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Pharma Fit Detection of micro organism by BAX pcr Rolf Hugen 19 04 2007 Salmonella? E coli 0157 H7 ? Listeria monocytogenes ? Listeria spp Enterobacter sakazakii ? Das Bax PCR System wird für den Nachweis pathogener Bakterien aus Lebensmitteln und Umgebungsproben eingesetzt Validierte PCR Testkits stehen für den Nachweis von Salmonellen, Listeria monocytogenes, Listeria spp , E colie O157 H7 und Enterobacter sakazakii zur Verfügung 272 bin Structure of DNA Double string molecule of antiparallel.
Detection of microorganism by BAX-pcr Rolf Hugen 19-04-2007 Enterobacter sakazakii ? Salmonella? E coli 0157:H7 ? Listeria monocytogenes ? Listeria spp Structure of DNA Snyder, L and Champness, W., Molecular Genetics of Bacteria (Washington, D.C.: ASM Press, 1997) • Double string molecule of antiparallel ordered Polynucleotide chains • Adenine Thymine • Cytosine Guanine Opening of DNA (Denaturation) • neuen PCR_system Annealing of primer Chain extention DNA Polymerase DNA Polymerase PCR: DNA amplification Animation pcranimatie gif BAX-PCR Culture and PCR Sample Enrichment Lysation Amplification Detection Results Sample preparation steps • µl enrichment in 200 µl Lyses-buffer • Lyses step Salmonella Listeria 20´at 37°C 60´at 55°C stopping of the reaction at 95°C; then cooling • No further DNA operating steps • Pipetting 50àl in the PCR tubes ã Place the samples in BAX and start the program The BAX-PCR tube Content of the tubes: Primer Nucleotide Polymerase Fluorescing dye Supplements Internal control A few pipetting steps so little chance of contamination Melting point of DNA and fluorescents signal >temperature signal no signal DNA melt curve 1000 6000 5000 4000 3000 2000 1000 800 600 400 200 70 75 80 85 90 95 Bax detection 70 75 80 85 90 95 Transformed result Internal control Results Speed of the test BPW 1d ND d BAX 0,5 d BPW d ND d TSA d BAX-PCR 2,5 d TSA d API d FLMV 6d Choice of primer • PCR primer most be specific • Molecular typing methods were used to search for specific dna sequences • With the RiboPrinter the first steps were made for primer designing Typing of micro-organism by the RiboPrinter genetic „Fingerprint“ Characterising and identifying of micro-organism by analysing the rRNA gene Specificity of the primer for the Salmonella BAX PCR • • RiboPrinting of 1572 Salmonella strains Demonstrating of the specificity with: • 194 Salmonella strains • 35 strains of other species Specificity = 100% Specificity of the primer for the Listeria BAX PCR • • RiboPrinting of 1572 L monocytogenes strains Demonstrating of the specificity with • 196 L monocytogens strains • 78 strains of other species specificity = 100% AOAC certificate Certification Report AOAC Research Institute Performance Tested MethodSM 100201 ofSalmonella the method = Detection 99,4% BAXTMAccuracy System with Automated PCR Assay for Screening Salmonella manufactered by Qualicon, Icn AOAC certificate Certification Report AOAC Research Institute Performance Tested MethodSM 070202 BAXTMAccuracy System with Automated of the method =Detection 98% L monocytogens PCR Assay for Screening L monocytogenes manufactered by Qualicon, Icn International certificates AOAC International Official Method Salmonella #2003.09; L Monocytogenes #2003.12 AOAC-RI Performance Tested Method Salmonella #100201; Listeria monocytogenes #070202; E Coli O157:H7 #010401 USDA-FSIS Adoption Salmonella #MLG 4C.00; Listeria monocytogenes #MLG 8A.00 Health Canada Certification Salmonella #MFLP-29; Listeria monocytogenes #MFLP-28 E coli O157:H7 #MFLP-30; Enterobacter sakazakii #MFLP-27 AFNOR Certification Salmonella #QUA 18/3-11/02 NordVal Certification Salmonella #2003-20-5408 Brazil MAPA Official Reference Method Salmonella #41 Japanese Ministry of Health, Labour and Welfare Listeria; Listeria monocytogenes Questions? ... primer designing Typing of micro-organism by the RiboPrinter genetic „Fingerprint“ Characterising and identifying of micro-organism by analysing the rRNA gene Specificity of the primer for the... PCR • • RiboPrinting of 1572 Salmonella strains Demonstrating of the specificity with: • 194 Salmonella strains • 35 strains of other species Specificity = 100% Specificity of the primer for the... with Automated of the method =Detection 98% L monocytogens PCR Assay for Screening L monocytogenes manufactered by Qualicon, Icn International certificates AOAC International Official Method