| 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。 |
| 关键词: 神经坏死病毒 地高辛 原位杂交 |
| DOI： |
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| 基金项目: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) |
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| Application of in situ hybridization in testing orange-sported grouper nervous necrosis virus |
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| Abstract: |
| 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 exeriment 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 hematoxy linandeosin (H&E) to compare with the result of in situ hybridization. We can look into the put rescence portion of the cells to make sure that the result is credible and nice. The established ISH for detecting OGNNV is highly sensitive, special and easily manipulated, and helpful to studies on virus location in tissue and pathogenesis. |
| Key words: nervous necrosis virus DIG in situ hybridization |
