进口锦鲤暴发病病原的nested-PCR鉴定

为了查明锦鲤暴发性疾病的病因，将患病锦鲤的脑，肝，脾和肾等组织悬液接种到CO，CK和EPS等鱼类细胞系中培养，均未发现细胞病变，从病灶中取样进行病原菌的分离和培养，证明锦鲤有副溶血弧菌和嗜水气单胞菌的感染，制备锦鲤疱疹病毒（KHV）的2对引物KHV9/5F和KHV9/5R，KHV1和KHV2，用嵌套式聚合酶链式反应（nested-PCR)在脑和脾脏组织的抽提物中扩增出长度为412bp的特异性的DNA片段，将该片段的nested-PCR拉增产物纯化后，克隆，测序，用NCBI-Blast软件将测序结果，在Genebank中进行搜寻，比较，结果发现与注册号为AF411803的KHV基因序列的一部分片段有99%的同源性，因此初步判断这次锦鲤暴发性疾病是由KHV引起的，副溶血弧菌和嗜水气单胞菌起着继发感染的作用。

Study on the Aetiology of Koi Epizootic Disease Using the Method of nested-Polymerase Chain Reaction Assay (nested-PCR)

In April, 2002, an outbreak of imported koi occurred in a farm in Guangdong Province with the mortality of 80% upward. In order to investigate the aetiology, the suspension of brain, kidney, spleen and liver were inoculated into fish cell lines of CO, CK and EPC, while no cytopathic effect was observed in these three cell lines. Bacteria were isolated from the loci and abdomen fluid and identified as Vibrio parahaemolyticus and Aeromonas hydrophila . Two pairs of primers were designed, labeled as KHV9/5F and KHV9/5R, KHV1 and KHV2. Specific fragment with the length of 412 bp was amplified with nested-polymerase chain reaction (PCR) assay from the extraction of brain and spleen. The amplified product was purified, cloned and sequenced. The sequence was compared with NCBI-Blast in Genebank and found to have the homogenicity of 99% with the fragment of koi herpesvirus (KHV) (coded as AF411803). It was deduced that the outbreak of koi occurred in Southern China was caused by KHV, and the bacteria of V. haemolyticus and A. hydrophila played a role of secondary infection.