한국식품과학회지 (Korean Journal of Food Science and Technology)

한국식품과학회 (Korean Society of Food Science and Technology)
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IMS-RT-PCR을 활용한 육성돈 분변으로부터 간염 A형 바이러스의 신속순수분리 및 검출법 연구

Use of IMS-RT-PCR for the Rapid Isolation and Detection of Hepatitis A Virus from the Swine Feces

  • 이희민 (전남대학교 농업생명과학대학 식품공학과 및 기능성식품연구센터) ;
  • 김두운 (전남대학교 농업생명과학대학 식품공학과 및 기능성식품연구센터)
  • Lee, Hee-Min (Department of Food Science and Technology and Functional Food Research Center, Chonnam National University) ;
  • Kim, Duwoon (Department of Food Science and Technology and Functional Food Research Center, Chonnam National University)
  • 투고 : 2012.08.03
  • 심사 : 2012.09.08
  • 발행 : 2012.10.31
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초록

돼지축사에서 채집해온 6개의 육성돈의 분변에서 식중독 유발 바이러스인 HAV와 HEV를 검출하였으며, HAV는 88.3%의 검출율을 보였으며, HEV는 33.3%의 검출율을 보였다. 결과에는 제시하지 않았으나, 염기서열 분석결과 HEV는 사람에게 전염이 가능한 유전자형인 III형이었으며, 실험적으로 사람의 간세포인 PLC/PRF/5에 접종하였을 때 증식이 됨을 확인하였다. 식중독 유발 바이러스인 HAV와 HEV는 오염된 식품이나 물을 섭취하거나 교차 오염에 의해 전염이 가능하기 때문에 돼지축사에서 위생상태의 개선뿐만 아니라 육류를 섭취하기 전인 운송 및 가공과정까지 식중독 유발 바이러스에 의한 교차오염을 막는 노력이 필요하다. HAV와 HEV 모두 검출된 분변에서 HAV를 순수분리하고 빠르게 검출하기 위해 IMS-RT-PCR을 적용하였으며, 항원-항체 반응에 의해 순수하게 HAV만을 분리할 수 있었다. 또한 HAV만이 순수분리 되었는지 재확인하기위해 세포감염을 통해 증식된 바이러스를 확보한 후 nested RT-PCR을 수행한 결과, HAV만을 순수 분리할 수 있음을 확인하였다. 이는 IMS 활용기술이 단순히 항체를 교체함으로써 다른 특정 식중독 유발 바이러스의 다양한 시료에서 바이러스 순수분리 및 검출에 활용 가능성이 있음을 확인하였다.

Human pathogenic viruses such as hepatitis A and E virus (HAV and HEV), which lead to acute liver failure and death, are foodborne pathogens associated with the consumption of virus-contaminated meats, filter-feeding bivalves, fruits, and salads. Two of the three swine farms examined in this study had HAV and HEV positive stool samples in a nested RT-PCR assay. The use of the immunomagnetic separation (IMS) facilitated the separation of HAV through interactions between the ligand on the virion surface and the antibody from the swine feces containing both HAV and HEV. The nested RT-PCR analysis was performed for the detection of HAV obtained from hepatocarcinoma cell line (PLC/PRF/5) contaminated with eluent fraction of IMS. This indicated that IMS has the potential to simultaneously isolate and concentrate target viruses by changing antibodies linked on the magnetic beads.

