| 摘要: |
| 于1994年5月-1995年7月，根据已克隆的草鱼出血病病毒GCHV-861株cDNA的部分序列，设计合成了两对PCR引物，采用RT-PCR技术对GCHV-861及GCHV-873两病毒株的dsRNA进行扩增。结果表明，两对引物仅能特异地检测出GCHV-861病毒株核酸的存在，而不能对GCHV-873病毒株的核酸进行特异扩增，该方法最小可检测出0.1Pg纯化的GCHV-861病毒dsRNA;采用该方法对GCHV-861人工感染的草鱼和稀有鲫组织进行RT-PCR检测，不仅能检测到发病期显症病鱼中GCHV-861病毒的存在，还能检测发病前期及愈后无出血病症状的病毒携带者中病毒的存在;利用该方法还从本所关桥实验场出血病流行季节收集的发病草鱼体内检测出GCHV的存在，说明所设计的引物具有一定的实际意义;利用该法还能检测出感染病毒的组织培养细胞系GCK和CIK上清液中GCHV的存在。还建立了简易的模板制备方法，采用该方法整个检测过程只需3-4h，可大大缩短检测时间。由此可见，RT-PCR技术是检测GCHV的快速、灵敏而特异的方法，发展和完善该技术对草鱼出血病的早期诊断和防治、抗病育种具有重要意义。 |
| 关键词: 草鱼出血病病毒 病毒检测 逆转录 聚合酶链反应 |
| DOI： |
| 分类号: |
| 基金项目:国家“八五”攻关课题，857220902。淡水生态与生物技术国家重点实验室和International foundation for science部分资助，A／2200-1号 |
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| DETECTION OF HEMORRHAGIC VIRUS OF GRASS CARP BY REVERSE TRANSCRIPTION—POLYMERASE CHAIN REACTION |
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Wang Tiehui1, Li Jun1, Yi Yonglan1, Zhou Liran2, Liu Hanqin1, Lu Renhou1, Chen Hongxi1
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1.Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072;2.School of Life Sciences.Wuhan University,Wuhan 430072
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| Abstract: |
| A rapid, sensitive and highly specific detection method for grass carp hemorrhagic virus (GCHV) based on reverse transcription—polymerase chain reaction was developed alter a May 1995 — July 1996 research. Two pairs of PCR primers were synthesized according to the cloned cDNA sequences of the GCHV-861 strain. For each primer combination only one specific major product was obtained when amplification was performed by using the genomic dsRNA of GCHV-861 strain The length of their expected products were 320 bp and 223 bp, respectively. No products were obtained when genomic nucleic acids other than GCHV-86l genomic dsRNA were used as RT-PCR templates. To assess the sensitivity of the method, dilutions of purified GCHV-86l genomic dsRNA (0.01 pg-1000 pg) were amplified and quantifies of as little as 0.1 pg of purified dsRNA were detected when the amplification product was analyzed by 1.5% agarose gel electrophoresis.
After about one-year-old grass carp (Ctenopharyngodon idell), and 3-5 cm rare minnow (Gobiotypris rarus), were artificially infected with GCHV-861 and GCHV-873 suspensions by the hyperosmotic immersion method, most of the fishes infected by GCHV-861 were dead during the 3rd - 7th day, 7 days later, no fish died. None was dead in the other group infected by GCHV-873. GCHV-861 strain could be detected not only in the diseased fishes with typical syndromes at developing period, but also in the carriers without any hemorrhage symptoms at pre- or post developing period. The virus could also be detected in the naturally diseased grass carp with hemorrhage symptoms collected from Guanqiao Fisheries Farm of our institute in 1996. GCHV-86l could also infect GCK and CIK cell cultures in vitro and could be detected from the fluids of their subcultures.
A simple, rapid and reliable dsRNA templates preparation method was developed. It was very suitable for detecting mass or very little samples, and the whole procedure could be finished within 3-4hs.
All the results show that the RT-PCR amplification method is a rapid, sensitive and highly specific method for detection of GCHV, and is of significant importance for early diagnosis, prophylaxis and treatment of hemorrhage of grass carp and GCHV- resistant breeding. |
| Key words: Hemorrhagic virus of grass carp (GCHV), Virus detection, Reverse transcription, Polymerase chain reaction |
