| 摘要: |
| 参照鱼类病毒性出血性败血症病毒序列, 根据PCR 引物设计的原则, 设计巢式PCR 引物,采用巢式逆转录聚合酶链反应(RT-PCR)方法, 对如何快速检测鱼类病毒性出血性败血症病的方法进行了较为系统的研究, 并对RT-PCR 的反应条件进行了优化。结果表明, 巢式RT-PCR 扩增获得279bp的特异性片断, 阴性对照无扩增条带。巢式RT-PCR 扩增出的特异性片段经测序分析, 结果证实与报道的序列完全一致。该方法最低可检测出0.1pg 的鱼类病毒性出血性败血症病毒RNA。初步建立了VHSV 的巢式RT-PCR 检测方法, 该方法灵敏、特异, 可为VHSV 的检测提供一个快速、有效的手段。
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| 关键词: 病毒性出血性败血症病毒, 巢式逆转录聚合酶链反应, 检测 |
| DOI：10.11693/hyhz200904015015 |
| 分类号: |
| 基金项目:宁波市重大科技攻关项目, 2008C10022 号; 国家科技支撑计划项目, 2007BAD43B05 号; 广西科技攻关项目, 桂科攻0718003-3 号 |
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| DETECTION OF FISH VIRAL HEMORRHAGIC SEPTICEMIA VIRUS (VHSV) BY NESTED REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION (RT-PCR) |
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NI Sui1, YU Xiao-Wei1, WANG Jian-Ping2, LEI Ai-Ying3, ZENG Di-Gang3, WU Xiong-Fei2
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1.Faculty of Life Science and Biotechnology, Ningbo University;2.Ningbo Academy of Ocean and Fishery;3.Guangxi Institute of Fisheries
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| Abstract: |
| Fish viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus in fish causing serious fish mortality. With the results of genomes sequence analysis on VHSV, two pairs of primers were designed. Under optimized RT-PCR conditions, target regions of VHSV glycoprotein gene were amplified using VHS1-2 and VHS3-4 primers. Products of 279bp segment were shown after the nested RT-PCR reaction. Negative control showed no amplification. The RT-PCR product was proved identical to previously reported VHSV sequence. The detection limit of the nested RT-PCR assay is 0.1pg. Therefore, the nested RT-PCR assay, a sensitive, specific, and quick tool for VHSV detection is established.
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| Key words: Viral hemorrhagic septicemia virus, Nested reverse transcription polymerase chain reaction, Detection |
