산업식품공학 (Food Engineering Progress)

  • 제15권4호
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  • Pages.370-375
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  • 2011
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  • 1226-4768(pISSN)
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  • 2288-1247(eISSN)
한국산업식품공학회 (Korean Society for Industrial Food Engineering)
권호 표지

PCR-DGGE를 이용한 유기농 채소의 유해 미생물 신속 검지

The Rapid Detection of Pathogens in Organically Grown Vegetables Using PCR-DGGE

  • 권오연 (호서대학교 식품생물공학과) ;
  • 손석민 (호서대학교 식품생물공학과)
  • Kwon, Oh Yeoun (Department of Food and Biotechnology, Hoseo University) ;
  • Son, Seok Min (Department of Food and Biotechnology, Hoseo University)
  • 투고 : 2011.10.07
  • 심사 : 2011.11.07
  • 발행 : 2011.11.30
⟨ 이전 논문 다음 논문 ⟩

초록

현재 식중독 미생물의 검사에 전통적인 배지법이 다수를 차지하고 있으나 PCR이나 면역 분석법과 같은 신속 검출 법들의 사용이 증가하는 추세이다. 그러나 대개 speciesspecific 프라이머를 이용한 PCR에 의한 DNA 증폭산물을 전기영동을 통해 확인하는 방법을 사용하고 있는데 이는 각종 균주에 대해 각기 다른 프라이머를 사용하여야하는 단점이 있다. 이러한 단점을 보완하기 위하여 본 연구에서 는 여러 가지 균주를 동시에 신속하게 검지할 수 있도록 PCR-DGGE 기술을 연구하게 되었다. 그 결과 6종(S. typhimurium, P. fluorescens, B. cereus, S. aureus, E. coli, L. monoytogenes)의 유해 미생물을 선정하였고 DGGE 상에서 확실한 분리능을 보여 유해미생물의 인공마커로 사용할 수 있음을 보였다. 또한 실제 PCR-DGGE 기법을 사용하기 위하여 채소에서의 유해미생물 검출 감도를 확인한 결과 B. cereus를 제외한 5종(S. typhimurium, P. fluorescens, S. aureus, E. coli, L. monoytogenes)의 균들은 모두 $10^1$ CFU/g까지 검출할 수 있었고 B. cereus는 $10^3$ CFU/g이상 채소에 존재할 때 검출할 수 있었다. 또한 균질화 방법에 따라서 식물 DNA와 유해 미생물 간의 경쟁적 증폭현상이 일어남도 확인할 수 있었다. 이로써 본 연구에서는 유해 미생물의 검지, 인공마커의 제조, 식물 DNA와 유해미생물간의 경쟁적 증폭 현상 방지책과 유해미생물의 검출 감도 확인 및 염기서열을 통한 동정을 통해 PCR-DGGE가 식품에서 유해 미생물의 신속 검지 방법으로써 활용할 수 있을 것으로 사료된다. 향후 국내 식품소비 패턴이 유기농 채소의 수요와 공급이 날로 증대 되고 있기 때문에 PCR-DGGE 기법이 유기농 채소들의 식중독 균에 대한 database를 구축하여 안전성을 확보하는데 기여할 수 있다고 생각된다.

In this study the polymerase chain reaction (PCR) combined with denaturing gradient gel electrophoresis (DGGE) was evaluated as a method permitting the rapid detection of pathogens in fresh originally grown vegetables. A universal primer (341GCf/534r) was selected for its ability to amplify the V3 region of 16S-rRNA genes in their target pathogens (Salmonella typhimurium, Pseudomonas fluorescens, Bacillus cereus, Listeria monoytogenes, Staphyloocus aureus, E. coli). The 194 bp fragments in PCR were successfully duplicated as expected. The amplified fragments of the same size from six different pathogens also showed good separation upon DGGE. The detection limit of PCR-DGGE for six pathogens in fresh-cut lettuces were over $10^{5}$ CFU/g when sampled by stomaching. However, when the sampling method was changed from stomaching to shaking, the detection limit of six pathogens in organic vegetables was shown to increase by over $10^{1}$ CFU/g, but only those of B. cereus were over $10^{3}$ CFU/g. Therefore, PCR-DGGE was shown to be a reliable method for the detection of pathogens in fresh-cut vegetables.

