the isolated KHV strain was determined to be CyHV-3.The above studies would lay foundation for molecular diagnosis and epidemiological investigation of KHVD.