1. PCR method for detecting pathogens in food and environmental samples
BAX® System Q7
PCR method 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?
BAX® System
Pathogen Detection
RiboPrinter® System
Bacterial Identification & Characterization

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 Target
Enrichment
Lysis
PCR
Total Time to Result
Salmonella
20-26 hr* (some need extra 3 hr regrowth)
30 min
3.5 hr
24-33 hr
E. coli O157:H7
14-24 hr
18-28 hr
E. coli O157:H7 MP
8-24 hr
3.5 hr (<3 hr for MP Express)
11-28 hr
Listeria monocytogenes
30-50 hr
80 min hr
Genus Listeria
22-30 hr hr
Enterobacter sakazakii
24 hr
28 hr
Yeast and Mold
44 hr
48 hr
Real-Time C. jejuni/coli/lari
24-48 hr
90 min
26-50 hr
Real-Time S. aureus
20-22 hr
90 minutes
23-24 hr
Reverse-Transcriptase Listeria spp
4 hr to enrich RNA + resuscitate cells
30-35 min
<3 hr
8 hr

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 # ; L. Monocytogenes #
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
NordVal – includes Q7
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. BAX® System Protocol Requires 3 Major Steps
On enriched/regrown or resuscitated samples…
1. Prepare samples
2. Process samples
3. Review results

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 results Use well view
Click on a well to see individual result.
Well 19
data

25. Review results Review 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 results Review melting curves
Each target has a unique melting curve
Salmonella positive
Genus Listeria positive
Reverse-transcriptase Listeria species positive
L. monocytogenes positive
E. coli O157: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. Real time assay: Intercalating dye chemistry “melts” fluorescent signal as temperature increases
Intercalating dye bound to double-stranded DNA emits signal
Unbound dye does not emit signal
Increasing temperature
Signal
No Signal

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

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

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)
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