大鲵虹彩病毒的PCR检测及初步应用  

Giant salamander Iridovirus PCR detection and preli minary application

作　　者：沈红保[1] 张建军[1] 张军燕[1] 李引娣[1] 陈媛媛[1] 

机构地区：[1]中国水产科学研究院黄河水产研究所,陕西西安710086

出　　处：《河北渔业》2012年第8期17-19,67,共4页

基　　金：国家科技基础条件平台建设项目(2006DKA 30470-011)

摘　　要：通过了解大鲵虹彩病毒(Giant salamander iridovirus,GSIV)在EPC细胞接毒情况和PCR的实际检测情况,为大鲵的病毒检测提供必要判断依据和检测方法。通过细胞接毒和组织分离分别提取病毒质粒,进行普通PCR扩增,电泳分析,比较病毒细胞培养和组织直接提取结果。Gsiv病毒体外培养:经传代48h的EPC细胞,25℃培养,12h后可观察到CPE出现,24h病变明显,CPE达15%,48h后CPE可达70%,72h后细胞完全脱落;PCR检测:对3个样本分别从组织提取和经细胞扩增分离的病毒进行普通PCR扩增检测,经细胞培养扩增的3个样本全部呈阳性;经组织提取的3个样本,2个呈阳性,一个呈阴性;电泳观察,病毒的扩增产物在500bp左右。在体外环境下GSIV是高病力的一种病毒,对EPC细胞在25℃时效应时间为3d,普通PCR经组织的检出率低,经细胞培养扩增可提高检出率但耗时久,不利于实际生产中快速、准确诊断疫情的需要。So understanding the culture situation of the giant salamander iridovirus in the EPC cell and their actual situation detected by PCR technique to get the judgment and test method for the detection of the disease in the giant alamander breeding. Get virus plasmids by two ways--extracted from the tissue and inoculation cells ,and then compared those virus plasmids which were analyzed by electro- phoresis and amplified by --PCR independently. Gsiv virus cultured in vitro. The EPC cells after pas- sage 48 h, keep cultivating at 25 ℃, after 12 h , could observed the CPE , after 24 h , CPE reached 15% , after 48 h ,CPE reached 70%and all the cells were completely off after 72 h. PCR test：Three samples were detected by PCR which were extracted from the tissue and separated from the inocula- tion cells. The samples of cell culture amplification ,were all positive. The samples extracted from the tissue were two positive,one negative; Electrophoresis suggested that virus amplification product of a- bout 500 bp. Gsiv in vitro environment is a higher disease force virus, EPC cells at 25℃, could keep- ing positive in three days, The detected rate of conventional PCR used to test the samples which were extracted from the tissue is low. The detected rate of conventional PCR used to test the samples sepa- rated from the inoculation cells is high, but it can take a very long time, could not meet the diagnostic accuracy and rapidity requirement of actual production.

关 键 词：大鲵 虹彩病毒 PCR 

分 类 号：S941[农业科学—水产养殖]