키워드

참고문헌

  1. Jung YK, Kim JH. Epidemiology and clinical features of acute hepatitis A: from the domestic perspective. Korean J. Hepatol. 15: 438-445 (2009) https://doi.org/10.3350/kjhep.2009.15.4.438
  2. Rodriguez-Manzano J, Miagostovich M, Hundesa A, Clemente- Casares P, Carratala A, Buti M, Jardi R, Girones R. Analysis of the evolution in the circulation of HAV and HEV in eastern Spain by testing urban sewage samples. J. Water Health 8: 346- 354 (2010) https://doi.org/10.2166/wh.2009.042
  3. Hollinger FB, Emerson SU. Hepatitis A virus. Vol. 1, pp. 799- 840. In: Fields Virology. Knipe DM, Howley PM (eds). Lippincott Williams & Wilkins, Philadelphia, PA, USA (2001)
  4. Provost PJ, Ittensohn OL, Villarejos VM, Arguedas JA, Hilleman MR. Etiologic relationship of marmoset-propagated CR326 hepatitis A virus to hepatitis in man. Proc. Soc. Exp. Biol. Med. 142: 1257-1267 (1973) https://doi.org/10.3181/00379727-142-37220
  5. Rakela J, Fay OH, Stevenson D, Gordon I, Mosley JW. Similarities of two hepatitis A virus strains. Bull. World Health Organ. 54: 561-564 (1976)
  6. Tam AW, Smith MM, Guerra ME, Huang C, Bradley DW, Fry KE, Reyes GR. Hepatitis E virus(HEV): Molecular cloning and sequence of the full-length viral genome. Virology 185: 120-130 (1991) https://doi.org/10.1016/0042-6822(91)90760-9
  7. Takahashi M, Kusakai S, Mizuo H, Suzuki K, Fujimura K, Masuko K, Sugai Y, Aikawa T, Nishizawa T, Okamoto H. Simultaneous detection of immunoglobulin A(IgA) and IgM antibodies against hepatitis E virus(HEV) is highly specific for diagnosis of acute HEV infection. J. Clin. Microbiol. 43: 49 - 56 (2005) https://doi.org/10.1128/JCM.43.1.49-56.2005
  8. Lu L, Li C, Hagedorn CH. Phylogenetic analysis of global hepatitis E virus sequences: Genetic diversity, subtypes, and zoonosis. Rev. Med. Virol. 16: 5-36 (2006) https://doi.org/10.1002/rmv.482
  9. Okamoto H. Genetic variability and evolution of hepatitis E virus. Virus Res. 127: 216-228 (2007) https://doi.org/10.1016/j.virusres.2007.02.002
  10. Lewis HC, Wichmann O, Duizer E. Transmission routes and risk factors for autochthonous hepatitis E virus infection in Europe: A systematic review. Epidemiol. Infect. 138: 145-166 (2010) https://doi.org/10.1017/S0950268809990847
  11. Meng XJ. Hepatitis E virus: Animal reservoirs and zoonotic risk. Vet. Microbiol. 140: 256-265 (2010) https://doi.org/10.1016/j.vetmic.2009.03.017
  12. Okamoto H, Takahashi M, Nishizawa T. Features of hepatitis E virus infection in Japan. Internal. Med. 42: 1065-1071 (2003) https://doi.org/10.2169/internalmedicine.42.1065
  13. Purcell RH, Emerson SU. Hepatitis E: an emerging awareness of an old disease. J. Hepatol. 48: 494-503 (2008) https://doi.org/10.1016/j.jhep.2007.12.008
  14. Tanaka T, Takahashi M, Kusano E, Okamoto H. Development and evaluation of an efficient cell-culture system for hepatitis E virus. J. Gen. Virol. 88: 903-911 (2007) https://doi.org/10.1099/vir.0.82535-0
  15. Monceyron C, Grinde B. Detection of hepatitis A virus in clinical and environmental samples by immunomagnetic separation and PCR. J. Virol. Methods 46: 157 - 166 (1994) https://doi.org/10.1016/0166-0934(94)90100-7
  16. Kim DW, Kim SR, Kwon KS, Lee JW, Oh MJ. Detection of hepatitis A virus from oyster by nested PCR using efficient extraction and concentration method. J. Microbiol. 46: 436-440 (2008) https://doi.org/10.1007/s12275-008-0131-1
  17. Kim SE, Kim MY, Kim DG, Song YJ, Jeong HJ, Lee SW, Lee JB, Park SY, Song CS, Oh SJ, Yoo HS, Choi IS. Determination of fecal shedding rates and genotypes of swine hepatitis E virus(HEV) in Korea. J. Vet. Med. Sci. 70: 1367-1371 (2008) https://doi.org/10.1292/jvms.70.1367
  18. Emerson SU, Purcell RH. Hepatitis E virus. Rev. Med. Virol. 13: 145-154 (2003) https://doi.org/10.1002/rmv.384

피인용 문헌

  1. Rapid detection of the hepatitis a virus from fresh lettuce using immunomagnetic separation and quantum dots assay vol.21, pp.2, 2014, https://doi.org/10.11002/kjfp.2014.21.2.170