Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
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Establishment of Elution and Concentration Procedure for Rapid and Sensitive Detection of Norovirus in Foods of Diverse Matrices

다양한 매트릭스가 혼합된 식품을 대상으로 노로바이러스 신속검출을 위한 탈리 및 농축방법 확립

  • Ahn, Jaehyun (Department of Biotechnology, Dongguk University-Gyeongju) ;
  • Kwon, Youngwoo (Department of Biotechnology, Dongguk University-Gyeongju) ;
  • Lee, Jeong-Su (Microbiology Division, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation) ;
  • Choi, Weon Sang (Department of Biotechnology, Dongguk University-Gyeongju)
  • 안재현 (동국대학교 생명공학과) ;
  • 권영우 (동국대학교 생명공학과) ;
  • 이정수 (식품의약품안전평가원 미생물과) ;
  • 최원상 (동국대학교 생명공학과)
  • Received : 2015.01.23
  • Accepted : 2015.04.17
  • Published : 2015.06.30
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Abstract

This article reports the development of an effective test procedure for detection of norovirus (NoV) in foods of diverse matrices. In this study, target foods included fermented milk, soybean paste, powders made from uncooked grains and vegetables, sesame leaves preserved in soy sauce, pickled mooli, and mooli. Viral recovery varied depending on the food matrices or elution buffers tested. Buffers were compared to determine effective elution buffers from artificially virus-contaminated foods. The conventional test procedure for concentrating viruses from food (elution-polyethylene glycol(PEG) precipitation-chloroform-PEG precipitation) was modified to save time by eliminating one PEG precipitation step. The modified procedure (elution-chloroform-PEG precipitation) was able to concentrate viruses more effectively than the conventional procedure. It also removed RT-PCR inhibitors effectively. The modified procedure was applied to target food for genogroup II NoV detection. NoV RNA was detected at the initial inoculum levels 3.125-12.5 RT-PCR units per 10-25 g tested food. The use of this newly established procedure should facilitate detection of low levels of norovirus in diverse foods.

본 연구는 다양한 식재료가 섞여있는 식품으로부터 노로바이러스를 효과적으로 검출하기 위한 시험법 개발에 관한 것이다. 각 식품이 가진 매트릭스가 매우 다르므로 모든 식품에 공통적으로 적용할 수 있는 표준화된 검출법이 현재로서는 없다. 본 연구에서는 발효식품(농후발효유, 된장), 절임식품(깻잎장아찌, 단무지)과 생식제품을 대상으로 실험을 진행하였다. PBS, beef extract, 아미노산-NaCl 용액 등을 이용하여 바이러스에 오염된 대상식품들로 부터 바이러스의 탈리율을 비교하였다. 이로부터 다양한 매트릭스가 혼합된 식품들에 적용 가능한 탈리용액을 선별하였다. 식품의약품안전처가 제안하여 현재 국내에서 굴, 야채, 과일 등을 대상으로 바이러스 농축에 널리 사용되고 있는 식중독 바이러스 검출법인 EPCP공정(탈리-PEG 침전-클로르포름 처리-PEG 침전)과 PEG 침전과정을 한번으로 줄인 수정된 ECP공정(탈리-클로르포름 처리-PEG침전)의 효능을 비교해 본 결과 ECP공정은 EPCP공정과 비슷하거나 나은 효율로 바이러스를 농축할 수 있었다. 또 바이러스 농축 후 QIAamp$^{(R)}$ Viral RNA Mini kit를 이용하여 RNA를 추출할 경우 식품에 존재하는 RT-PCR방해 물질들이 효과적으로 제거되어 추가적인 처리가 더 필요하지 않음을 알 수 있었다. 수정된 공정을 이용하여 무를 추가한 6가지 식품을 대상으로 검출한계를 조사해 본 바 10-25 g 식품으로부터 3.125-12.5 RT-PCR unit까지 검출이 가능하였다. 이는 기존에 보고된 방법들의 검출한계보다 더 우수한 것으로 장차 다양한 식품을 대상으로 효과적인 바이러스 검출이 가능할 것으로 기대된다.

