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A novel strategy to obtain quantitative data for modelling: Combined enrichment and real-time PCR for enumeration of salmonellae from pig carcasses
DTU Technical University of Denmark, Denmark.ORCID iD: 0000-0001-6492-1245
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2011 (English)In: International Journal of Food Microbiology, ISSN 0168-1605, E-ISSN 1879-3460, Vol. 145, no SUPPL. 1, p. S86-S95Article in journal (Refereed) Published
Abstract [en]

Salmonella is a major zoonotic pathogen which causes outbreaks and sporadic cases of gastroenteritis in humans worldwide. The primary sources for Salmonella are food-producing animals such as pigs and poultry. For risk assessment and hazard analysis and critical control point (HACCP) concepts, it is essential to produce large amounts of quantitative data, which is currently not achievable with the standard cultural based methods for enumeration of Salmonella. This study presents the development of a novel strategy to enumerate low numbers of Salmonella in cork borer samples taken from pig carcasses as a first concept and proof of principle for a new sensitive and rapid quantification method based on combined enrichment and real-time PCR. The novelty of the approach is in the short pre-enrichment step, where for most bacteria, growth is in the log phase. The method consists of an 8h pre-enrichment of the cork borer sample diluted 1:10 in non-selective buffered peptone water, followed by DNA extraction, and Salmonella detection and quantification by real-time PCR. The limit of quantification was 1.4 colony forming units (CFU)/20cm2 (approximately 10g) of artificially contaminated sample with 95% confidence interval of±0.7 log CFU/sample. The precision was similar to the standard reference most probable number (MPN) method. A screening of 200 potentially naturally contaminated cork borer samples obtained over seven weeks in a slaughterhouse resulted in 25 Salmonella-positive samples. The analysis of salmonellae within these samples showed that the PCR method had a higher sensitivity for samples with a low contamination level (<6.7CFU/sample), where 15 of the samples negative with the MPN method was detected with the PCR method and 5 were found to be negative by both methods. For the samples with a higher contamination level (6.7-310CFU/sample) a good agreement between the results obtained with the PCR and MPN methods was obtained. The quantitative real-time PCR method can easily be applied to other food and environmental matrices by adaptation of the pre-enrichment time and media. © 2010 Elsevier B.V.

Place, publisher, year, edition, pages
2011. Vol. 145, no SUPPL. 1, p. S86-S95
Keywords [en]
Enumeration, Mini-MPN, MPN, Quantification, Real-time PCR, Salmonella, bacterial DNA, peptone, water, accuracy, article, bacterium contamination, bacterium detection, carcass, colony forming unit, dilution, DNA extraction, nonhuman, quantitative analysis, real time polymerase chain reaction, swine, Abattoirs, Animals, Humans, Meat, Models, Biological, Polymerase Chain Reaction, Risk Assessment, Sus scrofa, Animalia, Suidae
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Natural Sciences
Identifiers
URN: urn:nbn:se:ri:diva-39024DOI: 10.1016/j.ijfoodmicro.2010.08.026Scopus ID: 2-s2.0-79953032774OAI: oai:DiVA.org:ri-39024DiVA, id: diva2:1324763
Note

Commission, FOOD-2006-CT-036272; Funding text 1: NK and CL were funded by the European Union funded Integrated Research Project BIOTRACER (contract FOOD-2006-CT-036272 ) under the 6th RTD Framework. BM was supported by the Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz (BMELV) . We gratefully thank Manuela Jaber, Gabi Berendonk, Johanna Ledwolorz for isolation, serotyping and phage typing Salmonella strains.

Available from: 2019-06-14 Created: 2019-06-14 Last updated: 2019-06-14Bibliographically approved

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Löfström, Charlotta

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