Comparison of Real-Time PCR and Conventional Culture Method for Detection of Cronobacter spp. in Powdered Foods

분말식품에서 Cronobacter spp. 검출을 위한 Real-Time PCR과 배지배양법의 비교검증

  • Chon, Jung-Whan (Department of Public Health, College of Veterinary Medicine, Konkuk University) ;
  • Song, Kwang-Young (Department of Public Health, College of Veterinary Medicine, Konkuk University) ;
  • Kim, Sun-Young (Department of Public Health, College of Veterinary Medicine, Konkuk University) ;
  • Hyeon, Ji-Yeon (Department of Public Health, College of Veterinary Medicine, Konkuk University) ;
  • Kim, Yun-Gyeong (Department of Public Health, College of Veterinary Medicine, Konkuk University) ;
  • Hwang, In-Gyun (Division of Microbiology, Korea Food and Drug Administration) ;
  • Kwak, Hyo-Sun (Division of Microbiology, Korea Food and Drug Administration) ;
  • Seo, Kun-Ho (Department of Public Health, College of Veterinary Medicine, Konkuk University)
  • Received : 2011.03.11
  • Accepted : 2011.03.21
  • Published : 2011.03.31

Abstract

The aim of this study was to compare the performance of conventional culture and real-time PCR for detection of Cronobacter spp. in powdered foods. Infant formula, baby food and Misugaru inoculated with Cronobacter were enriched in distilled water as first enrichment step, followed by incubating in Enterobacteriaceae enrichment (EE) broth as second enrichment step. A loopful of enriched sample was streaked onto Druggan-Forsythe-Iversen agar, followed by incubating at $37^{\circ}C$ for 24 h. One milliliter of the enriched distilled water and EE broth were used in real-time PCR assay. No statistical differences were observed in the number of positive samples between culture method and real-time PCR (p>0.05) in all types of food samples. The number of positives of real-time PCR was higher in the first enrichment media (distilled water) than the second enrichment media (EE broth), though there was no significant difference (p>0.05). It appears that some components of the second enrichment broth, EE broth, inhibit the reaction of real-time PCR. These results show that real-time PCR using a single enrichment with distilled water could be useful as an effective screening method for detection of Cronobacter while saving much time and labor compared to conventional culture method.

본 연구에서는 분말 식품에서 real-time PCR과 배지배양법을 사용하여 Cronobacter spp.를 검출하는 방법이 비교검증 되었다. 조제분유, 이유식, 미숫가루에 Cronobacter를 인위적으로 접종시킨 후, 식품공전의 방법에 따라 멸균증류수와 Enterobacteriaceae enrichment (EE) broth에서 각각 1, 2차 증균배양 하였으며, Druggan-Forsythe-Iversen에 선택배양하여 Cronobacter를 검출하였다. Real-time PCR은 멸균증류수 및 EE broth에서 1 ml을 채취한 후 DNA를 추출하여 시행하였다. 실험결과 모든 식품에서 배지배양법과 real-time PCR간에는 통계학적 유의차가 존재하지 않았다(p>0.05). 한편 모든 실험회차에서 real-time PCR 수행 시, 1차 증균액인 멸균증류수에서의 양성검출율이 2차 증균액인 EE broth에서보다 높았는데, 이는 2차 증균액 내의 구성성분 중 일부분이 real-time PCR의 반응을 저해했기 때문으로 사료된다. 연구결과를 종합해 볼 때, 1차 증균 후, real-time PCR을 통해 Cronobacter를 검출하는 방법은 정확한 민감도를 보이면서도 시간과 노동력을 절감할 수 있는 효과적인 방법으로 사료된다.

