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31 participants will strengthen the bridge between the Nordic food industry, research institutes and universities in the field of microbiological food safety with special focus on campylobacters and/or molecular techniques Project Manager: Peter Rådström, Lund University, Sweden (Coordinator) Country coordinators: Jeffrey Hoorfar, DFVF, Denmark (Deputy coordinator) Elisabeth Borch, SIK, Sweden Knut Rudi, MATFORSK, Norway Sigrun Gumundsdottir, Icelandic Fisheries Lab (IFL), Iceland Marja-Liisa Hänninen, Helsinki University, Finland http//www.CampyFood.org C AMPY F OOD A Molecular Safety Approach for Campylobacter
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Activities We will strengthen ongoing research at the participating laboratories in order to ensure synergy and transfer the technology to the food industry *mobility of research personnel *a project homepage *hands-on demonstrations *newsletters *workshops http//www.CampyFood.org C AMPY F OOD
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Clostridium botulinum Salmonella Clostridium botulinum Salmonella Virulence expression Rapid methods I II Food safety will be increased by the application of molecular-based techniques http//www.CampyFood.org C AMPY F OOD
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Botulism – rare but deadly An intoxication in which 30ng neurotoxin can be lethal Consumption 0.1g contaminated food can result in botulism High fatality rate (~10% cases) 7 serotypes of the neurotoxin on chromosome: types A, B, E, F on bacteriophage: types C, D on plasmid: type G Clostridium botulinum Toxin, 150 kDa
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Neurotoxin Formation Relative expression and quantification of bontB (mRNA) qRT-PCR BoNT/B production (protein) ELISA Biological activity of BoNT/B (active toxin) Mouse Bioassay
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protein Virulence factors mRNA DNA Virulence expression Environmental factors
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C. botulinum type B Time (h) 0510152025303540455055 OD (620 nm) -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative expression 0 2 4 6 8 10 12 BoNT/B (ng/ml) 0 500 1000 1500 2000 2500 3000 <2h4h5h20h
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Control in foods Preservative Effect CO 2 Inhibition of growth, Inhibition of outgrowth of spores NaClInhibition of growth, Inhibition of outgrowth of spores NaNO 2 Inhibition of growth
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Effect of NaCl 10% CO 2 0% NaCl 0 ppm NaNO 2 10% CO 2 2.5% NaCl 0 ppm NaNO 2 Time (h) 010203040 Relative expression 0 2 4 6 8 10 12 14 OD (620 nm) 0 1 2 3 4 5 Time (h) 010203040 Relative expression 0 2 4 6 8 10 12 14 OD (620 nm) 0 1 2 3 4 5 27 ng*ml -1 *OD -1 * 47 ng*ml -1 *OD -1 *
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Effect of NaNO 2 10% CO 2 0% NaCl 0 ppm NaNO 2 10% CO 2 0% NaCl 75 ppm NaNO 2 Time (h) 010203040 Relative expression 0 2 4 6 8 10 12 14 OD (620 nm) 0 1 2 3 4 5 Time (h) 0102030405060 Relative expression 0 2 4 6 8 10 12 14 OD (620 nm) 0 1 2 3 4 5 27 ng*ml -1 *OD -1 * 30 ng*ml -1 *OD -1 *
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Effect of CO 2 10% CO 2 0% NaCl 0 ppm NaNO 2 70% CO 2 0% NaCl 0 ppm NaNO 2 Relative expression Time (h) 010203040 0 2 4 6 8 10 12 14 OD (620 nm) 0 1 2 3 4 5 Time (h) 010203040 Relative expression 0 2 4 6 8 10 12 14 OD (620 nm) 0 1 2 3 4 5 27 ng*ml -1 *OD -1 * 126 ng*ml -1 *OD -1 *
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Effect of CO 2, NaCl and NaNO 2 Time (h) 0510152025303540 OD (620 nm) 0 1 2 3 4 5 Relative expression 0 2 4 6 8 10 12 14 10% CO 2 0% NaCl 0 ppm NaNO 2 27 ng*ml -1 *OD -1 * Time (h) 020406080100120 OD (620 nm) 0 1 2 3 4 5 Relative expression 0 2 4 6 8 10 12 14 70% CO 2 1.25% NaCl 75 ppm NaNO 2 154 ng*ml -1 *OD -1 *
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LPD= m =b 0 +b 13 *[CO 2 ]*[NaNO 2 ]+b 22 *[NaCl] 2 +e log(RE)=b 0 +b 11 *[CO 2 ] 2 +b 22 *[NaCl] 2 +e Traditional food preservatives (CO2, NaCl and NaNO2) stimulates the neurotoxin formation increasing the risk for food borne botulism
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FOOD MICROB HUMAN Virulence expression a step towards formulating new strategies for food preservation, predictive modelling and risk assessment.