Keywords

References

  1. Koopmans M., von Bonsdorff C., Vinje J., de Medici D., Monroe S.: Foodborne viruses. FEMS Microbiol. Rev. 26, 187-205 (2002). https://doi.org/10.1111/j.1574-6976.2002.tb00610.x
  2. Lopman B.A., Reacher M.H., Duijnhoven Y., Hanon F., Brown D., Koopmans M.: Viral gastroenteritis outbreaks in Europe, 1995-2000. Emerging Infect. Dis. 9, 90-96 (2003). https://doi.org/10.3201/eid0901.020184
  3. http://www.mfds.go.kr/
  4. Koopmans M., Duizer E.: Foodborne viruses: an emerging problem. Int. J. Food Microbiol. 90, 23-41 (2004). https://doi.org/10.1016/S0168-1605(03)00169-7
  5. Patel M.M., Hall A.J., Vinje J., Parashar U.D.: Noroviruses: A comprehensive review. J. Clin. Virol. 44, 1-8 (2009). https://doi.org/10.1016/j.jcv.2008.10.009
  6. CDC Surveillance for foodborne disease outbreaks-United States, 1998-2008. MMWR, 62 (2) (2013).
  7. Kingsley D.H.: An RNA extraction protocol for shellfishborne viruses. J. Virol. Methods 141, 58-62 (2007). https://doi.org/10.1016/j.jviromet.2006.11.027
  8. Schultz A.C., Saadbye P., Hoorfar J., Norrung B.: Comparison of methods for detection of norovirus in oysters. Int. J. Food Microbiol. 114, 352-356 (2007). https://doi.org/10.1016/j.ijfoodmicro.2006.09.028
  9. Comelli H.L.,Rimstad E., Larsen S., Myrmel M.: Detection of norovirus genotype I.3b and II.4 in bioaccumulated blue mussels using different virus recovery methods. Int J. Food Microbiol. 127, 53-59 (2008). https://doi.org/10.1016/j.ijfoodmicro.2008.06.003
  10. Ha S.H., Woo G.J., Hwang I.K., Choi W.S.: Development of a virus elution and concentration procedure for detecting norovirus in oysters. Food Sci. Biotechnol. 18, 1150-1154 (2009).
  11. Bidawid S., Farber J.M., Sattar S.A.: Rapid concentration and detection of hepatitis A virus from lettuce and strawberries. J. Virol. Methods. 88, 175-185 (2000). https://doi.org/10.1016/S0166-0934(00)00186-5
  12. Dubois E., Hennechart C., Deboosere N., Merle G., Legeay O., Burger B., Le Calve M., Lombard B., Ferre V., Traore O.: Intra-laboratory validation of a concentration method adapted for the enumeration of infectious F-specific RNA coliphage, enterovirus, and hepatitis A virus from inoculated leaves of salad vegetables. Int J. Food Microbiol. 108, 164-171 (2006). https://doi.org/10.1016/j.ijfoodmicro.2005.11.007
  13. Dubois E., Hennechart C., Merle G., Burger C., Hmila N., Ruelle S., Perelle S., Ferre V.: Detection and quantification by real-time RT-PCR of hepatitis A virus from inoculated tap waters, salad vegetables, and soft fruits: Characterization of the method performances. Int J. Food Microbiol. 117, 141-149 (2007). https://doi.org/10.1016/j.ijfoodmicro.2007.02.026
  14. Butot S., Putallaz T., Sanchez G.: Procedure for rapid concentration and detection of enteric viruses from berries and vegetables. Appl. Environ. Microbiol. 73, 186-192 (2007). https://doi.org/10.1128/AEM.01248-06
  15. Papafragkou E., Plante M., Mattison K., Bidawid S., Karthikeyan K., Farber J.M., Jaykus L.A.: Rapid and sensitive detection of hepatitis A virus in representative food matrices. J. Virol. Methods. 147, 177-187 (2008). https://doi.org/10.1016/j.jviromet.2007.08.024
