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Rapid Enumeration of Salmonella spp. in Contaminated Pork Meat Using Competitive PCR  

Moon, Ae-Rie (Department of Biotechnology, Dongguk University)
Choi, Weon-Sang (Department of Biotechnology, Dongguk University)
Publication Information
Journal of Food Hygiene and Safety / v.22, no.4, 2007 , pp. 248-256 More about this Journal
Abstract
In this study, the competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Salmonella spp. in pork meat. After comparing three DNA extraction methods, the modified guanidine thiocyanate-phenol-chloroform method was chosen for Salmonella DNA extraction in artificially inoculated pork meat. The previously reported 284-bp invA gene (Rahn et al. Mol. Cell. Probes 1992) was tested for specificity, and 57 Salmonella strains and 24 non-Salmonella strains were evaluated. All Salmonella strains tested were invA positive, and all non-Salmonella strains produced no false positive amplification products. The detection limit achieved was as low as 1,460 colony-forming units (cfu) per 0.1g of pork meat. For cPCR, the invA gene, which features a 82 bp-deletion, was cloned in the pGEM-4Z vector. A known amount of competitor DNA, which has the same primer binding sites, was co-amplified with Salmonella chromosomal DNA from the artificially inoculated pork meat. The cell-number determined by cPCR was approximately equal to the cfu from the most probable number (MPN) method. Finally, the whole procedure took only 5 hr.
Keywords
Detection; Enumeration; Salmonella spp.; PCR;
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1 Chevrier, D., Popoff, M.Y., Dion, M.P., Hermant, D. and Guesdon, J.L.: Rapid detection of Salmonella subspecies I by PCR combined with nonradioactive hybridization using covalently immobilized oligonucleotide on a microplate. FEMS Immunol. Med. Microbiol. 10, 245-252 (1995)   DOI
2 Lim H., Lee, K.H., Hong, C.-H., Bahk, G.-J. and Choi, W.S.: Comparison of four molecular typing methods for the differentiation of Salmonella spp. International J. Food Microbiol. 105, 411-418 (2005)   DOI   ScienceOn
3 Myint, M.S., Johnson, Y.J., Tablante, N.L. and Heckert, R.A.: The effect of pre-enrichment on the sensitivity and specificity and specificity of PCR for detection of naturally contaminated Salmonella in raw poultry compared to conventional culture. Food Microbiol. 23, 599-604 (2006)   DOI   ScienceOn
4 Andrews, W.H., Poelma, P.L. and Wilson, C.R.: Isolation and identification of Salmonella species. In Bacterial Analytical manual, 6th Ed. (US Food and Drug Administration) Association of Official Analytical Chemists, Arlington, VA (1984)
5 Botteldoorn, N., Coillie, E.V., Grijspeerdt, K., Werbrouck, H., haesebrouck, F., Donn, E., D'Haese, E., Heyndrickx, M., Pasmans, F. and Herman, L.: Real-time reverse transcription PCR for the quantification of the mntH expression of Salmonella enterica as a function of growth phase and phagosomelike conditions. J. Microbiol. methods 66, 125-135 (2006)   DOI   ScienceOn
6 Doran, J.L., Collinson, S.K., Buria, J., Sarlos, G., Todd, E.C.D., Munro, C.K., Kay, C.M., Banser, P.A., Peterkin, P.I. and Kay, W.W.: DNA-based diagnostic testsfor Salmonella species targeting agfA, the structural gene for thin, aggregative fimbriae. J. Clin. Microbiol. 31, 2263-2273 (1993)
7 Hashimoto, Y., Itho, Y., Fuginaga, Y., Khan, A.Q., Sultana, F., Miyake, m., Horose, K., Yamamoto, H. and Ezaki, T.: Development of nested PCR based on the viaB sequence to detect Salmonella typhi. J. Clin. Microbiol. 33, 775-777 (1995)
8 Mahon, J. and Lax, A.J.: A quantitative polymerase chain reaction method for the detection in avian feces of Salmonella carrying the spvR gene. Epidemiol. Infect. 111, 455-464 (1993)   DOI
9 Rahn, K, De Grandis, S.A., Clarke, R.C., McEwen, S.A., Galan, J.E., Ginnocchio, C., Curtiss III, R. and Gyles, C.L.: 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, (1992)   DOI
10 Way, J.S., Josephson, K.L., Pillai, S.D., Abbaszadegan, M., Gerba, C.P. and Pepper, I.L.: Specific detection of Salmonella spp. by multiplex polymerase chain reaction. Appl. Environ. Microbiol. 59, 1473-1479 (1993)
