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

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.

Keywords

Vibrio parahaemolyticus;real-time PCR;culture method;comparison

Acknowledgement

Supported by : 식약청

References

  1. Joseph SW, Cowell RR, Kaper JB. Vibrio parahaemolyticus and related halophilic vibrios. Crit. Rev. Microbiol. 10: 77-123 (1983)
  2. Su YC, Liu C. Vibrio parahaemolyticus: A concern of seafood safety. Food Microbiol. 24: 549-558 (2007) https://doi.org/10.1016/j.fm.2007.01.005
  3. Morris JG. Cholera and other types of vibriosis: A story of human pandemics and oysters on the half shell. Clin. Infect. Dis. 37: 272-280 (2003) https://doi.org/10.1086/375600
  4. DePaola A, Hopkin LH, Peeler JT, Wentz B, McPhearson RM. Incidence of Vibrio parahaemolyticus in US coastal waters and oysters. Appl. Environ. Microb. 56: 2299-2302(1990)
  5. Jung SH, Hur MJ, Ju JH, Kim KA, Oh SS, Go JM, Kim YH, Im JS. Microbiological evaluation of raw vegetables. J. Food Hyg. Safety, 21: 250-257 (2006)
  6. Kim MH, Shin WS. Microbiological quality of raw and cooked foods in middle and high school food service establishments. J. Korean Soc. Food Sci. Nutr. 37: 1343-1356 (2008) https://doi.org/10.3746/jkfn.2008.37.10.1343
  7. Korea Food and Drug Administration. Food & Drug Statistical Yearbook for 2009. Available from http://www.kfda.go.kr/index.kfda?mid=96&pageNo=1&seq=7181&cmd=v, Accessed Nov. 10, 2009.
  8. Korea Food and Drug Administration. Food code. Available from http://safefood.kfda.go.kr/RS/food_menu.jsp, Accessed Nov. 21, 2009.
  9. Ward LN, Bej AK. Detection of Vibrio parahaemolyticus in shellfish by use of multiples real-time PCR with $TaqMan^{\circR}$ fluorescent probes. Appl. Environ Microb. 72: 2031-2042 (2006) https://doi.org/10.1128/AEM.72.3.2031-2042.2006
  10. Yang C, Jiang Y, Huang K, Zhu C, Yin Y. Application of realtime PCR for quantitative detection of Campylobacter jejuni in poultry, milk, and environmental water. FEMS Immunol Med Mic. 38: 265-271 (2003) https://doi.org/10.1016/S0928-8244(03)00168-8
  11. Honda T, Iida T. The pathogenicity of Vibrio parahaemolyticus and the role of the thermostable direct hemolysin and related hemolysins. Rev. Med. Microbiol. 4: 106-113 (1993) https://doi.org/10.1097/00013542-199304000-00006
  12. Nordstrom JL, Vickery MC, Blackstone GM, Murray SL, DePaola A. Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters. Appl. Environ Microb. 73: 5840-5847 (2007) https://doi.org/10.1128/AEM.00460-07
  13. Hondo S, Goto I, Minematsu I, Ikeda N, Asano N, Ishibashi M, Kinoshita Y, Nishibuchi N, Honda T, Miwatani T. Gastroenteritis due to Kanagawa negative Vibrio parahaemolyticus. Lancet 1: 331-332 (1987)
  14. Thompson CA, Vanderzant C. Serological and hemolytic characteristics of Vibrio parahaemolyticus from marine sources. J. Food Sci. 41: 150-151(1976)
  15. Dileep V, Kumar HS, Kumar Y, Nishibuchi M, Karunasagar I, Karunasagar I. Application of polymerase chain reaction for the detection of Vibrio parahaemolyticus associated with tropical seafoods and coastal environment. Lett. Appl. Microbiol. 36: 423-427 (2003) https://doi.org/10.1046/j.1472-765X.2003.01333.x
  16. Kim YB, Okuda J, Matsumoto C, Takahashi N, Hashimoto S, Nishibuchi M. Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the tox R gene. J. Clin. Microbiol. 37: 1173-1177(1999)
  17. Kawatsu K, Ishibashi M, Tsukamoto T. Development and evaluation of a rapid, simple, and sensitive immunochromatographic assay to detect thermostable direct hemolysin produced by Vibrio parahaemolyticus in enrichment cultures of stool specimens. J. Clin. Microbiol. 44: 1821-1827 (2006) https://doi.org/10.1128/JCM.44.5.1821-1827.2006
  18. Blackstone GM, Nordstrom JL, Vickery MC, Bowen MD, Meyer RF, DePaola A. Detection of pathogenic Vibrio parahaemolyticus in oyster enrichment by real-time PCR. J. Microbiol. Methods 53: 149-155 (2003) https://doi.org/10.1016/S0167-7012(03)00020-4
  19. Hyeon JY, Hwang IG, Kawk HS, Park JS, Heo S, Choi IS, Park CK, Seo KH. 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. Anim. Resour. 29: 506-512 (2009) https://doi.org/10.5851/kosfa.2009.29.4.506
  20. Seo KH, Brackett RE. Rapid, specific detection of Enterobacter sakazakii in infant formula using a real-time PCR assay. J. Food Protect. 68: 59-63 (2005)
  21. Vold L, Holck A, Wasteson Y, Nissen H. High levels of background flora inhibits growth of Escherichia coli O157:H7 in ground beef. Int. J. Food Microbiol, 56: 219-225 (2000) https://doi.org/10.1016/S0168-1605(00)00215-4
  22. Malorny B, Paccassoni E, Fach P, Bunge C, Martin A, Helmuth R. Diagnostic real-time PCR for detection of Salmonella in food. Appl. Environ Microb. 70: 7064-7052 (2004)
  23. Bolinches J, Romalde JL, Toranzo AE. Evaluation of selective media for isolation and enumeration of vibrios from estrarine waters. J. Microbiol. Meth. 8: 151-160 (1988) https://doi.org/10.1016/0167-7012(88)90016-4
  24. Alam MJ, Tomochika K, Miyoshi S, Shinoda S. Analysis of seawaters for the recovery of culturable Vibrio parahaemolyticus and some other vibrios. Microbiol. Immunol. 45: 393-397 (2001)
  25. Bottone EJ, Robin T. Vibrio parahaemolyticus: Suspicion of presence based on aberrant biochemical and morphologicsl features. J. Clin. Microbiol. 8: 760-763 (1978)