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Comparison of Standard Culture Method and Real-time PCR Assay for Detection of Staphylococcus aureus in Processed and Unprocessed Foods

가공식품과 비가공식품에서의 황색포도상구균 검출을 위한 배지법과 Real-time PCR법의 비교

  • Lee, Jae-Hoon (Department of Public Health, College of Veterinary Medicine, Konkuk University) ;
  • Song, Kwang-Young (Department of Public Health, College of Veterinary Medicine, Konkuk University) ;
  • Hyeon, Ji-Yeon (Department of Public Health, College of Veterinary Medicine, Konkuk University) ;
  • Hwang, In-Gyun (Korea Food and Drug Administration, National Institute of Food and Drug Safety Evaluation) ;
  • Kwak, Hyo-Sun (Korea Food and Drug Administration, National Institute of Food and Drug Safety Evaluation) ;
  • Han, Jeong-A (Korea Food and Drug Administration, National Institute of Food and Drug Safety Evaluation) ;
  • Chung, Yun-Hee (Korea Consumer Agency) ;
  • Seo, Kun-Ho (Department of Public Health, College of Veterinary Medicine, Konkuk University)
  • 이재훈 (건국대학교 수의과대학) ;
  • 송광영 (건국대학교 수의과대학) ;
  • 현지연 (건국대학교 수의과대학) ;
  • 황인균 (식품의약품안전청 식품의약품안전평가원) ;
  • 곽효선 (식품의약품안전청 식품의약품안전평가원) ;
  • 한정아 (식품의약품안전청 식품의약품안전평가원) ;
  • 정윤희 (한국소비자원) ;
  • 서건호 (건국대학교 수의과대학)
  • Received : 2009.11.23
  • Accepted : 2010.03.19
  • Published : 2010.06.30

Abstract

Staphylococcus aureus is one of the major pathogens that can cause staphylococcal infection and food poisoning. In this study, we compared conventional culture methods and real-time PCR for detection of S. aureus in artificially inoculated milk, sausage, raw pork, and vegetable salad. The performance of a coagulase test for confirming S. aureus was also compared with a colony PCR test. Bulk food samples (500 g each) were artificially inoculated with S. aureus and divided into 20 samples (25 g or mL each). All samples were added to tryptic soy broth (225 mL/sample) with 10% NaCl and incubated at $37^{\circ}C$ for 24 h. After the enrichment, broth cultures were streaked onto Baird-Parker (BP) agar with egg yolk tellulite, and incubated at $37^{\circ}C$ for 24 h. In addition, 1 mL of broth cultures was collected to perform real-time PCR. Two suspicious colonies from the BP agar were picked up and plated on nutrient agar and incubated at $37^{\circ}C$ for 24 h followed, by a coagulase confirmation test and a colony PCR analysis. There were no statistical differences between culture methods and realtime PCR in food samples with low background microflora, such as milk and sausage. However, a significant statistical difference was found between the culture methods and real-time PCR for raw pork and vegetable salad. Furthermore, the colony PCR test of the presumptive colonies on BP agar for confirming S. aureus is more accurate and efficient than the coagulase test for unprocessed foods.

황색포도상구균은 사람과 동물에서 화농성 질환과 위장관계 질환을 일으키는 세균으로 자연계에 널리 상재하며 식품으로의 감염이 일어날 수 있어 식중독의 원인이 된다. 이에 신속하고 정확한 황색포도상구균의 검출이 요구되는 실정이다. 본 연구에서는 상재균의 오염도가 다른 다양한 축산 가공식품과 비가공식품에서 황색포도상구균의 검출을 위해 표준 시험법인 배지법과 자체 개발한 real-time PCR법의 검출도를 비교하였고 황색포도상구균 coagulase 확인시험과 real-time PCR법을 활용한 colony PCR 확인 시험을 비교하여 균 동정 능력을 비교하였다. 상재균 수가 적은 식품인 우유와 소시지 그리고 상대적으로 상재균수가 많은 생 돼지고기와 야채 샐러드를 샘플로 선정하여 인위적으로 500 g 혹은 mL의 샘플을 25 g씩 20개의 시료샘플로 나누어 실험하였을 때에 최소한 한 개 이상 양성 결과가 나오도록 황색포도상구균을 접종하여 배지법과 realtime PCR법을 시행하여 검출능력을 비교하였다. 배지법에서 획득한 의심 집락의 확인 동정은 coagulase 시험과 realtime PCR법을 활용한 colony PCR 확인시험을 병행하여 비교하였으며 각각의 결과를 real-time PCR법으로 신속 검출한 결과와 비교하였다. 식품 내 상재균 수가 적은 축산 가공식품인 우유나 소시지 등에서 황색포도상구균을 검출할 경우에는 배지법의 coagulase 확인시험과 colony PCR 확인시험과의 유의차가 없었으며 colony PCR 확인시험을 이용한 확인동정 결과와 real-time PCR법을 사용한 24시간 신속 검출 결과 사이에도 역시 유의차가 없었다. 반면에 상대적으로 상재균수가 많은 비가공식품인 생 돼지고기나 야채 샘플에서는 coagulase 시험법을 사용한 황색포도상구균의 확인동정은 유효성이 매우 떨어졌으며 colony PCR 확인시험 결과와 유의적인 차이를 보였다. Real-time PCR법을 사용한 24시간 신속 검출법은 배지법의 coagulase 시험법의 양성 수보다 낮은 검출률을 보였으나 colony PCR 확인시험보다 높은 검출률을 보여 상재균 수가 높은 샘플에서도 배지법을 대체해서 사용할 수 있는 효율적인 방법임을 보여주었다. 이러한 결과를 종합해 볼 때 본 연구에서 개발한 realtime PCR법은 기존 배지법을 대체하여 24시간 이내에 신속하게 황색포도상구균을 검출할 수 있고 coagulase 확인 시험을 대체할 수 있는 방법으로 여겨진다.