11 Choi, W.S.: Detection of Salmonella in milk by polymerase chain reaction. J. Food Hyg. Safety 15, 262-266 (2000)
12 Choi, W.S.: Detection of Yersinia enterocolitica in milk by polymerase chain reaction. Food Sci. Biotechnol. 10, 451-454 (2001)
13 Novinscak, A., Surette, C. and Filion, M.: Quantification of Salmonella spp. in composted biosolids using a TaqMan qPCR assay. J. Microbiol. Methods 70, 119-126 (2007)   DOI   ScienceOn
14 Ellingson, J.L.E., Anderson, J.L., and Carlson, S.A. and Sharma, V.K.: Twelve hour real-time PCR technique for the sensitive and specific detection of Salmonella in raw and ready-to0eat meat products. Mol. Cell. Probes 18, 51-57 (2004)   DOI   ScienceOn
15 Klerks, M.M., Van Bruggen, A.H.C., Zijlstra, C. and Donnikov, M.: Comparison of methods of extraction for Salmonella enterica Serovar enteritidis DNA from environmental substrates and quantification of organism by using a general internal procedural control. Appl. Environ. Microbiol. 72, 3879-3886 (2006)   DOI   ScienceOn
16 Collazo, C.M. and Galn, J.F.: The invasion-associated type III protein secretion system in Salmonella.-a review. Gene 192, 51-59 (1997)   DOI   ScienceOn
17 Galn, J.E. and Curtis III, R.: Distribution of the invA, -B, -C, and -D genes of Salmonella typhimurium among other Salmonella serovars: invA mutants of Salmonella typhi are deficient for entry into mammalian cells. Infect. Immun. 59, 2901-2908 (1991)
18 Isogai, E., Makungu, C., Yabe, J., Sinkala, P., Nambota, A., Isogai, H., Fukushi, H., Silungwe, M., Mubita, C., Syakalima, M., Hang'ombe, M., Kozaki, S. and Yasuda, J.: Detection of Salmonella invA by isothermal and chimeric primerinitiated amplification of nucleic acids (ICAN) in Zambia. Coparative Immunology, Microbiology & Infectious Diseases 28, 363-370 (2005)   DOI   ScienceOn
19 Cohen, H.J., Mechanda, S.M. and Lin, W.: PCR amplification of the fimA gene sequence of Salmonella spp. Appl. Environ. Microbiol. 12, 4303-4308 (1996)
20 Van Leusden, F.M., Van Schothorst, M. and Beckers, H.J.: The standard Salmonella isolation method. In Isolation and Identification Methods for Food Poisoning Organisms (Corry, J.E.L., Roberts, D., and Skinner, F.A.) Academic Press, London, pp. 35-49 (1982)
21 Wang, X., Jothikumar, N. and Griffiths, M.W.: Enrichment and DNA extraction protocols for the simultaneous detection of Salmonella and Listeria monocytogenes in raw sausage meat with multiplex real-time PCR. J. Food Prot. 67, 189-192 (2004)   DOI
22 Nam, H.-M., Srinivasan, V., Gillespie, B.E., Murinda, S.E. and Oliver, S.P.: Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples. International J. Food Microbiol. 102, 161-171 (2005)   DOI   ScienceOn
23 Baumler, A.J., Heffron, F. and Reissbrodt, R.: Rapid detection of Salmonella enterica primers specific for iroB. J. Clin. Microbiol. 35, 1224-1230 (1997)
24 Yeh, K.-S.Chen, T.-H., Liao, C.-W. and Lo, H.-C.: PCR amplification of the Salmonella typhimurium fimY gene sequence to detect the Salmonella species. International J. Food Microbiol. 78, 227-234 (2002)   DOI   ScienceOn
25 Hein, I., Flekna, G., Krassnig, M. and Wagner, M.: Real-time PCR for the detection of Salmonella spp. in food: An alternative approach to a conventional PCR system suggested by the FOOD-PCR project. J. Microbiol. Methods 66, 538-547 (2006)   DOI   ScienceOn
26 Malorny, B., Bunge, C. and Helmuth, R.: A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs. J. Microbiol. methods 70, 245-251 (2007)   DOI   ScienceOn
27 Li, Y., Zhuang, S. and Mustapha, A.: Application of a multiplex PCR for the simultaneous detection of Escherichia coli O157:H7, Salmonella and Shigella in raw and ready-to-eat meat products. Meat Science 71, 402-406 (2005)   DOI   ScienceOn
28 Jacobsen, C.S. and Holben, W.E.: Quantification of mRNA in Salmonella sp. seeded soil and chicken manure using magnetic capture hybridization RT-PCR. J. Microbiol. Methods 69, 315-321 (2007)   DOI   ScienceOn
29 Hanai, K., Satake, M., Naakanishi, H. and Venkateswaran, K.: Comparison of commercially available kits for detection of Salmonella strains in foods. Appl. Environ. Microbiol. 63, 775-778 (1997)
30 Hoorfar, J., Baggesen, D.L. and Porting, P.H.: A PCR-based strategy for simple and rapid identification of rough presumptive Salmonella isolates. J. Microbiol. Methods 35, 77-84 (1999)   DOI   ScienceOn
