한국어병학회지 (Journal of fish pathology)

  • 제25권3호
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  • Pages.257-262
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  • 2012
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  • 1226-0819(pISSN)
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  • 2233-5412(eISSN)
한국어병학회 (The Korean Society of Fish Pathology)
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Infectious hematopoietic necrosis virus (IHNV)-검출 Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) 법의 평가

Evaluation of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detection of infectious hematopoietic necrosis virus (IHNV)

  • 김위식 (전남대학교 수산과학연구소) ;
  • 전찬혁 (강릉원주대학교 해양자원육성학과) ;
  • 김정호 (강릉원주대학교 해양자원육성학과) ;
  • 오명주 (전남대학교 수산생명의학과)
  • Kim, Wi-Sik (The Fisheries Science Institute, Chonnam National University) ;
  • Jeon, Chan-Hyeok (Department of Marine Bioscience, Gangneung-Wonju National University) ;
  • Kim, Jeong-Ho (Department of Marine Bioscience, Gangneung-Wonju National University) ;
  • Oh, Myung-Joo (Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University)
  • 투고 : 2012.10.17
  • 심사 : 2012.11.13
  • 발행 : 2012.12.31
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초록

본 연구에서는 어체내 IHNV 모니터링에 LAMP법의 사용이 가능 하는지를 검토하기 위해 IHNV를 무지개송어에 인위적으로 감염시킨 후 시간 경과에 따라 LAMP법과 어류세포를 사용한 분리배양법을 이용하여 IHNV 검사를 실시하였다. IHNV를 $10^{6.5}\;TCID_{50}$/fish, $10^{5.5}\;TCID_{50}$/fish, $10^{4.5}\;TCID_{50}$/fish로 복강 주사한 결과, 40%, 0%, 0%의 누적폐사율이 관찰되었다. 폐사어 및 IHNV 접종 후 16일과 28일째에 각 실험구에서 채집한 생존어 5마리를 대상으로 한 IHNV 검사 결과, 폐사어에서 IHNV가 100% (8/8 마리) 분리되었고 (감염가: $10^{4.3}-10^{6.8}\;TCID_{50}/ml$), RT-LAMP법에서도 100% 검출되었다. 16일째 생존한 개체를 대상으로 한 IHNV 검사 결과에서는 $10^{6.5}\;TCID_{50}$/fish, $10^{5.5}\;TCID_{50}$/fish, $10^{4.5}\;TCID_{50}$/fish의 IHNV로 접종한 실험구에서 각각 60% (3/5 마리, 감염가: $10^{2.8}-10^{5.05}\;TCID_{50}/ml$), 20% (1/5 마리, $10^{1.05}\;TCID_{50}/ml$), 60% (3/5 마리, $10^{1.05}-10^{4.8}\;TCID_{50}/ml$) 의 검출율을 보였으나 LAMP법에서는 20% (1/5 마리), 0% (0/5 마리), 20% (1/5 마리) 의 검출율을 나타내었다. 28일째 생존한 개체 및 대조구의 어류에서는 IHNV가 분리 검출되지 않았다. 이상의 연구결과로 LAMP법은 IHNV-생존어에서 바이러스를 모니터링 하는데 한계가 있으나 병어로부터 IHNV를 검출하는데 유용하게 사용될 수 있을 것으로 사료되었다.

A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was evaluated to monitor infectious hematopoietic necrosis virus (IHNV) from artificially infected rainbow trout Oncorhynchus mykiss. The cumulative mortalities of fish challenged with IHNV at $10^{6.5}\;TCID_{50}$/fish, $10^{5.5}\;TCID_{50}$/fish and $10^{4.5}\;TCID_{50}$/fish were 40%, 0% and 0%, respectively. Dead fish and survivors at 16 and 28 d post-challenge in each group were employed for IHNV detection by RT-LAMP assay and virus isolation using BF-2 cells. IHNV from $10^{4.3}$ to $10^{6.8}\;TCID_{50}/ml$ was isolated from all the dead fish and also detected in all of the examined dead fish by RT-LAMP assay. In survivors at 16 d, 60% (3/5 fish, $10^{2.8}-10^{5.05}\;TCID_{50}/ml$), 20% (1/5 fish, $10^{1.05}\;TCID_{50}/ml$) and 60% (3/5 fish, $10^{1.05}-10^{4.8}\;TCID_{50}/ml$) were found to be IHNV-positive by virus isolation in fish challenged with IHNV at $10^{6.5}\;TCID_{50}$/fish, $10^{5.5}\;TCID_{50}$/fish and $10^{4.5}\;TCID_{50}$/fish, respectively, while 20% (1/5 fish), 0% (0/5 fish) and 20% (1/5 fish) were IHNV-positive by RT-LAMP assay. No IHNV was detected in the survivors at 28 d and control fish. These results indicate that the RT-LAMP assay is useful for detection of IHNV in diseased fish although it is not enough to monitor virus in IHNV-survivors.

키워드

참고문헌

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