Journal of the Korean Society of Food Science and Nutrition (한국식품영양과학회지)

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
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Rapid Detection of Salmonella spp. in Fresh-Cut Cabbage by Real-Time PCR

Real-Time PCR을 이용한 신선편이 양배추에서 Salmonella spp.의 신속검출

  • Bang, Mi-Kyung (Food Safety Team, Korea Food Research Institute) ;
  • Park, Seung-Ju (Food Safety Team, Korea Food Research Institute) ;
  • Kim, Yun-Ji (Food Safety Team, Korea Food Research Institute) ;
  • Kim, Ji-Gang (National Institute of Horticultural and Herbal Science) ;
  • Oh, Se-Wook (Dept. of Food and Nutrition, Kookmin University)
  • 방미경 (한국식품연구원 안전성연구단) ;
  • 박승주 (한국식품연구원 안전성연구단) ;
  • 김윤지 (한국식품연구원 안전성연구단) ;
  • 김지강 (국립원예특작과학원) ;
  • 오세욱 (국민대학교 식품영양학과)
  • Received : 2010.07.16
  • Accepted : 2010.08.11
  • Published : 2010.10.31
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Abstract

This study was conducted to find out the minimal time needed for detection of Salmonella spp. which exist at very low concentration in foods by using real-time PCR. The sal-F and sal-R sequences were used as primers and sal-P was used as a probe. The detection limit of Salmonella spp. was $3.77{\times}10^2\;cfu/mL$ in buffered peptone water (BPW). Microbial growth was monitored after artificially inoculated Salmonella spp. into BPW. The obtained growth curve was well fitted with the equation, y＝$0.0127x^2$+0.5927x-0.4317 ($R^2$=0.99), if assuming that 1 cell exists in 25 g sample (0.04 cfu/mL). The microbial concentration will be reduced to 10 fold by adding BPW during sample treatment, so actual initial concentration at the starting point of enrichment is 0.004 cfu/mL. At this condition, real-time PCR detection would be possible only when microbial concentration increase occurs to exceed the detection limit (377 cfu/mL). The time needed for microbial increase was calculated from the growth curve equation as 7 hours and 20 minutes. Therefore the total time required for detection was less than 10 hours including the PCR operating time.

식품에 극소량 존재하는 Salmonella spp.를 real-time PCR로 검출 시 필요한 최소시간을 구하고자 하였다. Sal-F, Sal-R 서열이 primer로 사용되었으며 sal-P 서열이 probe로 사용되었다. BPW에서 산출된 검출한계는 $3.77{\times}10^2\;cfu/mL$로 측정되었다. BPW에 인위적으로 Salmonella spp.를 접종하였으며 성장곡선을 구하였다. 성장곡선은 y＝$0.0127x^2$+0.5927x-0.4317($R^2$=0.99) 식을 나타내었다. 25 g 시료에 1 cell의 Salmonella spp.가 존재한다고 가정할 경우, 시료처리 과정에서 10배로 희석되므로 초기 균 농도는 0.004 cfu/mL 이며 이 농도에서 검출한계까지의 균수 증가가 발생해야 real-time PCR로 검출이 가능할 것으로 판단되었다. 성장곡선에서 균수 증가에 필요한 시간을 측정해 본 결과 7시간20분으로 산출되었으며 따라서 PCR 수행시간을 포함하여 10시간이면 검출이 가능함을 알 수 있었다. 식품에서의 실증실험을 위하여 시중에서 판매되고 있는 신선편이 양배추를 구하여 25 g에 1~10 cfu의 수준으로 S. Typhimurium을 인위접종한 후 real-time PCR 방법과 식품공전방법으로 검출하였을 때 두 방법 모두 동일하게 30개중 29개 시료에 대하여 음성을 나타내었다.