31 Kim, J., Demeke, T., Clear, R.M. and Patrick, S.K.: Simultaneous detection by PCR of Escherichia coli, Listeria monocytogenes and Salmonella typhimurium in artificially inoculated wheat grain. International J. Food Microbiol. 111, 21-25 (2006)   DOI   ScienceOn
32 Sambrook, J. and Russel, D.W.: Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Press, Cold Spring Harbor, NY, USA (2001)
33 Murphy, N.M., McLauchlin, J., Ohai, C. and Grant K.A.: Construction and evaluation of a microbiological positive process internal control for PCR-based examination of food samples for Listeria monocytogenes and Salmonella enterica. International J. Food Microbiol. doi:10.1016/j.ijfoodmicro. 2007.06.006
34 Malorny, B., Hoorfar, J., Bunge, C. and Helmuth, R.:Multicenter validation of the analytical accuracy of Salmonella PCR: towards an international standard. Appl. Environ. Microbiol. 69, 290-296 (2003)   DOI
35 Rexach, L., Dilassier, F. and Fach, P.: Polymerase chain reaction for Salmonella virulence-associated plasmid genes: a new tool in Salmonella epidemiology. Epidemiol. Infect. 112, 33-43 (1994)   DOI
36 Zhu, Q., Lim, C.K. and Chan, Y.N.: Detection of Salmonella typhi by polymerase chain reaction. J. Appl. Bacteriol. 80, 244-251 (1996)   DOI
37 Aznar, E., Alarcon, T., Buendia, Garcia-Penuela and Lopez- Brea, M.: Detection of decreased susceptibility to fluoroquinolones in Salmonella spp. by five different methods including real-time PCR. International J. Antimicrobial Agents 30, 67-71 (2007)   DOI   ScienceOn
38 Cortez, A.L.L., Carvalho, A.C.F.B., ikuno, A.A. and Vidal- Martins, A.M.C.: identification of Salmonella spp. isolates from chiken abattoirs by mutiplex-PCR. Research in Veterinary Science 81, 340-344 (2006)   DOI   ScienceOn
39 Cano, R.J., Ramussen, S.R., Fraga, G.S. and Palomares, J.C.: Fluorescent detection-polymerase chain reaction (FD-PCR) assay on microwell plates as a screening test for Salmonellas in foods. J. Appl. Bacteriol. 75, 247-253 (1993)   DOI   ScienceOn
40 Shannon, K.E., Lee, D.-Y., Trevors, J.T. and Beaudette, L.A.: Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment. Science of the Total Environment 382, 121-129 (2007)   DOI   ScienceOn
41 Patel, J.R., Bhagwat, A.A., Sanglay, G.C. and Solomon, M.B.: Rapid detection of Salmonella from hydrodynamic pressured-treated poultry using molecular beacon real-time PCR. Food Microbiol. 23, 39-46 (2006)   DOI   ScienceOn
42 Choi, W.S. and Hong C.-H.: Rapid enumeration of Listeria monocytogenes in milk using competitive PCR. International J. Food Microbiol. 84, 79-85 (2003)   DOI   ScienceOn
43 Lim, H., Hong, C.-H. and Choi, W.S.: Rapid enumeration of Listeria monocytogenes in pork meat using competitive PCR. Food Sci. Biotechnol. 14, 387-391 (2005)
44 Farrell, J.J., Doyle, L.J., Addison, R.M., Reller, L.B., Hall, G.S. and Procop, G.W.: Broad-range (Pan) Salmonella and Salmonella serotype typhi-specific real-time PCR assayspotential tools for the clinical microbiologist. Am. J. Clin. Pathol. 123, 339-345 (2005)   DOI
45 Kwang, J., Littledike, E.T. and Keen, J.E.: Use of the polymerase chain reaction for Salmonella detection. Lett. Appl. Microbiol. 22, 46-51 (1996)   DOI   ScienceOn
46 Nowak, B., von Mffling, T., Chaunchom, S. and Hartung, J.: Salmonella contamination in pigs at slaughter and on the farm: A field study using an antibody ELISA test and a PCR technique. International J. Food Microbiol. 115, 259-267 (2007)   DOI   ScienceOn
47 Bhagwat, A.A.: Rapid detection of Salmonella from vegetable rinse-water using real-time PCR. Food Microbiol. 21, 73-78 (2004)   DOI   ScienceOn
48 Olsen, J.E., Aabo, S., Hill, W., Notermans, S., Wernars, K., Granum, P.E., Popovic, T., Rasmussen, H.N. and Olsvik, O.: Probes and polymerase chain reaction for the detection of food-borne bacterial pathogens. International J. Food Microbiol. 28, 1-78 (1995)   DOI   ScienceOn
49 Fluit, A.C., Widjojoatmodjo, M.N., Box, A.T.A., Torensma, R. and Verhoef, J.: Rapid detection of Salmonellae in poultry with the magnetic immunopolymerase chain reaction assay. Appl. Environ. microbiol. 59, 1342-1346 (1993)