应用聚合酶链式反应(PCR)检测锦鲤疱疹病毒

为建立并优化锦鲤疱疹病毒检测方法,针对目前国内集约化养殖鲤鱼大面积流行的病死现象进行有针对性的检测,对在疫区采集到的病鱼肾脏、肝胰脏、肠等组织,提取基因组DNA,应用锦鲤疱疹病毒特异性引物进行PCR反应,同时设立阴性对照(TE buffer)。通过1%琼脂糖凝胶进行鉴定,与阴性对照比较,在484bp处出现特异性目的条带。经序列测定,所得PCR产物的基因序列与NCBI上锦鲤疱疹病毒的同源率达到99%以上。对同一批次的样品进行重复检测,结果表明该方法具有较好的可重复性,可据此判定结果。通过对锦鲤疱疹病毒的检测,确定了疫区引起框镜鲤(Cyprinus carpio)和建鲤(Cyprinus carpio var Jian)大批死亡的病原为锦鲤疱疹病毒(Koi Herpesvirus,KHV),从而对疫区控制、防治方案的制定提供了理论依据。建议尽快在鲤鱼主要养殖区开展KHV流行病学、诊断与检测及免疫预防等方面的研究。

Detection of Koi herpes virus by polymerase chain reaction (PCR)

A polymerase chain reaction(PCR) was used for detecting the koi herpesvirus(KHV) with the specificity primers and DNA template purifying from kidney,hepatopancreas,intestine of sick Cyprinus carpio and Cyprinus carpio var Jian.Compared with the negative control(TE buffer),a specific 484 base-paired segment was amplified,which has above 99 percent homology with the KHVs from GenBank.For the same batch of samples,the results can be repeatability with the method and showed that the pathogen causing sickness and considerable number of deaths to Cyprinus carpio and Cyprinus carpio var Jian is Koi Herpesvirus,which provides evidence to prevent and control the disease and emphasizes the necessity of reinforcing the KHV surveillance,diagnosis and prevention.