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