Keywords

References

  1. David M, Nancy RR, Richard L. 2005. Essentials of Food Safety and Sanitation. Pearson Prentice Hall, New Jersey, USA. p 50.
  2. Oliveira SD, Rodenbusch CR, Ce MC, Rocha SL, Canal CW. 2003. Evaluation of selective and non-selective enrichment PCR procedures for Salmonella detection. Lett Appl Microbiol 36: 217-221. https://doi.org/10.1046/j.1472-765X.2003.01294.x
  3. http://e-stat.kfda.go.kr
  4. Kim J, Mosana M, Castell-Perez E. 2010. Simulation of pathogen inactivation in whole and fresh-cut cantaloupe (Cucumis melo) using electron beam treatment. J Food Eng 97: 425-433. https://doi.org/10.1016/j.jfoodeng.2009.10.038
  5. Bohaychuk VM, Gensler GE, McFall ME, King RK, Renter DG. 2007. A real-time PCR assay for the detection of Salmonella in a wide variety of food and food animal matrices. J Food Prot 70: 1080-1087. https://doi.org/10.4315/0362-028X-70.5.1080
  6. Melissa OP, Maria C, Martha ES, Frank J, Peter D. 1999. Sensitivity, specificity, and predictive values of three Salmonella rapid detection kits using fresh and frozen poultry environmental samples versus those of standard plating. Appl Environ Microbiol 65: 1055-1060.
  7. Yan Z, Zhou L, Zhao Y, Wang J, Huang L, Hu K, Liu H, Wang H, Guo Z, Song Y, Huang H, Yang R. 2006. Rapid quantitative detection of Yersinia pestis by lateral-flow immunoassay and up-converting phosphor technology-based biosensor. Sens Actuators B 119: 656-663. https://doi.org/10.1016/j.snb.2006.01.029
  8. Krascsenicsova K, Piknova L, Kaclikova E, Kuchta T. 2008. Detection of Salmonella enterica in food using two-step enrichment and real-time polymerase chain reaction. Lett Appl Microbiol 46: 483-487. https://doi.org/10.1111/j.1472-765X.2008.02342.x
  9. Hein I, Flekna G, Krassnig M, Wagner M. 2006. Real-time PCR for the detection of Salmonella spp. in food: An alternative approach to a conventional PCR system suggested by the FOOD-PCR project. J Microbiol Methods 66: 538-547. https://doi.org/10.1016/j.mimet.2006.02.008
  10. Catarame TMG, O'Hanlon KA, McDowell DA, Blair IS, Duffy G. 2006. Comparison of a real-time polymerase chain reaction assay with a culture method for the detection of Salmonella in retail meat samples. J Food Safety 26: 1-15. https://doi.org/10.1111/j.1745-4565.2005.00025.x
  11. Malorny B, Paccassoni E, Fach P, Bunge C, Martin A, Helmuth R. 2004. Diagnostic real-time PCR for detection of Salmonella in food. Appl Environ Microbiol 70: 7046-7052. https://doi.org/10.1128/AEM.70.12.7046-7052.2004
  12. McGuinness S, McCabe E, O'Regan E, Dolan A, Duffy G, Burgess C, Fanning S, Barry T, O'Grady J. 2009. Development and validation of a rapid real-time PCR based method for the specific detection of Salmonella on fresh meat. Meat Sci 83: 555-562. https://doi.org/10.1016/j.meatsci.2009.07.004
  13. Malorny B, Bunge C, Helmuth R. 2007. A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs. J Microbiol Methods 70: 245-251. https://doi.org/10.1016/j.mimet.2007.04.013
  14. Liming SH, Bhagwat AA. 2004. Application of a molecular beacon-real-time PCR technology to detect Salmonella species contaminating fruits and vegetables. Int J Food Microbiol 95: 177-187. https://doi.org/10.1016/j.ijfoodmicro.2004.02.013
  15. Rodriguez-Lazaro D, Hernandez M, Esteve T, Hoorfar J, Pla M. 2003. A rapid and direct real time PCR-based method for identification of salmonella spp. J Microbiol Methods 54: 381-390. https://doi.org/10.1016/S0167-7012(03)00071-X
  16. Bhagwat AA. 2002. Simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains by real-time PCR. Int J Food Microbiol 84: 217-224.
  17. Jung BY, Lim HS. Jung SC. 2003. Development of differential media and multiplex PCR assays for the rapid detection of Listeria monocytogenes. Korean J Vet Res 43: 231-237.
  18. Korea Food & Drug Administration. 2008. Food code. Seoul, Korea.
  19. Luke TD, William JB, James CM, Margaret SN, Lynn AC, Williams BH, Linda G, Riggins WS. 2002. Real-time PCR detection of Salmonella in suspect foods from a gastroentritis outbreak in Kerr County, Texas. J Clin Microbiol 40: 3050-3052. https://doi.org/10.1128/JCM.40.8.3050-3052.2002
  20. Prentice N, Murray JS, Scott MF, Coombs JP, Rarton A. 2006. Rapid isolation and detection of Escherchia coli O157:H7 in fresh produce. J Rapid Methods Auto Microbiol 14: 299-308. https://doi.org/10.1111/j.1745-4581.2006.00069.x
  21. Hyeon JY, Hwang IG, Kwak HS, Park JS, Heo S, Choi IS, Park C, Seo KH. 2009. Evaluation of an automated ELISA $(VIDAS^{(R)})$ 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