한국축산식품학회지 (Food Science of Animal Resources)

  • 제33권5호
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  • Pages.610-616
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  • 2013
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  • 2636-0772(pISSN)
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  • 2636-0780(eISSN)
한국축산식품학회 (Korean Society for Food Science of Animal Resources)
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분유에 오염된 Cronobacter sakazakii 검출을 위한 중합효소연쇄반응, 실시간중합효소연쇄반응, 등온검출법의 비교

Comparison of Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, and Loop-Mediated Isothermal Amplification for the Detection of Cronobacter sakazakii in Milk Powder

  • 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)
  • 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)
  • 투고 : 2013.04.26
  • 심사 : 2013.08.22
  • 발행 : 2013.10.31
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초록

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.

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.

키워드

참고문헌

  1. Alex, P. and Georg, E. S. (1998) Structure of the outer membrane protein A transmembrane domain. Nat. Struct. Biol. 5, 1013-1017. https://doi.org/10.1038/2983
  2. Arad, I., Baras, M., Gofin, R., Bar-Oz, B., and Peleg, O. (2001) Does parity affect the neonatal outcome of very low birth weight infants. Eur. J. Obstet. Gynecol. Reprod. Biol. 94, 283-288. https://doi.org/10.1016/S0301-2115(00)00308-0
  3. Derzelle, S., Dilasser, F., Maladen, V., Soudrie, N., Leclercq, A., Lombard, B., and Lafarge, V. (2007) Comparison of three chromogenic media and evaluation of two molecular-based identification systems for the detection of Enterobacter sakazakii from environmental samples from infant formulae factories. J. Food Prot. 70, 1678-1684.
  4. Enosawa, M., Kageyama, S., Sawai, K., Watanabe, K., Notomi, T., Onoe, S., Mori, Y., and Yokomizo, Y. (2003) Use of loop-mediated isothermal amplification of the IS900 sequence for rapid detection of cultured Mycobacterium avium subsp. paratuberculosis. J. Clin. Microbiol. 41, 4359-4365. https://doi.org/10.1128/JCM.41.9.4359-4365.2003
  5. Farmer, J. J., Asbury, M. A., Hickman, F. W., and Brenner, D. J. (1980) Enterobacter sakazakii: A new species of "Enterobacteriaceae" isolated from clinical specimens. Int. J. Syst. Bacteriol. 30, 569-584. https://doi.org/10.1099/00207713-30-3-569
  6. Friedemann, M. (2007) Enterobacter sakazakii in food and beverages (other than infant formula and milk powder). Int. J. Food Microbiol. 116, 1-10. https://doi.org/10.1016/j.ijfoodmicro.2006.12.018
  7. Guillaume-Gentil, O., Sonnard, V., Kandhai, M. C., Marugg, J. D., and Joosten, H. (2005) A Simple and rapid cultural method for detection of Enterobacter sakazakii in environmental samples. J. Food Prot. 68, 64-69.
  8. Hara-Kudo, Y., Nemoto, J., Ohtsuka, K., Segawa, Y., Takatori, K., Kojima, T., and Ikedo, M. (2007) Sensitive and rapid detection of vero toxin-producing Escherichia coli using loop-mediated isothermal amplification. J. Med. Microbiol. 56, 398-406. https://doi.org/10.1099/jmm.0.46819-0
  9. Hara-Kudo, Y., Yoshino, M., Kojima, T., and Ikedo, M. (2005) Loop-mediated isothermal amplification for the rapid detection of Salmonella. FEMS Microbiol. Lett. 253, 155-161. https://doi.org/10.1016/j.femsle.2005.09.032
  10. Hu, L., Zhang, W., Zhang, X., Yuan, Y., Zhang, Y., Zhang, H., Ma, X., and Su, X. (2009) Development of a loop-mediated isothermal amplification assay for rapid detection of Enterobacter sakazakii in powdered infant formula. Wei Sheng Wu Xue Bao. 49, 378-382.
