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Rapid Quantification of Salmonella in Seafood Using Real-Time PCR Assay

  • Kumar, Rakesh (Microbiology, Fermentation and Biotechnology Division, Central Institute of Fisheries Technology) ;
  • Surendran, P.K. (Poothuvallil, Dr. Surendran Lane) ;
  • Thampuran, Nirmala (Microbiology, Fermentation and Biotechnology Division, Central Institute of Fisheries Technology)
  • Received : 2009.04.24
  • Accepted : 2009.11.01
  • Published : 2010.03.31

Abstract

A quantitative detection method for Salmonella in seafood was developed using a SYBR Green-based real-time PCR assay. The assay was developed using pure Salmonella DNA at different dilution levels [i.e., 1,000 to 2 genome equivalents (GE)]. The sensitivity of the real-time assay for Salmonella in seeded seafood samples was determined, and the minimum detection level was 20 CFU/g, whereas a detection level of 2 CFU/ml was obtained for pure culture in water with an efficiency of ${\geq}85%$. The real-time assay was evaluated in repeated experiments with seeded seafood samples and the regression coefficient ($R^2$) values were calculated. The performance of the real-time assay was further assessed with naturally contaminated seafood samples, where 4 out of 9 seafood samples tested positive for Salmonella and harbored cells <100 GE/g, which were not detected by direct plating on Salmonella Chromagar media. Thus, the method developed here will be useful for the rapid quantification of Salmonella in seafood, as the assay can be completed within 2-3 h. In addition, with the ability to detect a low number of Salmonella cells in seafood, this proposed method can be used to generate quantitative data on Salmonella in seafood, facilitating the implementation of control measures for Salmonella contamination in seafood at harvest and post-harvest levels.

