A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples

• Published in: Food microbiology 2009-05, Vol.26 (3), p.311-316
• Main Authors: D'Urso, Oscar Fernando, Poltronieri, Palmiro, Marsigliante, Santo, Storelli, Carlo, Hernández, Marta, Rodríguez-Lázaro, David
• Format: Article
• Language: English
• Subjects: bacterial contamination; Biological and medical sciences; Colony Count, Microbial; discriminant analysis; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; Filtration; Filtration - methods; Filtration - standards; Food Contamination - analysis; Food engineering; Food industries; Food Microbiology; food pathogens; Foodborne pathogens; foods; Fundamental and applied biological sciences. Psychology; General aspects; isolation; linear models; Listeria; Listeria monocytogenes; Listeria monocytogenes - isolation & purification; microbial detection; Molecular detection methods; molecular epidemiology; new methods; polymerase chain reaction; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; predictive microbiology; quantitative analysis; Real-time PCR; Reproducibility of Results; Salmonella; Salmonella enterica; Salmonella enterica - isolation & purification; Sensitivity and Specificity; Species Specificity; viability; Viable detection; Yogurt - microbiology
• ISSN: 0740-0020; 1095-9998
• DOI: 10.1016/j.fm.2008.12.006

We developed a novel filtration-based method that can eliminate dead or severally damaged Salmonella enterica and Listeria monocytogenes in food samples. This new method can recover all viable bacteria in less than 30 min, and can be coupled with a subsequent bacterial DNA extraction and real-time PCR. No statically significant differences ( p < 0.01) were found between real-time PCR results obtained separately from S. enterica and L. monocytogenes when different ratios of living and dead cells were used. The analytical sensitivity in both cases was 1 genome equivalent (GE), and the quantification was linear ( R 2 > 0.9969) over a 5-log dynamic range with PCR efficiencies >0.9754. When compared with the standard microbiological methods for the detection of these foodborne pathogens, the relative accuracy was excellent ranging from 95.72% to 104.48%. Finally, we applied the pre-treatment method to the direct detection of viable forms of these foodborne pathogens in food samples using yogurt as a model, the results being similar to those obtained using pure cultures.