한국수산과학회지 (Korean Journal of Fisheries and Aquatic Sciences)

  • 제46권1호
  • /
  • Pages.46-52
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  • 2013
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  • 0374-8111(pISSN)
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  • 2287-8815(eISSN)
한국수산과학회 (The Korean Society of Fisheries and Aquatic Science)
권호 표지
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Negatively Charged Membrane을 이용한 해수 중 어류질병바이러스의 검출

Detection of Fish Pathogenic Viruses in Seawater Using Negatively Charged Membranes

  • 지보영 (국립수산과학원 수산생물방역과) ;
  • 김광일 (농림수산식품부 농림수산검역검사본부) ;
  • 이순정 (국립수산과학원 수산생물방역과) ;
  • 김기홍 (부경대학교 수산생명의학과) ;
  • 진지웅 (부경대학교 수산생명의학과) ;
  • 정현도 (부경대학교 수산생명의학과)
  • Jee, Bo Young (Aquatic life disease control division, National Fisheries Research & Development Institute) ;
  • Kim, Kwang Il (Animal, Plant and Fisheries Quarantine and Inspection Agency, Ministry for Food, Agriculture, Forestry and Fisherises) ;
  • Lee, Soon Jeong (Aquatic life disease control division, National Fisheries Research & Development Institute) ;
  • Kim, Ki Hong (Department of Aquatic Life Medicine, Pukyong National University) ;
  • Jin, Ji Woong (Department of Aquatic Life Medicine, Pukyong National University) ;
  • Jeong, Hyun Do (Department of Aquatic Life Medicine, Pukyong National University)
  • 투고 : 2012.11.01
  • 심사 : 2013.02.04
  • 발행 : 2013.02.28
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초록

After an outbreak of viral disease in an aquafarm, release of virus (es) from infected fish into environmental seawater has been suspected. In the present study, we utilized a negatively charged membrane (HA type) as an efficient method for concentration and detection of fish pathogenic viruses, specifically, megalocytivirus and viral hemorrhagic septicemia virus (VHSV) present in field-collected seawater samples or inoculated into seawater artificially. Positively charged viruses adsorbed onto the negatively charged membrane and were eluted with 1 mM NaOH (pH 10.5) following rinsing with 0.5 mM $H_2SO_4$ (pH 3.0). Megalocytivirus and VHSV particles isolated using anegatively charged HA membrane from seawater inoculated with each virus at a concentration of 10 viral particles/mL were of sufficient quantity to show positive results in atwo-step PCR (or RT two-step PCR); however, despite it being negatively charged, a cellulose acetate (CA) membraneshowed negative results. In quantitative PCR, the detection limits of the HA membrane for megalocytivirus and VHSV in seawater were 1.20E+00 viral particles/mL and 1.22E+01 viralparticles/mL, respectively. The calculated mean recovery yields from 1 L seawater spiked with known concentrations of megalocytivirus and VHSV particles were 28.11% and 23.00%, respectively. The concentrate of a 1-L sample of culturing seawater from the aquatank of flounder suffering from VHSV showed clear positive results in PCR when isolated with an HA, but not a CA, membrane. Thus, viral isolation using an HA membrane is a practical and reliable method for detection of fish pathogenic viruses in seawater.

키워드

참고문헌

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