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Sample & Assay Technologies
PCR Based Methods for Successful Food Safety Testing from a Variety of Sample Matrices and Surfaces
Dr. Marcia ArmstrongDr. Marcia ArmstrongDr. Marcia ArmstrongDr. Marcia ArmstrongScientific Affairs ManagerFood Safety TestingGlobal Applied SciencesQIAGEN GmbH
QIAGEN - McGill Food Safety & Quality Program 1
Sample & Assay Technologies What is food safety?
Farm animal health � clean food
Testing to ensure safe food
Safe and unadulterated food for our plates
2QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
3
Food Supply Chain from Farm to Fork
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
� Image … Meaning reputation� Big international groups can‘t afford any mistake� In a world with 7 billion consumers and 1,5 billion food producers, no more than 500 companies
control 70 percent of food choice.
Among these companies, an elite group of ten food and beverage giants have consolidated immense market power. "The Big 10"
Collectively they generate revenue, more than $450 billion per year, equivalent to the GDP (Gross domestic product ) of the world's low-income countries combined
What drives the food testing market
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
� Regulation… Because they HAVE to…
1 Follow international and national guidances…
What drives the food testing market
Regulatory agencies
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
use the correct methods… based on official methods, like ISO or BAM, or based on alternative methods, such as EN ISO 16140 or AOAC
What drives the food testing market
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
8
Food safetytesting
Government / public labs•FDA•USDA•Federal institute food safety
Contract (private)
service labs*•Silliker•SGS•Eurofins
Food industry• Danone• Nestlé• Kraft
Food safety testing: Who does the testing?
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies Segments of the food safety testing market
Food-borne pathogen detection
� Specific detection of bacterial DNA
Testing for DNA from GMOs
� Qualitative and quantitative testing
Verifying ingredient authenticity
� Specific detection ofplant and animal DNA
Screening forallergens
� Specific detectionof DNA from allergens
The food safety testing marketThe food safety testing marketThe food safety testing marketThe food safety testing market
QIAGEN - McGill Food Safety & Quality Program 9
Sample & Assay Technologies The needs of food safety testing
10
� Pathogen detection � All major food pathogens, e.g., Salmonella spp., Listeria
spp., E. coli, Campylobacter
� GMO detection: Genetically modified organism � DNA introduced into plants for pesticide qualities or
herbicide resistance � In regulated countries various foods (e.g. plants, seeds,
and processed food products) are tested for GMOs (threshold for label declaration is 0.9%)
� Ingredient authentication � Testing for food alteration, dilution, fraud / exchange of
expensive raw materials with cheaper components� Usually no health threat, except
� Allergy (cow milk)� Animal protein in feed (since BSE)� Overpriced product� Wrong labels� Ethnic concerns (e.g. pork in halal food)
The globalized food market calls for more stringent safety and quality testing
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies Comparison of workflows for food pathogen detection
11
Real-time PCR
Immunoassay
Traditional culture
Real-time PCR workflow yields rapid, sensitive and highly specific results
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies QIAGEN: Full Circle – One Core Focus
Disseminating Technologies into Four Markets
12
Academia
Food TestingHuman ID &Forensics
Veterinary Testing
Applied Testing
SAMPLETech.
ASSAYTech.
Pharma
MolecularDiagnostics
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies QIAGEN at a Glance
13
Leading Provider of Sample & Assay Technologies
(1) Adjusted figures, excluding items such as restructuring and acquisition-related costs, share-based compensation and amortization of intangible assets. For more information on adjusted figures, please refer to the reconciliation tables in QIAGEN’s Q4 and fiscal year 2013 earnings release. (2) CER = at constant exchange rates. (3) As of January 1, 2014, QIAGEN has implemented two changes to its presentation of adjusted results. First, share-based compensation will be included as a cost in adjusted results. Furthermore, also as of January 1, 2014, costs for restructuring will only be adjusted for those related to business integration and acquisition-related activities. For further information on adjustments, please refer to the reconciliation tables in QIAGEN's Q4 2013 earnings release or appendices.
� >500 consumable products to collect, separate, purify, stabilize, store and amplify target analytes in samples(DNA, RNA, proteins, etc.)
