BAX® System Q7

1. BAX® System Q7 PCRmethod for detecting pathogens in food and environmental samples Amy B. Smith, Ph.D. – Technical Applications and Regulatory Support Specialist Shawn Gartside – Technical Sales Account Manager, Northeastern U.S. Marita Blackwell – Technical Support, Customer Service

2. -DuPont Qualicon Products-answering critical microbial questions Which organism is it? Have I seen it before?..where?..when? Where is it coming from? Is it there? RiboPrinter® System Bacterial Identification & Characterization BAX® System Pathogen Detection

3. DuPont Qualicon RiboPrinter® System Automated Bacterial Identification& Strain Level Characterization

4. An Identification System - and more • Identification capability • Characterization capability • Fully automated process • Results in 8 hours • Fully automated data analysis • no subjective data interpretation • flexible search, sort features • & reports • Electronic data sharing

5. BAX® System Advanced DNA-based detection of bacteria and other microbes in food and environmental samples. Provides food safety and food quality testing on the same platform.

6. BAX® System Start-Up Package • Everything you need to get started • Equipment • Heating blocks • Cooling blocks • Pipettes • Capping tools • Tube racks • Supplies (192 tests) • Pipette caps • Cluster tubes with caps • Nitrile gloves • Onsite training

7. BAX® Systems are easy to use. • Standard laboratory techniques require no advanced training • Tableted reagents minimize multiple transfers and calculations • Wizards and screen prompts lead you through the protocol • Closed-tube system minimizes amplicon contamination • Automated processing lets you load a rack and walk away • Electronic record of data provides LIMS-compatible storage, retrieval and printing

8. BAX® Systems are supported. • World-class customer service keeps your operation running smoothly. • Full one-year warranty on thermal cycler and computer • Extended service agreements available • Telephone helpline open 24/7 • Supported by product specialists worldwide

9. BAX® System PCR Assays for Screening Available today: • Salmonella • E. coli O157:H7 • E. coli O157:H7 MP (multiplex) • Genus Listeria • Listeria monocytogenes • Enterobacter sakazakii • Yeast and mold • Real-Time Campylobacter jejuni/coli/lari • Real-Time Staphylococcus aureus • Reverse-Transcriptase Listeria species • Each kit contains 96 tests: • PCR tubes with tablets • Optical caps • Lysis reagents

10. DuPont™ StatMedia™ Soluble Packets Enrichment media for Listeria • Just add packets to sterile water • and mix for instant enrichment media. Single, selective enrichment broth optimized for use with BAX® system assays to detect Listeria in food industry environmental samples. No autoclaving necessary.

11. BAX® Systems are quick. • Next-day results for most assays

12. BAX® Systems are flexible. Accommodates the workflow in your lab. • Up to 96 results per batch • Universal protocol allows you to “mix and match” many bacterial targets in the same batch • Facilitates high-volume workloads (200 or more tests per day) • New real-time assays can use probe-based chemistry for information beyond yes-or-no

13. BAX® System Approvals, Adoptions and Certifications • AOAC International Official Method • Salmonella #2003.09; L. Monocytogenes #2003.12 • AOAC-RI Performance Tested Method– includes Q7 • Salmonella #100201; Listeria monocytogenes #070202; E. Coli O157:H7 #010401; E. coli O157:H7 MP #050501; Genus Listeria #030502; Campylobacter jejuni/coli/lari; Listeria species-Reverse Transcriptase #030801 • USDA-FSIS Adoption • Salmonella #MLG 4C.00; Listeria monocytogenes #MLG 8A.00; E. coli O157:H7 and E. coli O157:NM #MLG 5A.00 – MP assay • USDA-APHIS NPIP Approval • Salmonella • Health Canada Certification– includes Q7 • Salmonella #MFLP-29; Listeria monocytogenes #MFLP-28; Genus Listeria #MFLP-15e; E. coli O157:H7 #MFLP-30; E. coli O157:H7 MP – Supp #2 to MFLP-30; Enterobacter sakazakii #MFLP-27 • AFNOR Certification– includes Q7 • Salmonella #QUA 18/3-11/02 • Danish Veterinary and Food Administration– includes Q7 • Salmonella • NordVal – includes Q7 • Salmonella • Brazil MAPA Official Reference Method • Salmonella MLG-4C.01; Listeria MLG-8A.01 • Japanese Ministry of Health, Labour and Welfare • Listeria; Listeria monocytogenes

14. Foods Tested with the BAX® System • Dairy • Meat • Seafood • Fruits/Vegetables/Salads • Grains • Herbs and spices • Chocolate • Pet food • Environmental samples • Other

15. BAX® System Assays Use Tableted PCR Reagents • Contain all the reagents needed for PCR • Taq polymerase • Nucleotides • Primers • Control reaction • Fluorescent dye • Packaged in PCR tubes • Provide for built-in process control • Eliminate multiple transfers • Reduce operator error • Include ingredients to overcome PCR inhibition • Closed-cap systems reduce potential for amplicon contamination • Three-year shelf life

