Food Science of Animal Resources (한국축산식품학회지)

Korean Society for Food Science of Animal Resources (한국축산식품학회)
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Comparison of Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, and Loop-Mediated Isothermal Amplification for the Detection of Cronobacter sakazakii in Milk Powder

분유에 오염된 Cronobacter sakazakii 검출을 위한 중합효소연쇄반응, 실시간중합효소연쇄반응, 등온검출법의 비교

  • Kim, Young-Joo (Department of Nutrition Education, Chung-Ang University) ;
  • Seo, Sheungwoo (School of Food Science and Technology, Department of Food and Nutrition, Chung-Ang University) ;
  • Wang, Xiaoyu (School of Food Science and Technology, Department of Food and Nutrition, Chung-Ang University) ;
  • Seo, Dong Joo (School of Food Science and Technology, Department of Food and Nutrition, Chung-Ang University) ;
  • Lee, Min Hwa (School of Food Science and Technology, Department of Food and Nutrition, Chung-Ang University) ;
  • Son, Na Ry (School of Food Science and Technology, Department of Food and Nutrition, Chung-Ang University) ;
  • Lee, Bog-Hieu (School of Food Science and Technology, Department of Food and Nutrition, Chung-Ang University) ;
  • Choi, Changsun (School of Food Science and Technology, Department of Food and Nutrition, Chung-Ang University)
  • 김영주 (중앙대학교 교육대학원 영양교육전공) ;
  • 서승우 (중앙대학교 식품공학부 식품영양학과) ;
  • 왕효우 (중앙대학교 식품공학부 식품영양학과) ;
  • 서동주 (중앙대학교 식품공학부 식품영양학과) ;
  • 이민화 (중앙대학교 식품공학부 식품영양학과) ;
  • 손나리 (중앙대학교 식품공학부 식품영양학과) ;
  • 이복희 (중앙대학교 식품공학부 식품영양학과) ;
  • 최창순 (중앙대학교 식품공학부 식품영양학과)
  • Received : 2013.04.26
  • Accepted : 2013.08.22
  • Published : 2013.10.31
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Abstract

Loop-mediated isothermal amplification (LAMP) is an emerging detection technology for the amplification of DNA under isothermal conditions. The aim of this study was to develop a rapid and reliable LAMP technique for the detection of Cronobacter sakazakii in milk powder. In order to enhance the sensitivity and specificity, LAMP primers targeting outer membrane protein A (ompA) gene of C. sakazakii were designed using Explorer V4 software. Thirty seven C. sakazakii strains and 13 pathogenic microorganisms were used for comparative detection of C. sakazakii using polymerase chain reaction (PCR), real-time PCR, and LAMP. LAMP developed in this study could specifically detect C. sakazakii strains without cross-reactivity with other foodborne pathogens. LAMP products amplified from ompA gene of C. sakazakii were digested with with HhaI and NruI enzyme. The specificity of LAMP was confirmed by restriction fragment length polymorphism (RFLP) analysis. LAMP could detect C. sakazakii within 1 h without bacterial culture and its detection limit was as low as 1 CFU/mL C. sakazakii in milk. In the comparison of the sensitivity, LAMP showed 10,000- and 100-times higher detection limit than PCR or real-time PCR, respectively. Therefore, this study can conclude that LAMP is a rapid and reliable detection technique for C. sakazakii contaminated in powdered milk.

본 연구에서는 영유아에게 치명적인 감염을 일으키는 C. sakazakii에 대하여 LAMP 검출법을 개발하였다. LAMP법에 의한 C. sakazakii의 검출율은 100%였으며 13개의 음성 지표군에 대해서는 모두 음성 반응을 보여 특이도가 매우 높은 것으로 판단되었다. 또한, HhaI과 NruI 두 개의 제한 효소를 LAMP product에 반응시킨 결과, 유전자의 특정 염기서열이 절단되는 것을 확인하였으며, 이를 통해 LAMP 검출법에 의해 증폭된 DNA가 C. sakazakii-specific ompA임을 확인하였다. 조제분유에 오염 된 C. sakazakii를 LAMP법으로 검출 시 검출한계는 $10^0$ CFU/mL이었으며 이는 기존의 PCR법이나 real-time PCR법에 비해 100-10,000배 높은 수준으로 민감도가 매우 높은 것으로 판단되었다. 이와 같이 높은 특이도와 민감도를 가진 LAMP 검출법은 C. sakazakii와 같은 급성 기회 감염균이나 병원성 미생물에 의한 식중독 발생시 현장에서 병원체를 간편하고 신속하게 검출할 수 있는 기술로 기대된다.

Keywords

References

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