Korean Journal of Food Science and Technology (한국식품과학회지)

Korean Society of Food Science and Technology (한국식품과학회)
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Comparison of DNA isolation methods for detection of foodborne pathogens by real-time PCR from foods

식품으로부터 식중독 세균 검출을 위한 Real-time PCR에 적합한 DNA 추출 방법 비교

  • Koo, Eun-Jeong (Department of Food and Nutrition, Kookmin University) ;
  • Kim, Dongho (Bio-Medical Science Co., Ltd.) ;
  • Oh, Se-Wook (Department of Food and Nutrition, Kookmin University)
  • 구은정 (국민대학교 자연과학대학 식품영양학과) ;
  • 김동호 (BMS(주)) ;
  • 오세욱 (국민대학교 자연과학대학 식품영양학과)
  • Received : 2016.05.17
  • Accepted : 2016.06.28
  • Published : 2016.08.31
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Abstract

This study was conducted to find out the most suitable DNA isolation methods for PCR detection of foodborne pathogens. Four DNA isolation methods including Universal Genomic DNA Extraction Kit (TaKaRa), PrepMan Ultra (Applied Biosystems), boiling method and alkaline lysis method (w/PEG) were tested and compared. The Universal Genomic DNA Extraction kit (TaKaRa) was considered as the more efficient isolation method for Escherichia coli O157:H7 and Staphylococcus aureus in lettuce, fish and beef. Meanwhile to detect the foodborne pathogens directly from foods without enrichment, the four different buffers such as double-distilled water, saline, glycine-saline, glycine-saline with Tween-20 and beef extract were also evaluated. As a result, saline was more suitable buffer for E. coli O157:H7. And double-distilled water was more suitable buffer than saline for S. aureus, respectively

본 연구에서는 다양한 식품으로부터 식중독 세균의 DNA를 추출하는 효율을 비교 하였다. 사용된 DNA 추출 방법은 TaKaRa Kit를 이용하는 방법, PrepMan reagent를 이용하는 방법, boiling method, PEG를 이용한 alkaline method가 사용되었다. 비용절감이나 시간절약 면에서 boiling method나 PrepMan method도 고려할 수 있지만, column kit를 이용하는 TaKaRa kit가 효율적이라고 판단되었다. 또한 정성 시험법에서 적은 양의 균을 검출하기 위해 증균배양을 거치게 되는데 이때 사용되는 증균배지의 성분이 DNA 추출 후에도 잔류하여 saline과 비교하였을 때 DNA 추출효율이 낮은 결과를 나타내었다. 따라서 증균배지의 성분을 제거한 뒤 DNA를 추출하는 것이 PCR의 효율을 높일 수 있을 것으로 예상된다. 정량 시험법에서는 증균과정을 거치지 않아 균을 검출할 때 DNA의 추출 효율이 중요하기 때문에 DNA 추출 효율을 높이기 위한 가장 좋은 버퍼를 선정하고자 하였다. 그 결과 E. coli O157:H7에서는 saline이, S. aureus에서는 증류수를 버퍼로 사용했을 때 DNA 추출 효율이 가장 높은 것으로 나타났다.

