Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
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Comparison of DNA Extraction Methods for the Detection of Foodborne Pathogenic Bacteria from Livestock Manure Composts

퇴비에서 식중독균 검출을 위한 DNA 추출 방법 비교

  • Kim, Sung-Youn (Division of Safety Analysis, Experiment & Research Institute National Agricultural Products Quality Management Service) ;
  • Seo, Dong-Yeon (Division of Safety Analysis, Experiment & Research Institute National Agricultural Products Quality Management Service) ;
  • Moon, Ji-Young (Division of Safety Analysis, Experiment & Research Institute National Agricultural Products Quality Management Service)
  • 김성연 (국립농산물품질관리원 시험연구소 안전성분석과) ;
  • 서동연 (국립농산물품질관리원 시험연구소 안전성분석과) ;
  • 문지영 (국립농산물품질관리원 시험연구소 안전성분석과)
  • Received : 2019.07.10
  • Accepted : 2019.11.28
  • Published : 2019.12.30
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Abstract

This study investigated the efficacy of DNA extraction methods for real-time PCR detection of foodborne pathogenic bacteria in livestock manure composts. Livestock manure composts were inoculated with Escherichia coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes, Bacillus cereus and incubated in enrichment broth. For DNA extraction, enriched samples were treated following boiling method, by chloroform, C18 powder, and proteinase K. As a result, 4 species of bacteria were detected by real-time PCR when subjected to boiling for 30 min and treated with proteinase K. These results suggest that detection of foodborne pathogens by real-time PCR from livestock manure composts could be applicable using effective DNA extraction methodology such as the boiling method or proteinase K.

본 연구에서는 가축분퇴비에 존재할 수 있는 식중독균의 검출을 위하여 기존의 배양을 이용한 방법을 대체할 수 있는 real-time PCR을 적용하고자 하였으며, 이에 따라 유전자 증폭에 영향을 미치는 DNA 추출 방법에 따른 식중독균 검출 효율을 비교하였다. 적용한 방법은 가열 처리, 유기용매 및 흡착제 처리, 효소 처리의 3가지로 구분할 수 있으며, 각 방법에 따른 DNA의 검출 효율을 실험 결과로 나타내었다. 가열 처리 방법에서는 가열 시간의 증가에 따라 DNA 검출 효율이 높아지는 경향을 나타냈으며, 유기용매 및 흡착제는 효과를 나타내지 않았고, 효소 처리의 경우에는 그람 양성균 보다는 그람 음성균의 DNA가 추출 효율이 더 높은 것으로 나타났다. 결론적으로 퇴비에서 30분 이상의 가열 처리와 효소의 처리를 통한 DNA 추출 방법은 real-time PCR을 적용한 식중독균 검출에 적합한 것으로 판단된다.

Keywords

References

  1. Code of hygienic practice for fresh fruits and vegetables. CXC 53-2003. Codex alimentarius, Rome, Italy (2017).
  2. Korean Establishment of the legal standards and the designation of fertilizers. Rural Development Administration, Wanju, Korea (2019).
  3. Korean Fertilizer Control Act. Minister of Agriculture, Food and Rural Affairs, Sejong, Korea (2019).
  4. Kang, C.Y., Park, H.T., Seo, D.S., Kang, S.P., 2014, Development strategy for the byproduct fertilizer industry. R720-2. Korea Rural Economic Institute, Naju, Korea.
  5. Jung, K.S., Heu, S.G., Roh, E.J., Lee, D.H., Yun, J.C., Kim, K.H., Prevalence of pathogenic bacteria in livestock manure compost and organic fertilizer. Korean J. Soil Sci. Fert., 44, 824-829 (2011). https://doi.org/10.7745/KJSSF.2011.44.5.824
  6. Lee, T., Kim, S.R., Han, S.H., 2009, Infection status of hazardous microbes in vegetables and analyze their infection route. Rural Development Administration, Wanju, Korea.
  7. Korean Examination of the quality of fertilizer and sampling standard. Rural Development Administration, Wanju, Korea (2019).
  8. Kim, H.J., Kim, Y.S., Chung, M.S., Oh, D.H., Bahk, G.J., Chun, H.S., Ha, S.D., Trends in rapid detection methods for food-borne pathogenic microorganisms by using new technologies. J. Food Hyg. Safety, 25, 376-387 (2010).
  9. Koo, E.J., Kim, D.H., Oh, S.W., Comparison of DNA isolation methods for detection of foodborne pathogens by realtime PCR from foods. Korean J. Food Sci. Technol., 48, 335-340 (2016). https://doi.org/10.9721/KJFST.2016.48.4.335
  10. Mafra, I., Silva, S.A., Moreira, E., Silva, C., Beatriz, M., Oliveira, P.P., Comparative study of DNA extraction methods for soybean derived food products. Food Control, 19, 1183-1190 (2008). https://doi.org/10.1016/j.foodcont.2008.01.004
  11. Pinto, A.D., Forte, V.T., Guastadisegni, M.C., Martino, C., Schena, F.P., Tantillo, G., A comparison of DNA extraction methods for food analysis. Food Control, 18, 76-80 (2007). https://doi.org/10.1016/j.foodcont.2005.08.011
  12. Cullen, D.W., Hirsch, P.R., Simple and rapid method for direct extraction of microbial dna from soil for pcr. Soil Biol. Biochem., 30, 983-993 (1998). https://doi.org/10.1016/S0038-0717(98)00001-7
  13. Yun, B.H., Jang, Y.J., Kim, Y.R., Kim, H.Y., Kim, W.I., Han, S.H., Kim, S.R., Ryu, J.G., Kim, H.J., Virulence profile and antimicrobial resistance of Escherichia coli from flies captured from agricultural environment. J. Food Hyg. Safety, 32, 147-153 (2017). https://doi.org/10.13103/JFHS.2017.32.2.147
  14. Elizaquivel, P., Aznar, R., Comparison of four commercial DNA extraction kits for PCR detection of Listeria monocytogenes, Salmonella, Escherichia coli O157:H7, and Staphylococcus aureus in fresh minimally processed vegetables. J. Food Prot., 71, 2110-2114 (2008). https://doi.org/10.4315/0362-028X-71.10.2110
  15. Wang, X., Jothikumar, N., Griffiths, M.W., Enrichment and DNA extraction protocols for the simultaneous detection of Salmonella and Listeria monocytogenes in raw sausage meat with multiplex real-time PCR. J. Food Prot., 67, 189-192 (2004). https://doi.org/10.4315/0362-028X-67.1.189