  16. Fumian T.M., G. Leite J.P.G., Marin V.A., Miagostovich M.P.: A rapid procedure for detecting noroviruses from cheese and fresh lettuce. J. Virol. Methods. 155, 39-43 (2009). https://doi.org/10.1016/j.jviromet.2008.09.026
  17. Moon A., Hwang I.G., Choi W.S.: Development of a virus elution and concentration procedure for detecting norovirus in cabbage and lettuce. Food Sci. Biotechnol. 18, 407-412 (2009).
  18. Moon A., Ahn J., Choi W.S.: Elution buffers for human enteric viruses in vegetables with applications to norovirus detection. J. Fd Hyg. Safety 28, 287-292 (2013). https://doi.org/10.13103/JFHS.2013.28.4.287
  19. Le Guyader F.S., Mittelholzer C., Haugarreau L., Hedlund K., Alsterlund R., Pommepuy M., Svensson L.: Detection of noroviruses in raspberries associated with a gastroenteritis outbreak. Int. J. Food Microbiol. 97, 179-186 (2004). https://doi.org/10.1016/j.ijfoodmicro.2004.04.018
  20. Rzezutka A., D'Agostino M., Cook N.: An ultracentrifugation-based approach to the detection of hepatitis A virus in soft fruits. Int. J. Food Microbiol. 108, 315-320 (2006).
  21. Kim H.Y., Kwak I.S., Hwang I.K., Ko G.P.: Optimization of methods for detecting norovirus on various fruit. J. Virol. Methods. 153, 104-110 (2008). https://doi.org/10.1016/j.jviromet.2008.07.022
  22. Schwab K.J., Neill F.H., Fankhauser R.L., Daniels N.A., Monroe S.S., Bergmire-Sweat D.A., Estes M.K., Atmar R.L.: Development of Methods To Detect "Norwalk-Like Viruses" (NLVs) and Hepatitis A Virus in Delicatessen Foods: Application to a Food-Borne NLV Outbreak. Appl. Environ. Microbiol. 66, 213-218 (2000). https://doi.org/10.1128/AEM.66.1.213-218.2000
  23. Love D.C., Casteel M.J., Meschke J.S., Sobsey M.D.: Methods for recovery of hepatitis A virus (HAV) and other viruses from processed foods and detection of HAV by nested RTPCR and TaqMan RT-PCR. Int. J. Food Microbiol. 126, 221-226 (2008). https://doi.org/10.1016/j.ijfoodmicro.2008.05.032
  24. Kim S.R., Kim D., kwon K., Hwang I., Oh M.: Detection of norovirus in contaminated ham by RT-PCR and nested PCR. Food Sci. Biotechnol. 17, 651-654 (2008).
  25. Shinohara M, Uchida K., Shimada S., Tonioka K., Suzuki N., Minegishi T., Kawahashi S., Yoshikawa Y., Ohashi N.: Application of simple method using minute particles of amorphorus calcium phosphate for recovery of norovirus from cabbage, lettuce, and ham. J. Virol. Methods 187, 153-158 (2013). https://doi.org/10.1016/j.jviromet.2012.09.025
  26. Croci L., De Medici D., Scalfaro C., Fiore A., Toti L.: The survival of Hepatitis A virus in fresh produce. Int. J. Food Microbiol. 73, 29-34 (2002). https://doi.org/10.1016/S0168-1605(01)00689-4
  27. Summa M., von Bonsdorff C., Maunula L.: Evaluation of four virus recovery methods for detecting norovirus on fresh lettuce, sliced ham, and frozen raspberries. J. Virol. Methods 183, 154-160 (2012). https://doi.org/10.1016/j.jviromet.2012.04.006
  28. Morales-Rayas R, Wolffs P.F.G., Griffiths M.W.: Simultaneous separation and detection of hepatitis A virus and norovirus in produce. Int. J. Food Microbiol. 139, 48-55 (2010). https://doi.org/10.1016/j.ijfoodmicro.2010.02.011
