Abstract
Conventional methods to detect Salmonella spp. in foodstuffs may take up to 1 wk. Methods for pathogen detection are required. Real-time detection of Salmonella spp. will broaden our ability to screen large number of samples in a short time. This chapter describes a step-by-step procedure using an oligonucleotide probe that becomes fluorescent upon hybridization to the target DNA (Molecular Beacon; MB) in a real-time polymerase chain reaction (PCR) assay. The capability of the assay to detect Salmonella species from artificially inoculated fresh- and fresh-cut produce as well as ready-to-eat meats is demonstrated. The method uses internal positive and negative controls which enable researchers to detect false-negative PCR results. The procedure uses the buffered peptone water for the enrichment of Salmonella spp. and successfully detects the pathogen at low level of contamination (2–4 cells/25 g) in <24 h.
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References
Bhagwat, A. A. (2006) In Microbiology of Fresh Produce (Matthews, K. R., ed.), pp. 121–165, American Society for Microbiology, Washington, DC.
Bhagwat, A. A. and Lauer, W. (2004) Food borne outbreaks in raw produce can be prevented. Food Quality 11, 62–63.
NACMCF (1999) National Advisory Committee on microbiological criteria for foods. Microbiological safety evaluations and recommendations on fresh produce. Food Control 10, 117–143.
Feng, P. (1997) Impact of molecular biology on the detection of foodborne pathogens. Molec. Biotechnol. 7, 267–278.
Bhagwat, A. A. (2003) Simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains by real-time PCR. Int. J. Food Microbiol. 84, 217–224.
Hill, W. E. (1996) The polymerase chain reaction: applications for the detection of foodborne pathogens. Crit. Rev. Food Sci. Nutr. 36, 123–173.
Bhagwat, A. A. (2004) Rapid detection of Salmonella from vegetable rinse-water using real-time PCR. Food Microbiol. 21, 73–78.
Hines, E. (2000) PCR-based testing: unraveling the mystery. Food Quality 7, 22–28.
Hoorfar, J. and Radstorm, P. (2000) Automated 5′ nuclease PCR assay for identification of Salmonella enterica. J. Clin. Microbiol. 38, 3429–3435.
Liming, S. H. and Bhagwat, A. A. (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.
Liming, S. H., Zhang, Y., Meng, J., and Bhagwat, A. A. (2004) Detection of Listeria monocytogenes in fresh produce using molecular beacon-real-time PCR technology. J. Food Sci. 69, 240–245.
Chen, W., Martinez, G., and Mulchandani, A. (2000) Molecular beacons: a real-time polymerase chain reaction assay for detecting Salmonella. Anal. Biochem. 280, 166–172.
Tyagi, S. and Kramer, F. R. (1996) Molecular beacon: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308.
Nogva, H. K. and Lillehaug, D. (1999) Detection and quantification of Salmonella in pure cultures using 5−-nuclease polymerase chain reaction. Int. J. Food Microbiol. 51, 191–196.
Kimura, B., Kawasaki, S., Fujii, T., Kusunoki, J., Itoh, T., and Flood, S. J. A. (1999) Evaluation of TaqMan PCR assay for detecting Salmonella in raw meat and shrimp. J. Food Protect. 62, 329–335.
McKllip, J. L. and Drake, M. A. (2000) Molecular beacon polymerase chain reaction detection of Escherichia coli O157:H7 in milk. J. Food Protect. 63, 855–859.
Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11, 1026–1030.
Chen, S., Yee, A., Griffiths, M., et al. (1997) The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities. Int. J. Food Microbiol. 35, 239–250.
Liao, C.-H. and Shollenberger, L. M. (2003) Detection of Salmonella by indicator agar media and PCR as affected by alfalfa seed homogenates and native bacteria. Lett. Appl. Microbiol. 36, 152–156.
Miller, D. N. (2001) Evaluation of gel filtration resins for the removal of PCR-inhibitory substances from soils and sediments. J. Microbiol. Methods 44, 49–58.
Heller, L. C., Davis, C. R., Peak, K. K., et al. (2003) Comparison of methods for DNA isolation from food samples for detection of Shiga toxin-producing Escherichia coli by real-time PCR. Appl. Environ. Microbiol. 69, 1844–1846.
Shearer, A. E. H., Strapp, C. M., and Joerfer, R. D. (2001) Evaluationof a polymerase chain reaction-based system for detection of Salmonella Enteritidis, Escherichia coli O157:H7, Listeria spp., and Listeria monocytogenes on fresh fruits and vegetables. J. Food Protect. 64, 788–795.
Patel, J. R., Bhagwat, A. A., Sanglay, G. C., and Solomon, M. B. (2006) Rapid detection of Salmonella from hydrodynamic pressure-treated poultry using molecular beacon real-time PCR. Food Microbiol. 23, 39–46.
Gawande, P. V. and Bhagwat, A. A. (2002) Inoculation onto solid surfaces protect Salmonella spp. during acid challenge: a model study using polyethersulfone membranes. Appl. Envron. Microbiol. 68, 86–92.
Andrews, W. H., June, G. A., Sherrod, P. S., Hammack, T. S., Amaguana, R. M., and Andrews, W. H. (1998) In Bacteriological Analytical Manual (Jackson, G. A., ed.), pp. 5.01–5.20, AOAC International, Gaithersburg, MD.
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Bhagwat, A.A., Patel, J., Chua, T., Chan, A., Cruz, S.R., Aguilar, G.A.G. (2008). Detection of Salmonella Species in Foodstuffs. In: Marx, A., Seitz, O. (eds) Molecular Beacons: Signalling Nucleic Acid Probes, Methods, and Protocols. Methods in Molecular Biology, vol 429. Humana Press. https://doi.org/10.1007/978-1-60327-040-3_3
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DOI: https://doi.org/10.1007/978-1-60327-040-3_3
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