Korean Journal of Food Science and Technology (한국식품과학회지)

Korean Society of Food Science and Technology (한국식품과학회)
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Comparison of the Standard Culture Method and Real-time PCR for the Detection of Vibrio parahaemolyticus in Seafoods and Vegetables

해산식품과 채소에서 Vibrio parahaemolyticus 검출을 위한 배지배양법과 real-time PCR의 비교검증

  • Chon, Jung-Whan (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 (Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Korea Food and Drug Administration) ;
  • Kwak, Hyo-Sun (Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Korea Food and Drug Administration) ;
  • Han, Jeong-A (Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Korea Food and Drug Administration) ;
  • Chung, Yun-Hee (Test and Research Center, Korea Consumer Protection Board) ;
  • Song, Kwang-Young (Department of Public Health, College of Veterinary Medicine, Konkuk University) ;
  • Seo, Kun-Ho (Department of Public Health, College of Veterinary Medicine, Konkuk University)
  • 천정환 (건국대학교 수의과대학) ;
  • 현지연 (건국대학교 수의과대학) ;
  • 황인균 (식품의약품안전청 식품미생물팀) ;
  • 곽효선 (식품의약품안전청 식품미생물팀) ;
  • 한정아 (식품의약품안전청 식품미생물팀) ;
  • 정윤희 (한국소비자원 시험검사소) ;
  • 송광영 (건국대학교 수의과대학) ;
  • 서건호 (건국대학교 수의과대학)
  • Received : 2009.12.24
  • Accepted : 2010.02.18
  • Published : 2010.06.30
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Abstract

Vibrio parahaemolyticus (V. parahaemolyticus), which is commonly found in raw seafood, causes gastroenteritis in humans. Rapid and effective methods have been developed as culture methods require up to 5-7 days. In this study, real-time PCR was compared with the standard culture method for detecting V. parahaemolyticus in seafood and radish sprout samples. Five hundred grams of the samples were artificially contaminated with V. parahaemolyticus then divided into 20 samples. The samples were incubated in alkaline peptone water and then streaked onto thiosulfate-citrate-bile saltssucrose agar. Biochemical tests for suspicious colonies were performed using the API 20NE strip. In parallel, real-time PCR was performed targeting the toxR gene using the enrichment broth. The real-time PCR was sensitive in discriminating V. parahaemolyticus from other foodborne pathogens. The detection limit of the real-time PCR was $10^3\;CFU/mL$ in phosphate-buffered saline. Although the real-time PCR detected more positive samples (76 out of 180, 42%) than the culture method (66 out of 180, 37%), there was no significant statistical difference (p>0.05) between the two methods. In conclusion, real-time PCR assays could be an alternative to the standard culture method for detecting V. parahaemolyticus in seafood and radish sprouts, which has many advantages in terms of detection time, labor, and sensitivity.

V. parahaemolyticus는 국내에서 여름과 가을철에 걸쳐 발생되며 생선이나 패류 등의 해산식품을 날것으로 섭취하거나 불완전하게 익혀 먹을 경우 식중독을 일으키는 병원균이다. 본 연구의 목적은 표준 검출기법인 배지배양법을 이용한 V. parahaemolyticus의 검출과 real-time PCR을 이용한 V. parahaemolyticus의 검출에 있어서 그 유효성과 효율성을 검증하는 것이다. V. parahaemolyticus의 발생기록과 발생가능성이 있는 여러 해산식품과 무순에 적절한 균량을 접종하고 APW로 증균배양하였다. 증균배양이 끝난 후 TCBS선택배지에 배양액을 획선도말하고, 동시에 증균배양액에서 1 mL을 채취하여 real-time PCR을 실시하였다. TCBS에서 초록색이 나온 집락을 1-3개 선별하여 TSI 배지에 접종하여 screening test를 거친 후 API 20NE strip을 사용하여 확인동정하였다. 또한 정상세균총이 목적균의 성장과 검출에 어느 정도의 영향을 미치는지 평가하기 위하여 25 g의 식품 내 정상세균총의 수준을 함께 측정하였다. 실험결과, 자체 제작한 real-time PCR 서열은 V. parahaemolyticus를 특이적으로 검출할 수 있었고 검출 한계는 PBS에서 $10^3\;CFU/mL$ 였다. 또한, 식품 내 정상세균총이 높을 경우 증균배양 시 목적균의 성장에 영향을 미칠 수 있다는 것을 확인하였다. Real-time PCR은 180개의 전체 샘플 중 76개의 양성 결과를 보여 66개의 양성 결과를 낸 배지배양법에 비해 더 많은 양성 검출율을 보였으나 통계학적인 유의차는 발견되지 않았다. Real-time PCR은 표준검출법인 배지배양법과 비교해 볼 때 동등하거나 우수한 검출력을 지닌 것으로 보이며 이러한 realtime PCR법은 24시간 이내에 확정동정까지 가능하여 시간과 노동력의 소모가 많은 배지배양법에 앞서 선별검사로 사용할 경우 시간, 비용, 노동력 절감에 매우 유효할 것으로 판단된다.

Keywords

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