16. BAX® System and PCR

17. 1. Prepare samples 2. Process samples 3. Review results BAX® System Protocol Requires 3 Major Steps On enriched/regrown or resuscitated samples…

18. Enrich food samples 8-48 hours • Follow standard protocols, such as BAM or ISO, for enriching the food type • Shorter enrichment times available with BAX® system proprietary media • E. coli O157:H7 MP • 8-20 hour broth for enriching raw beef samples • Listeria • Standard assay uses single 24-hour broth for enriching environmental samples • Reverse-transcriptase assay uses 4-hr cell resuscitation buffer • 24 hour enrichment for food samples – beta trials

19. Perform lysis • Mix lysis reagent and add to cluster tubes. • Add samples to cluster tubes. • Heat cluster tubes in two stages. • Transfer cluster tubes to cooling block. • 8-hour assays • Mix resuscitated sample with lysing agents, then heat. • Dilute lysate in dilution buffer.

20. Hydrate PCR tablets Arrange PCR tubes in cooling block Hydrate PCR tablets with lysate Secure optical caps

21. Load PCR tubes into the cycler

22. Technologies - BAX Q7 Assays • SYBR Green – intercalates to all double stranded DNA • Melting curve analysis • Computer algorithm provides + or – • Salmonella, E. coli O157:H7, Listeria Genus, L. mono, Y&M, E. sakazakii • Real Time PCR – uses labeled probes • 5 dye channels allow for a variety of probes to be mixed in a single reaction • Qualitative and quantitative • C. coli/jejuni/lari, S. aureus • Reverse Transcriptase (RevTran) – targets RNA, with SYBR green • No Enrichment!! • Ribosomal RNA is targeted • DNA is transcribed • Melting curve analysis (as above) • 8 hour Listeria spp

23. Review results for standard PCR assays • Negative • Positive • Indeterminate • Signal error • Note: Reverse-transcriptase assays also produce melting curves.

24. Review resultsUse well view • Click on a well to see individual result. Well 19 data

25. Review resultsReview melting curves Melting curves display peaks within a set temperature range for the control and target (if present). Strong positive result Target peak Positive control peak Negative result

26. Review resultsReview melting curves • Each target has a unique melting curve Salmonella positive Reverse-transcriptase Listeria species positive Genus Listeria positive L. monocytogenes positive E. coliO157:H7 positive E. coli O157:H7 MP positive E. sakazakii positive

27. Review results for real-time PCR assays • Negative • Positive • Indeterminate • Signal error

28. Intercalating dye bound to double-stranded DNA emits signal Unbound dye does not emit signal Real time assay: Intercalating dye chemistry “melts” fluorescent signal as temperature increases Increasing temperature Signal No Signal

29. Extension progresses – Probe is cleaved Extension complete – probe is removed Extension progresses – Probe is displaced Polymerization begins – Probe is intact R R Q R Target-specific Reporter dye Q Q Quencher dye 5’ 5’ 5’ 5’ 5’ 5’ Forward Primer 3’ 3’ 3’ Q R Probe 5’ 5’ 5’ 5’ 3’ 3’ 3’ 5’ 3’ 5’ 5’ 5’ 5’ 3’ 5’ Reverse Primer Reporter dye separates from Quencher dye and emits fluorescent signal Fluorescent signal intensity is proportional to amount of amplicon produced. Reporter dye begins to move away from Quencher dye Quencher dye greatly reduces fluorescent signal from Reporter dye Real time assay: Probe-based chemistry allows for multi-target detection in same sample.

30. Ribosomes with Ribosomal RNA Messenger RNA Chromosomal DNA Bacterial cell Small RNAs Multiple RNA copies for every one DNA copy, depending on the state of the cell Reverse-Transcriptase PCR

31. Reverse-Transcriptase PCR • Reverse Transcriptase (RT) synthesizes a complimentary DNA (cDNA) sequence from the RNA template • The reaction is heated to inactivate the RT and activate the Taq Polymerase • Primers bind to cDNA and Taq extends, forming double-stranded DNA. • The BAX® system standard PCR process then amplifies the newly synthesized double-stranded DNA.

32. BAX® System Q7 Thermal Cycler • Allows for new Q7 assays with advanced PCR capabilities • Flexible chemistry (probes or intercalating dye) • Up to five probes can be labeled in same reaction –allows for multiple targets in one sample • Faster processing time for new assays– 40 PCR cycles in about 90 minutes • With all the advantages of the original BAX® system cycler

33. Listeria Assays (BAX PCR) Current methods • Listeria genus single stage environmental • Next day results • Listeria genus single stage for product (24e) • Next day results • L. mono two stage food • Third day results • Rev Trans Listeria environmental • 8 hour result

34. Rev Tran details – video for workshop • Insert RevTran link here……. • ..\BAX docs\Listeria Rev Tran\8-Hour_listeria.exe

35. BAX® Systems for the Competitive Edge Get reliable DNA-based results Release product faster Reduce inventory costs Minimize pathogen risk The essential tool Improve micro response time Eliminate costly delays due to false positives for all your business needs Have more confidence in product safety Incorporate the best micro tools with HACCP program Be prepared for regulatory inspections