摘要
针对近年来由虹彩病毒所引起的海水养殖鱼类疾病呈日趋严重的态势,该文在证实了引发我国大黄鱼大规模流行病的病原为一种虹彩病毒及测定病毒全基因组序列(111,760bp;GenBankaccessionnumber:AY779031)的基础上,通过与已报道虹彩病毒核酸序列进行分析比较,结合生物信息学手段,确定了以虹彩病毒ATPase基因保守区序列(295bp)作为扩增靶序列,设计合成了一对特异性引物,通过改进PCR模板的制备方法和优化扩增条件,建立了大黄鱼虹彩病毒PCR快速检测技术,并开发成简便、快速、实用的检测试剂盒,该试剂盒的检测灵敏度相当于30个病毒粒子,模板制备时间约30min、回收率为52%、半个工作日即可得到准确的结果,无非特异性扩增带,适用于大黄鱼虹彩病毒病的早期快速诊断、苗种的检疫及水质环境的监测,目前正在推广应用。
A rapid and sensitive PCR-based method for detection of large yellow croaker iridovirus (LYCIV) was described, which involved the amplification of a 295 bp fragment of the LYCIV ATPase gene from DNAs isolated from naturally infected fish spleens. The specificity and sensitivity of the PCR procedure were tested on the virus- infected fish, the DNA fragment with expected size was detected from spleen DNA samples of infected fish, whereas no fragments were amplified from healthy fish spleen DNA, white spot syndrome baculoviruses (WSBV) DNA and pseudorabies virus (PRV) DNA. Detection limit of this method was 10 -7 ng positive plasmid DNA containing target sequence, equal to about 30 virions. The time for preparation of DNA template was less than 30 minutes, and the good results could be obtained in a half working-day. Finally a simple and rapid PCR-based Kit for detection of LYCIV was developed and has been applying to the practical mari-culture industry.
出处
《生物技术》
CAS
CSCD
2005年第3期38-40,共3页
Biotechnology
基金
国家"863"计划项目(2001AA620603)
广东省科技计划项目(2003C20312)资助
