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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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