PCR-baserad diagnostik och toxinanalys Peter Rådström LTH, Lunds universitet Diagnostic PCR Amplification + Specificity Rapid Automation - Detection Limit Robustness No Isolate Neurotoxins Clostridium botulinum Mycotoxins Fusarium graminearum
PCR as a complement to culturebased methods Diagnostic PCR Why do we need new methods? *High specificity and accuracy (no false-negative/-positive results) *High Rapidity (at-line and on-line analysis) Amplification *Quantitative analysis (food spoilage micro-organisms) + Specificity Rapid Automation - Detection Limit Robustness No Isolate *Low detection limit (less than one pathogen per 5 gram) *Simplicity (user-friendly and automation) *High robustness (inter-lab reproducibility) *Acceptance (validation and standardisation) *Low cost (number of test) *Sample matrix flexibility (no interference)
Salmonella and natural feed samples Sample type No of samples No of positive samples NMKL PCR Faeces and intestines Fish meal 1 Maize gluten 1 1 Meat meal 1 Mixed feed Rape meal 8 Soya 59 3 3 Soya, acidified 36 1 7 Total 155 1
Expressionsanalys - toxinbildning DNA protein toxin mrna Milöfaktorer
Clostridium botulinum Clostridium botulinum beskrivs som en sporbildande och nervgiftproducerande bakterie Bakterien är vanligt förekommande i jord och sediment samt återfinns i tarmsystemet hos fiskar och djur
Neurotoxin Formation Relative expression and quantification of bontb (mrna) qrt-pcr (Real-Time PCR) Microarray BoNT/B production (protein) ELISA Biological activity of BoNT/B (active toxin) Mouse Bioassay
C. botulinum type B 3 1. 1 BoNT/B (ng/ml) 5 15 1 5 OD (6 nm) 1..8.6... 1 8 6 Relative expression -. Lövenklev M, Holst E, Borch E, Rådström P () Appl Environ Microbiol. 7:919-97 Lövenklev M, Artin I, Hagberg O, Borch E, Holst E, Rådström P () Appl Environ Microbiol. 7:98-93 5 1 15 5 3 35 5 5 55 h 5h h Time (h) <h
Livsmedelskontroll Konserverings medel CO (modifierad atmosfärförpackning) Effekt Hämmar bakteriens växt och sporbildning NaCl Hämmar bakteriens växt och sporbildning NaNO Hämmar bakteriens växt
Effect of NaNO Relative expression 1 1 1 8 6 * 7 ng*ml -1 *OD -1 5 3 1 OD (6 nm) 1% CO % NaCl ppm NaNO 1 3 Relative expression 1 1 1 8 6 Time (h) 3 ng*ml -1 *OD -1 * 5 3 1 OD (6 nm) 1% CO % NaCl 75 ppm NaNO 1 3 5 6 Time (h)
Effect of NaCl Relative expression Relative expression 1 5 * 1 1 7 ng*ml -1 *OD -1 8 3 6 1 1 3 1 1 1 8 6 Time (h) 7 ng*ml -1 *OD -1 1 3 Time (h) * 5 3 1 OD (6 nm) OD (6 nm) 1% CO % NaCl ppm NaNO 1% CO.5% NaCl ppm NaNO
Effect of CO Relative expression Relative expression 1 1 1 8 6 * 7 ng*ml -1 *OD -1 1 3 Time (h) 1 5 1 1 * 8 3 6 16 ng*ml -1 *OD -1 1 1 3 Time (h) 5 3 1 OD (6 nm) OD (6 nm) 1% CO % NaCl ppm NaNO 7% CO % NaCl ppm NaNO
OD (6 nm) OD (6 nm) 5 3 1 Effect of CO, NaCl and NaNO 5 1 15 5 3 35 5 3 1 7 ng*ml -1 *OD -1* Time (h) 15 ng*ml -1 *OD -1 Time (h) * 1 1 1 6 8 1 1 8 6 1 1 1 8 6 Relative expression Relative expression 1% CO % NaCl ppm NaNO 7% CO 1.5% NaCl 75 ppm NaNO
Matematisk behandling som visar hur de olika konserveringsmedlen påverkar bakteriens växt och nervgiftbildning LPD= m =b +b 13 *[CO ]*[NaNO ]+b *[NaCl] +e log(re)=b +b 11 *[CO ] +b *[NaCl] +e Lövenklev M, Rådström P () A novel approach to food safety. New Food 1:19- Lövenklev M, Holst E, Borch E, Rådström P () Appl Environ Microbiol. 7:919-97 Lövenklev M, Artin I, Hagberg O, Borch E, Holst E, Rådström P () Appl Environ Microbiol. 7:98-93
Molecular detection and monitoring methods for mycotoxigenic fungi 1. Analytical methods. Plate count methods 3. PCR. Real Time PCR (qpcr) 5. Microarray Detection of single gene fragments Analysis and detection of the expression of single genes Analysis of mycotoxin biosynthetic gene clusters
Methods for the analysis of mycotoxins in food samples analytical methods (HPLC, GC, MS, TLC) molecular methods (real time PCR, microarray) endpoint control monitoring of gene expression threshold value critical control points based on molecular data rejection of contaminated food prediction and prevention of contamination
Environmental parameter which influences expression of mycotoxin biosynthetic genes ph regulation catabolit repression regulatory gene gene cluster water activity a w ph temperature substrat inhibitory substances
General correlation between growth and mycotoxin biosynthesis 1 1 growth idiophase cfu/aflatoxin 1 1 1 trophophase expression data analytical data 1 1 aflatoxin [µg/g] mycotoxin biosynthesis Time [d] cfu/ml aflatoxin [µg/g] Rolf Geisen, Federal Research Centre for Nutrition and Food (BFEL), Germany
Fusarium graminearum H 3 C O 9 8 1 7 H H H O 1 11 3 13 O 6 5 1 CH 3 15 A B C H H Layout des Mycochip ( blau = Trichothecengene ) Monitoring der Expression Trichothecenbiosynthetischer Gene H CH 3 1 OOC CH CH CH 3 Allgemeine Grundstruktur der Trichothecene Technologie Lizenz-Buro (TLB)
MycoChip fumonisin genes control genes, positive patulin genes aflatoxin genes trichothecene genes control genes, negativ ochratoxin genes Technologie Lizenz-Buro (TLB)
Indicative expression pattern for trichothecene biosynthesis of Fusarium poae MycoChip expression profile MGA medium trichothecene production: not detectable maize medium trichothecene production: moderate YES medium trichothecene production: high similar expression pattern indicates active trichothecene biosynthesis, but can be determined before trichothecenes are produced in considerable amounts
History of development of molecular systems for mycotoxin producing fungi Microarray analysis A. flavus F. avenaceum F. cerealis F. sambucinum F. solani F. venenatum S. chartrarum A. alternata P. expansum F. verticillioides F. graminearum multiplex PCR Fusaria, Identification Aflatoxin, Fumonisin expression analysis Mycochip A. ochraceus (pks) 96 97 98 99 1 3 5 F. graminearum P. roqueforti A. flavus A. parasiticus A. versicolor F. avenaceum/tricinctum F. culmorum F. poae P. nordicum P. verrucosum A. carbonarius A. ochraceus P. expansum trichothecene chemotypes aflatoxins trichothecenes patulin ochratoxin PCR/multiplex PCR qpcr RT-qPCR microarray originally compiled by L. Niessen, 5