Keywords

References

  1. Alarcon, B., Vicedo, B., and Aznar, R. (2006) PCR-based procedure for detection and quantification of Staphylococcus aureus and their application in foods. J. Appl Microbiol. 100, 352-364. https://doi.org/10.1111/j.1365-2672.2005.02768.x
  2. Ellender, R. D., Huang, L., Sharp, S. L., and Telleton, R. P. (1995) Isolation, enumeration, and identification of Grampositive cocci from frozen crabmeat. J. Food Prot. 58, 853- 857.
  3. Goto, M., Takahashi, H., Segawa, Y., Hayashidani, H., Takatori, K., and Hara-Kudo, Y. (2007) Real-time PCR method for quantification of Staphylococcus aureus in milk. J. Food Prot. 70, 90-96.
  4. Han, S. R., Hyeon, J. Y., Kim, H. Y., Park, J. S., Heo, S., Shin, H. S., and Seo, K. H. (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. 286, 16-622. https://doi.org/10.5851/kosfa.2008.28.5.616
  5. Hyeon, J. Y., Hwang, I. G., Kwak, H. S., Park, J. S., Heo, S., Choi, I. S., Park, C. K., and Seo, K. H. (2009) Evaluation of an automated ELISA($VIDAS^{{\circledR}}$) and real-time PCR by comparing with a conventional culture methods 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
  6. Ingham, S. C., Becker, K. L., and Fanslau, M. A. (2003) Comparison of the Baird-Parker agar and 3M Petrifilm Staph Express Count plate methods for enumeration of Staphylococcus aureus in naturally and artificially contaminated foods. J. Food Prot. 66, 2151-2155.
  7. Kim, M. H., Kim, W. J., Shin, W. S., Shon, D. H., and Cha, S. K. (2003) Feasibility study on the use of liposomes for detecting food-borne pathogenic bacteria. Korean J. Food Sci. Ani. Resour. 23, 278-283.
  8. Marin, M. E., de la Rosa, M. C., and Cornejo, I. (1992) Enterotoxigenicity of Staphylococcus strains isolated from Spanish dry-cured hams. Appl. Environ. Microbiol. 58, 1067- 1069.
  9. Moon, J. Y., Lee, E. J., and Kim, Y. B. (2004) Rapid detection of methicilin resistant Staphylococcus aureus by multiplex PCR. J. Bacteriol. Virol. 34, 91-100.
  10. Much, P., Pichler, J., Kasper, S. S., and Allerberger, F. (2009) Foodborne outbreaks, Austria 2007. Wien Klin Wochenschr. 121,77-85. https://doi.org/10.1007/s00508-008-1125-z
  11. Palomares, C., Torres, M. J., Torres, A., Aznar, J., and Palomares, J. C. (2003) Rapid detection and identification of Staphylococcus aureus from blood culture specimens using real-time fluorescence PCR. Diagn. Microbiol. Infect. Dis. 45, 183-189. https://doi.org/10.1016/S0732-8893(02)00542-4
  12. Rall, V. L. M., Vieira, F. P., Rall, R., Vieitis, R. L., Fernandes, Jr., Candeias, J. M. G., Cardoso, K. F. G., and Araujo, Jr. J. P. (2008) PCR detection of Staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk. Vet. Microbiol. 132, 408-413. https://doi.org/10.1016/j.vetmic.2008.05.011
  13. Riyaz-Ul-Hassan, S., Verma, V., and Qazi, G. N. (2008) Evaluation of three different molecular markers for the detection of Staphylococcus aureus by polymerase chain reaction. Food Microbiol. 25, 452-459. https://doi.org/10.1016/j.fm.2008.01.010
  14. Suarez, M. J., Arias, M. L., and del Mar Gamboa, M. (2008) Staphylococcus aureus enterotoxin A detection using the polymerase chain reaction (PCR) and its correlation with coagulase and thermonuclease tests. Arch. Latinoam. Nutr. 58, 59-63.
  15. Yoon, J. H., Lee, S. J., and Hong, K. W. (2008) A multiplex PCR assay for the direct detection of Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus in food. Food Eng. Prog. 12, 24-31.

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