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Cost-effective optimization of real-time PCR-based detection of Campylobacter and Salmonella with inhibitor tolerant DNA polymerases
DTU Technical University of Denmark, Denmark.
DTU Technical University of Denmark, Denmark.
DTU Technical University of Denmark, Denmark.
DTU Technical University of Denmark, Denmark.
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2015 (English)In: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 119, no 5, p. 1391-1402Article in journal (Refereed) Published
Abstract [en]

Aims: The aim of this study was to cost-effectively improve detection of foodborne pathogens in PCR inhibitory samples through the use of alternative DNA polymerases. Methods and Results: Commercially available polymerases (n = 16) and PCR master mixes (n = 4) were screened on DNA purified from bacterial cells in two validated real-time PCR assays for Campylobacter and Salmonella. The five best performing (based on: limit of detection (LOD), maximum fluorescence, shape of amplification curves and amplification efficiency) were subsequently applied to meat and faecal samples. The VeriQuest qPCR master mix performed best for both meat and faecal samples (LODs of 102 and 104 CFU ml-1 in the purest and crudest DNA extractions respectively) compared with Tth (LOD = 102-103 and 105-106 CFU ml-1). AmpliTaqGold and HotMasterTaq both performed well (LOD = 102-104 CFU ml-1) with meat samples and poorly (LOD = 103-106 CFU ml-1/not detected) with faecal samples. Conclusions: Applying the VeriQuest qPCR master mix in the two tested real-time PCR assays could allow for simpler sample preparation and thus a reduction in cost. Significance and Impact of the Study: This work exemplifies a cost-effective strategy for optimizing real-time PCR-based assays. However, a DNA polymerase suitable for one assay and sample type is not necessarily optimal for other assays or sample types.

Place, publisher, year, edition, pages
Blackwell Publishing Ltd , 2015. Vol. 119, no 5, p. 1391-1402
Keywords [en]
Salmonella, Campylobacter, Detection, DNA polymerase, Faeces, Inhibitors, Minced meat, Sample preparation, locked nucleic acid, Taq polymerase, bacterial DNA, DNA directed DNA polymerase, bacterium, cost analysis, detection method, DNA, enzyme activity, feces, inhibition, inhibitor, meat, optimization, polymerase chain reaction, real time, Article, bacterial cell, bacterium culture, bacterium detection, colony forming unit, controlled study, cost effectiveness analysis, DNA extraction, feces analysis, food contamination, limit of detection, nonhuman, real time polymerase chain reaction, Salmonella enterica serovar Typhimurium, animal, chemistry, economics, evaluation study, genetics, isolation and purification, metabolism, microbiology, procedures, Bacteria (microorganisms), Animals, DNA, Bacterial, DNA-Directed DNA Polymerase, Real-Time Polymerase Chain Reaction
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Natural Sciences
Identifiers
URN: urn:nbn:se:ri:diva-39003DOI: 10.1111/jam.12937Scopus ID: 2-s2.0-84944675291OAI: oai:DiVA.org:ri-39003DiVA, id: diva2:1324977
Available from: 2019-06-14 Created: 2019-06-14 Last updated: 2019-07-03Bibliographically approved

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Löfström, Charlotta

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