� Bioinformatics software and instrumentation� Molecular diagnostics and research test kits
� >1,270 issued patents� >990 pending patents
� Molecular Diagnostics (hospitals, labs)� Pharma (pharmaceutical & biotech companies)� Academic Research
(academia, research institutes)� Applied Testing (vet., human ID, food testing, etc.)
� >4,000 employees worldwide
Outlook 2014 2013 2012 Change
Net Sales (m $) +4-5% CER² 1.306 1.255+4%
(+5% CER2)
Net. Income, adj.1 (m $) - 275,1 260,7 +6%
EPS, adj.1 ($) ~$1.07-1.093 $1.14 $1.08
Product RangeIntellectual Property (as of Dec 31, 2013)
> 500,000 Customers Employees (as of December 31, 2013)
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies Success Factor: Global Reach
Serving more than 500,000 customers worldwide
Hilden, Hamburg and Stockach
Hombrechtikon
“Gaitherstown”, Frederick
“Gaitherstown“Regional HQ Americas
Manchester Hilden
Hamburg
StockachHombrechtikon
Germantown
Frederick
Gaithersburg
Venlo
Shanghai
Shenzhen
R&D Facilities
Shenzhen
Manchester
Marseille
HildenOperative HQ
ShanghaiRegional HQ Asia
14
Marseille
Over 35 sites in more than 20 countries
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
15QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
16QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies Sample and Assay Technologies
Complexsample
Golgi apparatus, Glycoproteins, Microtubules, Mitochondria, Mitochondrial nucleic acids, Vacuoles, Talin, Nucleolus, Polymerases, Ceramides, Chromosomes, Chromatin, mRNA, Cytoplasm, Leucocytes, Sugars, Lipids, Salts, Urea, Carbonic acids, Cofactors, Precursors, Hemoglobins, Erythrocytes, Monocytes, Smooth endoplasmatic reticulum, Macrophages, Thrombocytes, Platelets, Lymphocytes, Basophils, Eosinophils, Neutrophils, Megacaryocytes, Plasma, Clotting factors, Actin, Microfilaments, Serum, Fibrin, Lysosomes, Ezrin, DNA, Hemaglobins, Heptaglobins, Transferrin, Fibrinogen, Serum albumin, tRNA, Salts, Polymerases, Centrioles, Immunoglobulins, DNA,, Cytokines, Angiotensins, Chemokines, Bradykines, Plasma membranes, Ribosomes, Actin, Vesicles, Complement components, Nuclei, Rough endoplasmatic reticulum, Nucleoli, Golgi apparatus, Glycoproteins, Microtubules, Mitochondria, Mitochondrial nucleic acids, Vacuoles, Talin, Nucleolus, Polymerases, Ceramides, Chromosomes, Chromatin, mRNA, Cytoplasm, Leucocytes, Sugars, Lipids, Salts, Urea, Carbonic acids, Cofactors, Precursors, Hemoglobins, Erythrocytes, Monocytes, Smooth endoplasmatic reticulum, Macrophages, Thrombocytes, Platelets, Lymphocytes, Basophils, Eosinophils, Neutrophils, Megacaryocytes, Plasma, Clotting factors, Actin, Microfilaments, Serum, Fibrin, Lysosomes, Ezrin, Hemaglobins, Heptaglobins, Transferrin, Fibrinogen, Serum albumin, tRNA, Carrier proteins
SampleTechnologies
Golgi apparatus, Glycoproteins, Microtubules, Mitochondria, Mitochondrial nucleic acids, Vacuoles, Talin, Nucleolus, Polymerases, Ceramides, Chromosomes, Chromatin, mRNA, Cytoplasm, Leucocytes, Sugars, Lipids, Salts, Urea, Carbonic acids, Cofactors, Precursors, Hemoglobins, Erythrocytes, Monocytes, Smooth endoplasmatic reticulum, Macrophages, Thrombocytes, Platelets, Lymphocytes, Basophils, Eosinophils, Neutrophils, Megacaryocytes, Plasma, Clotting factors, Actin, Microfilaments, Serum, Fibrin, Lysosomes, Ezrin, DNA, Hemaglobins, Heptaglobins, Transferrin, Fibrinogen, Serum albumin, tRNA, Salts, Polymerases, Centrioles, Immunoglobulins, DNA,, Cytokines, Angiotensins, Chemokines, Bradykines, Plasma membranes, Ribosomes, Actin, Vesicles, Complement