  11. Iversen, C. and Forsythe, S. J. (2003) Risk profile of Enterobacter sakazakii, an emergent pathogen associated with infant milk formula. Trends Food Sci. Tech. 14, 443-454. https://doi.org/10.1016/S0924-2244(03)00155-9
  12. Iversen, C., Mullane, N., McCardell, B., Tall, B. D., and Lehner, A. (2008) Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomospecies 1, and of three subspecies, Cronobacter dublinensis subsp. dublinensis subsp. nov., Cronobacter dublinensis subsp. lausannensis subsp. nov. and Cronobacter dublinensis subsp. lactaridi subsp. nov. Int. J. Syst. Evol. Microbiol. 58, 1442-1447. https://doi.org/10.1099/ijs.0.65577-0
  13. Liu, C., Zheng, W., Zhang, H., Hou, Y., and Liu, Y. (2009) Sensitive and rapid detection of Enterobacter sakazakii in infant formula by loop-mediated isothermal amplification method. J. Food Safety 29, 83-94. https://doi.org/10.1111/j.1745-4565.2008.00142.x
  14. Liu, Y., Cai, X., Zhang, X., Gao, Q., Yang, X., Zheng, Z., Luo, M., and Huang, X. (2006) Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula. J. Microbiol. Meth. 65, 21-31. https://doi.org/10.1016/j.mimet.2005.06.007
  15. Mohan Nair, M. K. and Venkitanarayanan, K. (2007) Role of bacterial OmpA and host cytoskeleton in the invasion of human intestinal epithelial cells by Enterobacter sakazakii. Pediatr. Res. 62, 664-669. https://doi.org/10.1203/PDR.0b013e3181587864
  16. Mori, Y., Nagamine, K., Tomita, N., and Notomi, T. (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun. 289, 150-154. https://doi.org/10.1006/bbrc.2001.5921
  17. Nagamine, K., Hase, T., and Notomi, T. (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell. Probe. 16, 223-229. https://doi.org/10.1006/mcpr.2002.0415
  18. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, E63. https://doi.org/10.1093/nar/28.12.e63
  19. Orlandi, P. A., and Lampel, K. L. (2000) Extraction-free, filter-based template preparation for rapid and sensitive PCR detection of pathogenic parasitic protozoa. J. Clin. Microbiol. 38, 2271-2277.
  20. Saito, R., Misawa, Y., Moriya, K., Koike, K., Ubukata, K., and Okamura, N. (2005) Development and evaluation of a loopmediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae. J. Med. Microbiol. 54, 1037-1041. https://doi.org/10.1099/jmm.0.46071-0
  21. Singh, S. P., Williams, Y. U., Miller, S., and Nikaido, H. (2003) The C-terminal domain of Salmonella enterica serovar typhimurium ompA is an immunodominant antigen in mice but appears to be only partially exposed on the bacterial cell surface. Infect. Immun. 71, 3937-3946. https://doi.org/10.1128/IAI.71.7.3937-3946.2003
  22. Yamazaki, W., Taguchi, M., Ishibashi, M., Kitazato, M., Nukina, M., Misawa, N., and Inoue, K. (2008) Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Campylobacter jejuni and Campylobacter coli. J. Med. Microbiol. 57, 444-451. https://doi.org/10.1099/jmm.0.47688-0
  23. Ye, Y., Wu, Q., Yao, L., Dong, X., Wu, K., and Zhang, J. (2009) A comparison of polymerase chain reaction and international organization for standardization methods for determination of Enterobacter sakazakii contamination of infant formulas from Chinese mainland markets. Foodborne Pathog. Dis. 6, 1229-1234. https://doi.org/10.1089/fpd.2008.0262
  24. Yoshida, A., Nagashima, S., Ansai, T., Tachibana, M., Kato, H., Watari, H., Notomi, T., and Takehara, T. (2005) Loop-mediated isothermal amplification method for rapid detection of the periodontopathic bacteria Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. J. Clin. Microbiol. 43, 2418-2424. https://doi.org/10.1128/JCM.43.5.2418-2424.2005