References

  1. Andrews, W. H. and T. S. Hammack. 2001. Salmonella, Chapter 5, In: Bacteriological Analytical Manual, U.S. Food and Drug Administration, 8th Ed., Revision A., AOAC International, Gaithersburg, MD.
  2. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Scidman, J. A. Smith, and K. Struhl. 1994. Current Protocols in Molecular Biology, pp 2.0.1-2.14.8. John Wiley and Sons, New York.
  3. Bean, N. H., J. S. Goulding, M. T. Daneils, and F. J. Angulo. 1997. Surveillance for foodborne disease outbreaks - United States, 1988-1992. J. Food Prot. 60: 1265-1286.
  4. Blodgett, R. 2006. Most probable numbers from serial dilutions. In: Bacteriological Analytical Manual, Appendix 2. Available at: http://www. cfsan.fda.gov/ebam/bam-a2.html.
  5. Boughton, C., J. Egan, G. Kelly, B. Markey, and N. Leonard. 2007. Quantitative examination of Salmonella spp. in the lairage environmental of a pig abattoir. Foodborne Pathog. Dis. 4: 26-32. https://doi.org/10.1089/fpd.2006.57
  6. D'Aoust, J-Y. 1989. Salmonella, pp. 327-445. In M. P. Doyle (ed.). Foodborne Bacterial Pathogens. Marcel, Inc., New York.
  7. Eyiger, A., K. T. Carli, and C. B. Unal. 2002. Implementation of real-time PCR to tetrathionate broth enrichment step of Salmonella detection in poultry. Lett. Appl. Microbiol. 34: 37-41. https://doi.org/10.1046/j.1472-765x.2002.01036.x
  8. Fegan, N., P. Vanderlinde, G. Higgs, and P. Desmarchelier. 2004. Quantification and prevalence of Salmonella in beef cattle presenting at slaughter. J. Appl. Microbiol. 97: 892-898. https://doi.org/10.1111/j.1365-2672.2004.02380.x
  9. Hatha, A. A. M. and P. Lakshmanaperumalsamy. 1997. Prevalence of Salmonella in fish and crustaceans from markets in Coimbatore, South India. Food Microbiol. 14: 111-114. https://doi.org/10.1006/fmic.1996.0070
  10. Heinitz, M. L., R. D. Ruble, D. E. Wagner, and S. R. Tatini. 2000. Incidence of Salmonella in fish and seafood. J. Food Protect. 63: 579-592.
  11. Jensen, A. N., G. Sorensen, D. L. Baggesen, R. Bodker, and J. Hoorfar. 2003. Addition of novobiocin in pre-enrichment step can improve Salmonella culture of modified semisolid Rappaport-Vassiliadis. J. Microbiol. Methods 55: 249-255. https://doi.org/10.1016/S0167-7012(03)00158-1
  12. Josefsen, M. H., M. Krause, H. Hansen, and J. Hoorfar. 2007. Optimization of a 12-hour TaqMan PCR-based method for detection of Salmonella bacteria in meat. Appl. Environ. Microbiol. 73: 3040-3048. https://doi.org/10.1128/AEM.02823-06
  13. Kumar, R., P. K. Surendran, and N. Thampuran. 2009. Distribution and genotypic characterization of Salmonella serovars isolated from seafood of Cochin, India. J. Appl. Microbiol. 106: 515-524. https://doi.org/10.1111/j.1365-2672.2008.04020.x
  14. Levin, R. E. 2004. The application of real-time PCR to food and agriculture systems: A review. Food Biotechnol. 18: 97-133. https://doi.org/10.1081/FBT-120030386
  15. Malorny, B., J. Hoorfar, M. Hugas, A. Heuvelink, P. Fach, L. Ellerbroek, C. Bunge, C. Dorn, and R. Helmuth. 2003. Interlaboratory diagnostic accuracy of a Salmonella specific PCR-based method. Int. J. Food Microbiol. 89: 241-249. https://doi.org/10.1016/S0168-1605(03)00154-5
  16. 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: 7046-7052. https://doi.org/10.1128/AEM.70.12.7046-7052.2004
  17. Malorny, B., C. Radstrom, M. Wagner, N. Kramer, and J. Hoorfar. 2008. Enumeration of Salmonella bacteria in food and feed samples by real-time PCR for quantitative microbial risk assessment. Appl. Environ. Microbiol. 74: 1299-1304. https://doi.org/10.1128/AEM.02489-07
  18. Martinez-Urtaza, J., M. Saco, J. de Novoa, P. Perez-Pioneiro, J. Peiteado, A. Lozano-Leon, and O. Garcia-Martin. 2004. Influence of environmental factors and human activity on the presence of Salmonella serovars in a marine environment. Appl. Environ. Microbiol. 70: 2089-2097. https://doi.org/10.1128/AEM.70.4.2089-2097.2004
  19. Nogva, H. K., K. Rudi, K. Naterstad, A. Holck, and D. Lillehaug. 2000. Application of 5'-nuclease PCR for quantitative detection of Listeria monocytogenes in pure cultures, water, skim milk, and unpasteurized whole milk. Appl. Environ. Microbiol. 66: 4266-4271. https://doi.org/10.1128/AEM.66.10.4266-4271.2000
  20. Rahn, K., S. A. De Grandis, R. C. Clarke, S. A. McEwen, J. E. Galan, C. Ginocchio, R. Curtiss III, and C. L. Gyles. 1992. Amplification of an invA gene sequence of Salmonella Typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol. Cell. Probes 6: 271-279. https://doi.org/10.1016/0890-8508(92)90002-F
  21. Rantsiou, K., V. Alessandria, R. Urso, P. Dolci, and L. Cocolin. 2008. Detection, quantification and vitality of Listeria monocytogenes in food as determined by quantitative PCR. Int. J. Food Microbiol. 121: 99-105. https://doi.org/10.1016/j.ijfoodmicro.2007.11.006
  22. Sambrook, J. and D. W. Russel, 2001. Molecular Cloning - A Laboratory Manual, pp. 9.1-9.92. Cold Spring Harbor Laboratory Press, New York.
  23. Seo, K. H., I. E. Velentin-Bon, and R. E. Brackett. 2006. Detection and enumeration of Salmonella Enteritidis in homemade ice cream associated with an outbreak: Comparison of conventional and real-time PCR methods. J. Food Prot. 69: 639-643.
  24. Wang, S. and R. E. Lewin. 2006. Rapid quantification of Vibrio vulnificus in clams (Protochaca staminae) using real-time PCR. Food Microbiol. 23: 757-761. https://doi.org/10.1016/j.fm.2006.01.007