| 摘要: |
| 于1995年7月在青岛市城阳对虾养殖场采集患暴发性流行病的中国对虾样品，提纯HHNBV并研制了一组地高辛标记的HHNBV核酸探针。采用核酸斑点杂交方法研究了此组核酸探针的灵敏度和特异性。结果表明，此组探针检测HHNBV DNA的灵敏度为62.5pg，对469ng病虾胃中的病毒可以检出；此组探针与3.75ug健康对虾组织和2ng健康对虾DNA均无交叉反应。1997年，应用此组特异性核酸探针检测了发病虾池对虾及紧急抢捕虾，检测结果显示为强烈的HHNBV阳性，表明此组核酸探针在对虾杆状病毒的检测、对虾暴发性流行病的诊断和抗特种病原对虾育种等方面具有很高的应用价值。 |
| 关键词: 中国对虾 核酸探针 斑点杂交 检测 杆状病毒 |
| DOI： |
| 分类号: |
| 基金项目:国家“八五”科技攻关项目（95年度加强），85-021-03-06号 |
附件 |
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| DETECTION OF HYPODERMAL AND HEMATOPOIETIC NECROSIS BACULOVIRUS (HHNBV) BY DIG-DNA PROBE DOT-BLOT HYBRIDIZATION |
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SHl Cheng-yin,SONG Xiao-ling,HUANG Jie,YANG Cong-hai
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Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Marine cultural Disease &Ecology Laboratory , Ministry of Agriculture，Qingdao，266071
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| Abstract: |
| Diseased shrimps, Penaeus chinensis, were collected during an occurrence of explosive epidemic disease at a shrimp farm of Chengyang, Qingdao in July 1995. The hypodermal and hematopoietic necrosis baculovirus (HHNBV) was isolated from these shrimps. Two different digoxigenin (DIG) labeled DNA probes were constructed. The sensitivity and specificity of these probes were detected by DNA dot-blot hybridization assay. The results show that the sensitivity was 62.5pg to HHNBV DNA; these probes were able to hybridize with 469ng stomach tissues of HHNBV-infected Penaeus chinensis, but not with HHNBV-uninfected P. chinensis tissues and DNA. These probes were used to detect the shrimps in diseased ponds and an urgent harvest pond in 1997. The detecting results were intensely positive. In contrast to PCR method, DIG DNA probe dot-blot hybridization had less procedures, which only needs simple reagents and apparatus, can detect hundreds specimen at one time, and there is no need to extract DNA from specimen. So it was easy to use. We have made a kind of dot-blot hybridization kit for HHNBV. As probes were easily synthesized the method of DNA probe can spread more easily than anti-serum method. Compared with PCR method, the DNA probe method has a lower sensitivity but higher specificity. The sequences of HHNBV DNA in clone C12 and C44 were known. We have designed several pairs of PCR primers according of these sequences. So we can amplify HHNBV DNA segments in specimen with PCR, then hybridize those products with DNA probe. The sensitivity would highly increase and the specificity remains well. So these DIG labeled DNA probes have a high-applied value in detecting HHNBV, diagnosing explosive epidemic disease and specific pathogen resistant (SPR) shrimp breeding. |
| Key words: Penaeus chinensis, DNA probe, Dot-blot hybridization, Detection, Baculovirus |