  29. Vega E., Smith J., Garland J., Matos A., Pillaii S.D.: Variability of virus attachment patterns to butterhead lettuce. J. Food Prot. 68, 2112-2117 (2005). https://doi.org/10.4315/0362-028X-68.10.2112
  30. Dubois E., Agier C., Traore O., Hennechart C., Merle G., Cruciere C., Laveran H.: Modified concentration method for the detection of enteric viruses on fruits and vegetables by reverse transcriptase-polymerase chain reaction or cell culture. J. Food Prot. 65, 1962-1969 (2002). https://doi.org/10.4315/0362-028X-65.12.1962
  31. Scherer K., Johne R., Schrader C., Ellerbroek L., Schulenburg K., Klein G.: Comparison of two extraction methods for viruses in food and application in a norovirus gastroenteritis outbreak. J. Virol. Methods 169, 22-27 (2010). https://doi.org/10.1016/j.jviromet.2010.06.008
  32. Sair A.I., D'Souza D.H., Moe C.L., Jaykus L.A.: Improved detection of human enteric viruses in foods by RT-PCR. J. Virol. Methods. 100, 57-69 (2002). https://doi.org/10.1016/S0166-0934(01)00397-4
  33. Abad F.X., Pinto R.M., Bosch A.: Survival of enteric viruses on environmental formites. Appl. Environ. Microbiol. 60, 3704-3710 (1994).
  34. Kurdziel A.S., Wilkinson N., Langton S., Cook N.: Survival of poliovirus on soft fruit and salad vegetables. J. Food Prot. 64, 706-709 (2001). https://doi.org/10.4315/0362-028X-64.5.706
  35. Baert L., Uyttendaele M., Debevere J.: Evaluation of viral extraction methods on a broad range of ready-to-eat foods with conventional and real-time RT-PCR for norovirus GII detection. Int. J. Food Microbiol. 123, 101-108 (2008). https://doi.org/10.1016/j.ijfoodmicro.2007.12.020
  36. Lees D.N., Henshilwood K., Dore W.J.: Development of a method for detection of enteroviruses in shellfish by PCR with poliovirus as a model. Appl. Environ. Microbiol. 60, 2999-3005 (1994).
  37. Jaykus L.A., Leon R.D., Sobsey M.D.: A virion concentration method for detection of human enteric viruses in oysters by PCR and oligoprobe hybridization. Appl. Environ. Microbiol. 62, 2074-2080 (1996).
  38. Leggitt P.R., Jaykus L.A.: Detection methods for human enteric viruses in the representative foods. J. Food Prot. 63, 1738-1744 (2000). https://doi.org/10.4315/0362-028X-63.12.1738
  39. Atmar R.L., Neill F.H., Romalde J.L., Le Guyader O.L., Woodley C.M., Metcalf T.G., Estes M.K.:Detection of Norwalk virus and Hepatitis A virus in shellfish tissues with the PCR. Appl. Environ. Microbiol. 61, 3014-3018 (1995).
  40. Schwab K.J., Estes M.K., Neill F.H., Atmar R.L.: Use of heat release and an internal RNA standard control in reverse transcription-PCR detection of Norwalk virus from stool samples. J. Clin. Microbiol. 35, 511-514 (1997).
  41. Dulbecco R. : The nature of viruses. In Virology, 2nd Ed. (Dulbecco R., and Ginsberg H.S. eds.) J.B. Lippincott Company, Philadelphia, pp. 1-26 (1988).
  42. Rueckert R.R. : Picornaviridae: The viruses and their replication. In Fields Virology, 3rd Ed. (Fields B.N., Knipe D.M., Howley P.M., Chanock R.M., Melnick J.L., Monath T.P., and Roizman B. eds.) Lippincott-Raven Publishers, Philadelphia.New York, pp. 609-654 (1996).