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
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-DuPont Qualicon Products-answering critical microbial questions
BAX® SystemBAX® SystemPathogen DetectionPathogen Detection
RiboPrinter® SystemRiboPrinter® SystemBacterial Identification & CharacterizationBacterial Identification & Characterization
Is it there?
Which organism is it?Have I seen it before?..where?..when?Where is it coming from?
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DuPont Qualicon RiboPrinterDuPont Qualicon RiboPrinter®® System System Automated Bacterial Identification Automated Bacterial Identification
& Strain Level Characterization& Strain Level Characterization
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An Identification System - and more
Identification capabilityCharacterization capabilityFully automated processResults in 8 hoursFully automated data analysis•no subjective data interpretation
•flexible search, sort features & reports
Electronic data sharing
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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.
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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
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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
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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
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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
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DuPont™ StatMedia™ Soluble Packets
Just add packets to sterile water
and mix for instant enrichment media.
No autoclaving necessary.
Enrichment media for Listeria
Single, selective enrichment broth optimized foruse with BAX® system assays to detect Listeriain food industry environmental samples.
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BAX® Systems are quick.Next-day results for most assaysTarget 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 30 min 3.5 hr 18-28 hr
E. coli O157:H7 MP 8-24 hr 30 min 3.5 hr(<3 hr for MP Express)
11-28 hr
Listeria monocytogenes 30-50 hr 80 min 3.5 hr 34.5-64.5 hr
Genus Listeria 22-30 hr 80 min 3.5 hr 26.5-34.5 hr
Enterobacter sakazakii 24 hr 30 min 3.5 hr 28 hr
Yeast and Mold 44 hr 30 min 3.5 hr 48 hr
Real-Time C. jejuni/coli/lari
24-48 hr 30 min 90 min 26-50 hr
Real-Time S. aureus 20-22 hr 80 min 90 minutes 23-24 hr
Reverse-Transcriptase Listeria spp
4 hr to enrich RNA + resuscitate cells
30-35 min
<3 hr 8 hr
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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
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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
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Foods Tested with the BAX® System
Dairy
Meat
Seafood
Fruits/Vegetables/Salads
Grains
Herbs and spices
Chocolate
Pet food
Environmental samples
Other
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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
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BAX® System and PCR
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BAX® System Protocol Requires 3 Major Steps
On enriched/regrown or resuscitated samples…
1. Prepare samples 2. Process samples 3. Review results
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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
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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.
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Hydrate PCR tablets
Arrange PCR tubes in cooling block
Hydrate PCR tablets with lysate
Secure optical caps
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Load PCR tubes into the cycler
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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
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Review results for standard PCR assays
Negative
Positive
Indeterminate
Signal error
Note: Reverse-transcriptase assays also produce melting curves.
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Review results
Use well view
Click on a well to see individual result.
Well 19data
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Review results
Review melting curves
Melting curves display peaks within a set temperature range for the control and target (if present).
Strong positive result
Negative result
Target peak
Positive control peak
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Review results
Review melting curves
Each target has a unique melting curve
Salmonella positive
E. coli O157:H7 MP positive
L. monocytogenes positive
Genus Listeria positive
E. sakazakii positive
Reverse-transcriptase Listeria species positive
E. coli O157:H7 positive
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Review results for real-time PCR assays
Negative
Positive
Indeterminate
Signal error
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Real time assay: Intercalating dye chemistry “melts” fluorescent signal as temperature increases
No SignalSignalIncreasing temperature
Intercalating dye bound to double-stranded DNA emits signal
Unbound dye does not emit signal
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Real time assay: Probe-based chemistry allows for multi-target detection in same sample.
5’3’
5’5’3’
5’
ForwardPrimer
ReversePrimer
ProbeR Q
Target-specific Reporter dye Quencher dye
Quencher dye greatly reduces fluorescent signal from Reporter dye
Polymerization begins – Probe is intact
5’3’
5’5’3’
5’
R
Q
Extension progresses – Probe is displaced
Reporter dye begins to move away from Quencher dye
5’3’
5’5’3’
5’
R
Q
Extension progresses – Probe is cleaved
Reporter dye separates from Quencher dye and emits fluorescent signal
5’3’
5’5’3’
5’
R Q
Extension complete – probe is removed
Fluorescent signal intensity is proportional to amount of amplicon produced.
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Reverse-Transcriptase PCR
Chromosomal DNAMessenger RNARibosomes with Ribosomal RNA
Bacterial cell
Multiple RNA copies for every one DNA copy, depending on the state of the cell
Small RNAs
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Reverse-Transcriptase PCR
1. Reverse Transcriptase (RT) synthesizes a complimentary DNA (cDNA) sequence from the RNA template
2. The reaction is heated to inactivate the RT and activate the Taq Polymerase
3. Primers bind to cDNA and Taq extends, forming double-stranded DNA.
4. The BAX® system standard PCR process then amplifies the newly synthesized double-stranded DNA.
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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
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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
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Rev Tran details – video for workshop
Insert RevTran link here…….
..\BAX docs\Listeria Rev Tran\8-Hour_listeria.exe
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BAX® Systems for the Competitive Edge
Get reliable DNA-based resultsRelease product faster
Minimize pathogen risk
Eliminate costly delays due to false positives
Incorporate the best microtools with HACCP program
Be prepared for regulatory inspections
Have more confidence in product safety
Improve microresponse time
Reduce inventory costs
The essential tool
for all your business needs
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