Keywords

References

  1. Padhye NV, Doyle MP. Escherichia coli O157: H7: Epidemiology, pathogenesis, and methods for detection in food. J. Food Protect. 55: 555-565 (1992) https://doi.org/10.4315/0362-028X-55.7.555
  2. Centers of Disease Control and Prevention. Outbreak of Escherichia coli O157: H7 and Campylobacter among attendees of the Washington county fair-New york, 1999. Morb. Mortal. Wkly Rep. 48: 803-805 (1999)
  3. Eaton KA, Friedman DI, Francis GJ, Tyler JS, Young VB, Haeger J, Abu-Ali G, Whittam TS. Pathogenesis of renal disease due to enterohemorrhagic Escherichia coli in germ-free mice. Infect. Immun. 76: 3054-3063 (2008) https://doi.org/10.1128/IAI.01626-07
  4. Alarcon B, Vicedo B, Aznar R. PCR-based procedures for detection and quantification of Staphylococcus aureus and their application in food. J.Appl. Microbiol. 100: 352-364 (2006) https://doi.org/10.1111/j.1365-2672.2005.02768.x
  5. Valle J, Gomez-Lucia E, Piriz S, Goyache J, Orden JA, Vadillo S. Enterotoxin production by staphylococci isolated from healthy goats. Appl. Environ. Microbiol. 56: 1323-1326 (1990)
  6. Hitchins AD, Feng P, Watkins WD, Rippey SR, Chandler LA. Escherichia coli and the coliform bacteria. FDA Bacteriological Analytical Manual 8: 4.01-4.29 (1998)
  7. QIA. Standards for Processing and Ingredients Specifications of Livestock Products. Quarntine and Inspection Agency, Gimcheon, Korea (2011)
  8. Warren BR, Parish ME, Schneider KR. Comparison of conventional culture methods and FTA filtration-nested PCR for the detection of Shigella boydii and Shigella sonnei on tomato surfaces. J. Food Protect. 8: 1606-1612 (2005)
  9. Pagadala S, Parveen S, Schwarz JG, Rippen T, Luchansky JB. Comparison of automated BAX PCR and standard culture methods for detection of Listeria monocytogenes in blue crabmeat (Callinectus sapidus) and blue crab processing plants. J. Food Protect. 11: 1930-1933 (2011)
  10. Malorny B, Huehn S, Dieckmann R, Krmer N, Helmuth R. Polymerase chain reaction for the rapid detection and serovar identification of Salmonella in food and feeding stuff. Food Anal. Method. 2: 81-95 (2009) https://doi.org/10.1007/s12161-008-9057-9
  11. Hwang BH, Lee JW, Cha HJ. Polymerase chain reaction-based detection of total and specific vibrio species. Appl. Biochem. Biotechnol. 162: 1187-1194 (2010) https://doi.org/10.1007/s12010-009-8853-z
  12. Daum LT, Barnes WJ, McAvin JC, Neidert MS, Cooper LA, Huff WB, Gaul L, Riggins WS, Morris S, Salmen A, Lohman KL. Real-time PCR detection of Salmonella in suspect foods from a gastroenteritis outbreak in Kerr County, Texas. J. Clin. Microbiol. 40: 3050-3052 (2002) https://doi.org/10.1128/JCM.40.8.3050-3052.2002
  13. Prentice N, Murray JS, Scott MF, Coombs JP, Parton A. Rapid isolation and detection of Escherichia coli O157: H7 in fresh produce. J. Rapid Methods Autom. Microbiol. 14: 299-308 (2006) https://doi.org/10.1111/j.1745-4581.2006.00069.x
  14. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 6: 986-994 (1996) https://doi.org/10.1101/gr.6.10.986
  15. Narayanan C, Dubey S, Wali SA, Shukla N, Kumar R, Mandal AK, Ansari SA. Comparative efficacy of different DNA extraction methods for PCR-based assay in Tectona grandis L.f. Indian J. Biotechnol. 7: 133-136 (2008)
  16. de Medici D, Croci L, Delibato E, di Pasquale S, Filetici E, Toti L. Evaluation of DNA extraction methods for use in combination with SYBR green I real-time PCR to detect Salmonella enterica serotype enteritidis in poultry. Appl. Environ. Microbiol. 69: 3456-3461 (2003) https://doi.org/10.1128/AEM.69.6.3456-3461.2003
  17. Chomczynski P, Rymaszewski M. Alkaline polyethylene glycolbased method for direct PCR from bacteria, eukaryotic tissue samples, and whole blood. Biotechniques 40: 454-458 (2006) https://doi.org/10.2144/000112149
  18. Heller LC, Davis CR, Peak KK, Wingfield D, Cannons AC, Amuso PT, Cattani J. Comparison of methods for DNA isolation from food samples for detection of shiga toxin-producing Escherichia coli by real-time PCR. Appl. Environ. Microbiol. 69: 1844-1846 (2003) https://doi.org/10.1128/AEM.69.3.1844-1846.2003
  19. 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 Protect. 71: 2110-2114 (2008) https://doi.org/10.4315/0362-028X-71.10.2110
  20. Hyeon JY, Hwang IG, Kwak HS, Park CK, Choi IS, Seo KH. Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella enteritidis using real-time PCR assay. J. Vet. Sci. 11: 143-149 (2010) https://doi.org/10.4142/jvs.2010.11.2.143
  21. Miller ND, Davidson PM, D'Souza DH. Real-time reverse-transcriptase PCR for Salmonella typhimurium detection from lettuce and tomatoes. LWT-Food Sci. Technol. 44: 1088-1097 (2011) https://doi.org/10.1016/j.lwt.2010.08.003
  22. Ibekwe AM, Watt PM, Grieve CM, Sharma VK, Lyons SR. Multiplex fluorogenic real-time PCR for detection and quantification of Escherichia coli O157: H7 in dairy wastewater wetlands. Appl. Environ. Microbiol. 68: 4853-4862 (2002) https://doi.org/10.1128/AEM.68.10.4853-4862.2002
  23. Sabet NS, Subramaniam G, Navaratnam P, Sekaran SD. Detection of methicillin-and aminoglycoside-resistant genes and simultaneous identification of S. aureus using triplex real-time PCR taqman assay. J. Microbiol. Meth. 68: 157-162 (2007) https://doi.org/10.1016/j.mimet.2006.07.008