原位杂交技术在斜带石斑鱼神经坏死病毒检测中的应用  

Application of in situ hybridization in testing orange-sported grouper nervous necrosis virus

作　　者：陈晓艳[1,2] 何建国[1] 

机构地区：[1]中山大学生命科学学院,广东广州510275 [2]暨南大学生物工程学系,广东广州510632

出　　处：《海洋科学》2008年第6期1-4,共4页Marine Sciences

基　　金：国家863计划项目(2001AA601010);暨南大学引进人才启动基金项目;广东省水产经济动物病原生物学及流行病学重点实验室开放基金项目(2007A004)

摘　　要：根据斜带石斑鱼(Epinephelus coioids)神经坏死病毒(orange-spotted nervous necrosis virus,OGNNV)的主衣壳蛋白(major capsid protein,MCP)基因的保守序列,设计一对引物,从感染OGNNV的斜带石斑鱼组织匀浆液提取RNA为模板进行RT-PCR扩增,得到426 bp的cDNA片断。用得到的RT-PCR产物加上地高辛(DIG)标记作为核酸探针。通过注射病毒提取液人工感染一组斜带石斑鱼,解剖感染病毒的斜带石斑鱼,从中分离出脑和眼睛,运用原位杂交技术检测组织中的OGNNV。实验表明,原位杂交具有较高的灵敏性和特异性,可以用原位杂交的办法来检测养殖的石斑鱼是否有携带该病毒,达到监控和预防神经坏死病爆发的目的。本实验还采用了H&E染色方法检测了感染NNV的石斑鱼脑部和眼部组织,观察细胞内的坏死部分。与原位杂交做对比,提高了检测的可靠性和准确性。本实验建立的石斑鱼神经坏死病毒的ISH检测方法有着较高的特异性和敏感性,易于操作,有助于石斑鱼神经坏死病毒的组织定位、发病机理的研究。According to the conserved region of the major capsid protein gene of orange-spotted grouper （Epinephelus coioids） nervous necrosis virus （OGNNV）, a pair of primers was designed. Using the primers, a RT-PCR product of 426 bp was cloned from the RNA template extracted from orange-spotted grouper infected by OGNNV. The RT-PCR product labeled by digoxigenin （DIG） was used for the non-radioactive nucleic acid probe. A group of orange-spotted groupers were artificialiy infected by injecting infected tissue filtrates and then the eyes and brain of moribund fish were fixed, and OGNNV was detected by in situ hybridization（ISH）. The experiment showed that in situ hybridization is very sensitive and special, So we can use this method to detect whether grouper is infected with OGNNV or not. It can be used in inspecting and preventing the disease of VNN. The eyes and brains from diseased orange-spotted grouper were also detected by hematoxylinand eosin （H＆E） to compare with the result of in situ hybridization. We can look into the putrescence portion of the cells to make sure that the result is credible and nice. The established ISH for de- tecting OGNNV is highly sensitive , special and easily manipulated, and helpful to studies on virus location in tissue and pathogenesis.

关 键 词：神经坏死病毒 地高辛 原位杂交 

分 类 号：S941[农业科学—水产养殖]