Acknowledgement

Supported by : 식약청

References

  1. Chon, J.H., J.Y. Hyeon, I.G. Hwang, H.S. Kwak, J.A. Han, Y.H. Chung, K.W. Song, and K.H. Seo. 2010. Comparison of standard culture method and real-time PCR for detection of Vibrio parahaemolyticus in seafoods and vegetables. Korean J. Food Sci. Technol. 42, 355-360.
  2. Chon, J.H., J.Y. Hyeon, I.G. Hwang, H.S. Kwak, J.A. Han, M.S. Kim, J.H. Kim, K.W. Song, and K.H. Seo. 2011. Comparison of real-time PCR and culture methods for detection of Campylobacter jejuni in various foods. Korean J. Food Sci. Technol. 43, 119-123. https://doi.org/10.9721/KJFST.2011.43.1.119
  3. Food and Agriculture Organization/World Health Organization. Joint FAO/WHO workshop on Enterobacter sakazakii and other microorganisms in powdered infant formula. Available from: http://www.who.int/foodsafety/publications/feb. 2004/en/print.html. Accessed Dec. 20, 2010.
  4. Han, S.R., J.Y. Hyeon, H.Y. Kim, J.S. Park, S. Heo, H.C. Shin, and K.H. Seo. 2008. Evaluation of conventional culture methods and validation of immunoassays for rapid detection of Listeria monocytogenes in dairy and processed foods. Korean J. Food Sci. Ani. Resour. 28, 616-622. https://doi.org/10.5851/kosfa.2008.28.5.616
  5. Heller, L.C., C.R. Davis, K.K. Peak, D. Wingfield, A.C. Cannons, P.T. Amuso, and J. Cattani. 2003. 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. https://doi.org/10.1128/AEM.69.3.1844-1846.2003
  6. Hyeon, J.Y., I.G. Hwang, H.S. Kawk, J.S. Park, S. Heo, I.S. Choi, C.K. Park, and K.H. Seo. 2009. Evaluation of an automated ELISA and real-time PCR by comparing with a conventional culture method for the detection of Salmonella spp. in steamed pork and raw broccoli sprouts. Korean J. food Sci. Ani. Resour. 29, 506-512. https://doi.org/10.5851/kosfa.2009.29.4.506
  7. Hyeon, J.Y., I.G. Hwang, H.S. Kawk, C. Park, I.S. Choi, and K.H. Seo. 2010. 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. https://doi.org/10.4142/jvs.2010.11.2.143
  8. Iversen, C. and S.J. Forsythe. 2003. Risk profile of Enterobacter sakazakii, an emergent pathogen associated with infant milk formula. Trends Food Sci. Technol. 11, 443-454.
  9. Jung, J.H. and S.Y. Lee. 2010. Microbial growth in dry grain food (Sunsik) beverages prepared with water, milk, soymilk, or honey-water. J. Food Sci. 75, 239-242.
  10. Kim, K.S., S.S. Jang, S.K. Kim, J.H. Park, S.G. Heu, and S.Y. Ryu. 2008. Prevalence and genetic diversity of Enterobacter sakazakii in ingredients of infant foods. Int. J. Food Microbiol. 29, 196-203.
  11. Kim, H.J., M.S. Koo, and S.W. Oh. 2010. Performance comparison of 3 differenct isolation media of Cronobacter sakazakii. J. Korean Soc. Food Sci. Nutr. 39, 764-768. https://doi.org/10.3746/jkfn.2010.39.5.764
  12. Korea Food and Drug Administration. Food code. Available from http://safefood.kfda.go.kr/RS/food_menu.jsp, Accessed Jan. 21, 2011.
  13. Lee, J.H., K.Y. Song, J.Y. Hyeon, I.G. Hwang, H.S. Kwak, J.A. Han, Y.H. Chung, and K.H. Seo. 2010. Comparison of standard culture method and real-time PCR assay for detection of Staphylococcus aureus in processed and unprocessed foods. Korean J. Food Sci. Ani. Resour. 30, 410-418. https://doi.org/10.5851/kosfa.2010.30.3.410
  14. Makowski, G.S., E.L. Davis, and S.M. Hopfer. 1996. The effect of storage on Guthrie cards: implications for deoxyribonucleic acid amplification. Ann. Clin. Lab. Sci. 26, 458-469.
  15. Malorny, B., E. Paccassoni, P. Fach, C. Bunge, A. Martin, and R. Helmuth. 2004. Diagnostic real-time PCR for detection of Salmonella in food. Appl. Environ. Microbiol. 70, 7064-7052.
  16. Muytjens, H.L., H.C. Zanen, H.J. Sonderkamp, L.A. Kollée, I.K. Wachsmuth, and J.J. Farmer. 1983. Analysis of eight cases of neonatal meningitis and sepsis due to Enterobacter sakazakii. J. Clin. Microbiol. 18, 115-120.
  17. Seo, K.H. and R.E. Brackett. 2005. Rapid, specific detection of Enterobacter sakazakii in Infant formula using a real-time PCR assay. J. Food Prot. 68, 59-63. https://doi.org/10.4315/0362-028X-68.1.59
  18. Thunberg, R.L., T.T. Tran, and M.O. Walderhaug. 2000. Detection of thermophilic Campylobacter spp. in blood-free enriched samples of inoculated foods by the polymerase chain reaction. J. Food Prot. 63, 299-303. https://doi.org/10.4315/0362-028X-63.3.299
  19. United States Department of Agriculture/Food Safety and Inspection Service. Microbiology Laboratory Guidebook. Available from http://www.fsis.usda.gov/Science/Microbiological_Lab_Guide book/index.asp. Accessed Jan. 28, 2011.
  20. Yang, C., Y. Jiang, K. Huang, C. Zhu, and Y. Yin. 2003. Application of real-time PCR for quantitative detection of Campylobacter jejuni in poultry, milk and environmental water. FEMS Immunol. Med. Microbiol. 38, 265-271. https://doi.org/10.1016/S0928-8244(03)00168-8