키워드

참고문헌

  1. Ampe F, Sirvent A, Zakhia N. 2001. Dynamics of the microbial community responsible for traditional sour cassava starch fermentation studied by denaturing gradient gel electrophoresis and quantitative rRNA hybridization. Int. J. Food Microbial. 65: 45-54. https://doi.org/10.1016/S0168-1605(00)00502-X
  2. Bennett A.R, Greenwood D, Tennant C, Banks, Bets R.P. 1998. Rapid and definitive detection of Salmonella in food by PCR. Lett. Appl. Microbial. 26: 437-441. https://doi.org/10.1046/j.1472-765X.1998.00368.x
  3. Booysen C, Dicks L.M, Melijering I, Ackermann A. 2002. Isolation, identification and changes in the composition of lactic acid bacteria during the malting of two different barely cultivars. Int. J. Food Microbial. 76: 63-73. https://doi.org/10.1016/S0168-1605(02)00007-7
  4. Cocolin L, Manzano M, Cantoni C, Comi G. 2001. Denaturing gradient gel electrophoresis analysis of the 16S rRNA gene V1 region to monitor dynamic changes in the bacterial population during fermentation of Italian sausages. Appl. Environ. Microbiol. 67: 5113-21. https://doi.org/10.1128/AEM.67.11.5113-5121.2001
  5. Como-Sabetti K, Reagan S, Ritter K, Parrott C.M, Simonds S, Hraboxy S, Ritter B. 1997. Outbreaks of E.coli O157:H7 associated with eating alfalfa sprouts- Michigan and Virginia. Morbidity and Mortality Weekly Report 46: 741-744.
  6. Dolye, M.P. 1990. Fruit and vegetable safety-microbiologicals. Hortscience 25: 1478-1481.
  7. Francis G.A, Thomas C, O'Berine D.O. 1999. The microbiological safety of minimally processed vegetables. Int. J. Food Sci. Tech. 34: 1-22. https://doi.org/10.1046/j.1365-2621.1999.00253.x
  8. Fasoli S, Marzotto M, Rizotti L, Rossi F, Dellaglio F, Torriani S. 2003. Bacterial composition of commercial probiotics products as evaluated by PCR-DGGE analysis. Int. J. Food Microbial. 82: 59- 70. https://doi.org/10.1016/S0168-1605(02)00259-3
  9. Kagimoto S. 1995. PCR-SSCP, direct sequencing. Japanese J. Clin. Med. 53: 310-315.
  10. Kobayashi K, Taguchi M, Seto K. 1994. Polymerase chain reaction (PCR) method for the detection of Salmonella by using Chelex resin after enrichment culture. Kansenshogaku Zasshi. 68: 1203- 1210.
  11. Meroth C.B, Walter J, Hertel C, Brandt M.J, Hammes W.P. 2003. Monitoring the bacterial population dynamics in sourdough fermentation processes by using PCR-denaturing gradient gel electrophoresis. Appl. Environ. Microbial. 69: 475-482. https://doi.org/10.1128/AEM.69.1.475-482.2003
  12. Muyzer G, EC de Waal, A Uitterlinden. 1993. Profiling of complex microbial populations using denaturating gradient gel electrophoresis analysis of polymerase chain reaction amplified gene coding for 16S rRNA. Appl. Environ. Microbial. 59: 695-700.
  13. Nachamkin I, Bohachick K, Patton, C.M. 1993. Flagellin gene typing of Campylobacter jejuni by restriction fragment length polymorphism analysis. J. Clin. Microbial. 31: 1531-1536.
  14. Niderhauser C, Hofelein C, Allmann M, Burkhalter P, Luthy J, Candrian, U. 1994. Random amplification of polymorphic bacterial DNA:Evalution of 11 oligonucleotides and application to food contaminated with Listeria monocytogenes. J. Appl. Bacteriol. 77: 574-582. https://doi.org/10.1111/j.1365-2672.1994.tb04404.x
  15. Nubel U, Garcia-Pichel F, Kuhl M, Muyzer G. 1999. Quantifying microbial diversity morphotypes, 16S rRNA genes, and carotenoids of oxygenic phototrophs in microbial mats. Appl. Environ. Microbial. 65: 422-430.
  16. Olsen T.E, Aabo S, Hill W, Notermans S, Wernars K, Ganum P.E, Poporic T, Rasmussen H.N, Slavik O. 1995. Probes and polymerase chain reaction for detection of food-borne bacterial pathogens. Int. J. Food Microbiol. 28: 1-78. https://doi.org/10.1016/0168-1605(94)00159-4
  17. Shin SY, Koo YJ. 1999. Specific detection of Listeria monocytogenes in foods by a Polymerase Chain Reaction. Korean J. Food Sci. Tech. 6: 1628-1634.
  18. van Beek S, Priest F.G. 2002. Evolution of the lactic acid bacterial community during malt whisky fermentation: a polyphasic study. Appl. Environ. Microbiol. 68: 297-305. https://doi.org/10.1128/AEM.68.1.297-305.2002