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EU 6th FP. Area: Food Quality and Safety (5.4.4 Area: Traceability processes along the production chain) T5.4.4.1 Origin and development of unintended micro- organisms in the food and feed chains (IP) The objective is to develop and improve methods for tracing the origin of biological agents contaminating (including as the result of a criminal act) food (also bottled or canned drinking water) and animal feed and to model their development (growth, proliferation and toxicogenesis) as a function of ambient (e.g. temperature and relative humidity) and processing conditions, and their point of entry into the food chain (including the home environment).
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Salmonella Rapid methods II
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Day 0 Day 1 Day 3 Day 4 25 g feed + 225 ml BPW Pre-enrichment Enrichment RVS Selective agar plates Confirmation Day 2 Conventional analysis of Salmonella
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Why do we need new methods? *Low detection limit (less than one pathogen per 25 gram) *High specificity and accuracy (no false-negative/-positive results) *High robustness (inter-lab reproducibility) *High Rapidity (at-line and on-line analysis) *Acceptance (validation and standardisation) *Low cost (number of test) *Simplicity (user-friendly and automation) *Sample matrix flexibility (no interference) *Quantitative analysis (food spoilage micro-organisms)
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Amplification Growth-based Viable Counts + Detection Limit Quantitative Simplicity - Specificity Rapid Laborious
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Rapid Methods 1. Cell counting methods Flow cytometry Direct epifluorescent microscopy 2. Modified and automated conventional methods Spiral plater Dipslides Chromogenic/fluorogenic media 3. Impedimetry 4. Bioluminescence 5. Immunological methods ELISA Immunocapture 6. Nucleic acid-based assays Hybridisation Amplification methods (PCR) Restriction fragment length polymorphism (RFLP) Random amplified polymorfic DNA (RAPD) RiboPrinter
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Challenges with Diagnostic PCR Risk of inhibition from biological samples Low concentration of target Reduce the size of the heterogeneous bulk sample to a homogeneous PCR sample
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PCR InhibitorMechanismRef. ProteinasesDegr. of Polym.Powell et al. 1994 IgGBinding to DNAAbu Al-Soud et al. 2000 PolysaccharidesBinding to Polym.Monteiro et al. 1997 LactoferrinRelease of iron ionsAbu Al-Soud, Rådström 2001 Calcium ionsCompeting with Mg 2+ Bickley et al. 1996 PhenolDenatur. of Polym.Katcher, Schwartz 1994 EDTAChelation of Mg2+Rossen et al. 1992 HeparinBinding to DNASatsangi et al. 1994 Taq DNA polymerase
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The importance of DNA polymerase and PCR facilitators in Diagnostic PCR
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Diagnostic PCR 1. Sampling 2. Sample preparation 3. DNA amplification 4. Detection of PCR products DNA polymerases PCR facilitators Pre-PCR Processing
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Internal control 284 bp Salmonella (invA gene) 150 bp Internal control Rahn et al. 1992
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1. Sampling 2. Sample preparation 3. DNA amplification 4. Detection of PCR products Feed in BPW 1:10 Homogenisation Pre-enrichment for 18 h @ 37ºC (isolate obtained!) Samples withdrawn after shaking No DNA extr. or cell lysis! Tth DNA polymerase Gel electrophoresis Enrichment PCR method
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PCR Method Detection limit
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No of positive samples Sample type No of samples NMKL PCR Faeces and intestines 22 0 0 Fish meal 4 0 1 Maize gluten 1 0 1 Meat meal 1 0 0 Mixed feed 24 0 0 Rape meal 8 0 0 Soya 59 3 3 Soya, acidified 36 1 7 Total 155 4 12 Evaluation of the developed diagnostic PCR protocol on natural samples
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Amplification Molecular Methods + Specificity Rapid Automation - Detection Limit Robustness Acceptance Immunological methods Polymerase Chain Reaction
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Acknowledgements Waleed Abu Al-Soud, 2000 Ingrid Artin, --- Halfdan Grage, 2002 Oskar Hagberg, 2005 Rickard Knutsson, 2001 Charlotta Löfström, 2005 Maria Lövenklev, 2003 Petra Wolffs, 2004
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