A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples.
We developed a novel filtration-based method that can eliminate dead or severely 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.
D'Urso
Oscar Fernando
OF
Department of Environmental and Biological Sciences, Institute of Physiology, University of Salento, Lecce, Italy.
Poltronieri
Palmiro
P
Marsigliante
Santo
S
Storelli
Carlo
C
Hernández
Marta
M
Rodríguez-Lázaro
David
D
D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005374:Filtration / Q000379:methods* / Q000592:standards; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D019779:Salmonella enterica / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D015014:Yogurt / Q000382:microbiology*
D004269:DNA, Bacterial