Evaluation of five DNA extraction methods in the detection of <i>Salmonella enterica</i> from meat using nested PCR

Abstract

Polymerase Chain Reaction (PCR) based detection methods have received significant attention in food borne microbial pathogen detection. However, reliability and sensitivity of these methods are highly depending on the extraction of adequate amount of pure DNA using appropriate extraction method. Hence, selection of appropriate DNA extraction method is very important in PCR based detection of microbial pathogens. In this study, the extraction efficiency of five commonly used DNA extraction methods was evaluated. Salmonella enterica was used as experimental organism and five extraction methods were tested for their ability to extract DNA from spiked pork meat samples. Pork meat samples were incubated for four hours after being added a dilution series (10 0 - 10 3 CFU/mL) of Salmonella enterica culture. Then DNA was extracted from those samples by the five commonly used DNA extraction methods. Using extracted DNA, fliC gene of Salmonella was amplified by Nested PCR. Out of those five methods, the modified Fontana and Kapperd methods were found to be more effective in DNA extraction. In addition, those methods gave high detection sensitivity of 10 1 CFU/mL in Nested PCR amplification. Keywords : DNA extraction; Nested PCR; Salmonella, Fontana, Kapperd DOI: http://dx.doi.org/10.4038/jas.v6i1.3809 The Journal of Agricultural Sciences , 2011 Vol.6(1) 24-31