components, Nuclei, Rough endoplasmatic reticulum, Nucleoli, Golgi apparatus, Glycoproteins, Microtubules, Mitochondria, Mitochondrial nucleic acids, Vacuoles, Talin, Nucleolus, Polymerases, Ceramides, Chromosomes, Chromatin, mRNA, Cytoplasm, Leucocytes, Sugars, Lipids, Salts, Urea, Carbonic acids, Cofactors, Precursors, Hemoglobins, Erythrocytes, Monocytes, Smooth endoplasmatic reticulum, Macrophages, Thrombocytes, Platelets, Lymphocytes, Basophils, Eosinophils, Neutrophils, Megacaryocytes, Plasma, Clotting factors, Actin, Microfilaments, Serum, Fibrin, Lysosomes, Ezrin, Hemaglobins, Heptaglobins, Transferrin, Fibrinogen, Serum albumin, tRNA, Carrier proteins
PureAnalyte
DNA
Assay Technologies
Target detected
Information
1 2 3 4
SAMPLE Technologies ASSAY Technologies
17QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies Sample and Assay Technologies
FoodSample/ Bacteriaenrichment
DNA exraction
PureDNA
DNA
Assay Technologies
Target detected
Information
1 2 3 4
SAMPLE Technologies ASSAY Technologies
18QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies Segments of the food safety testing market
Food-borne pathogen detection
� Specific detection of bacterial DNA
Testing for DNA from GMOs
� Qualitative and quantitative testing
Verifying ingredient authenticity
� Specific detection ofplant and animal DNA
Screening forallergens
� Specific detectionof DNA from allergens
The food safety testing marketThe food safety testing marketThe food safety testing marketThe food safety testing market
QIAGEN - McGill Food Safety & Quality Program 19
Sample & Assay Technologies
Automation
Consumables
Sample disruption DNA purification PCR analysis
QIAgility
QIAcube
QIAsymphony Rotor-Gene Q
TissueLyser LT
TissueLyser II
Tissue
Ruptor
mericon DNA extraction portfolio
� QIAsymphony mericon Bacteria kit� DNeasy mericon Food Kit
� mericon DNA Bacteria Kit
� mericon DNA Bacteria Plus Kit
mericon PCR Assays portfolio
� Pathogen detection assays
� Ingredient authentication
� GMO detection
� GMO quantification
The complete QIAGEN mericon Food Portfolio
QIAGEN - McGill Food Safety & Quality Program 20
Sample & Assay Technologies mericon assay portfolio overview
� Salmonella spp.� Listeria spp.� Listeria monocytogenes� Campylobacter spp.� Escherichia coli� Cronobacter spp.� Staphylococcus aureus� Quant Legionella spp.� Quant Legionella pneumophila� Shigella spp.� Yersinia enterocolitica� Vibrio triple� Coming soon!
�E. coli O:157 PLUS�O157:H7 and Big 6
Forpathogen detection
� Cattle� Ruminant� Chicken� Pig� Turkey� Sheep� Goat� Apricot Kernels� Corn� Soy
� Horse new
For ingredientauthentication
ForGMO
detection
mericon real-time PCR assay portfolio April 2013
�Screen 35S�Screen Nos�Quant MON 810�Quant RR Soy
�Screen 35S-pat�Screen bar�Screen CTP2-CP4EPSPS�MON 810 Corn�RR Soy
32 assays
21QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies GMO Detection workflow
22
DNA purification with the DNeasy mericon Food Kit
GMO Detection with PCR analysis using the mericonGMO kits
Reliable and efficientanalysis of results
� GMO
� Positive control
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies Ingredient Authentication workflow
23
DNA purification with the DNeasy mericon Food Kit
Animal and Plant Ingredient Identification with PCR analysis using the mericonIngredient Authentication kits
Reliable and efficientanalysis of results
� Plant or Animal
� Positive control
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies QIAGENs CTAB Protocol
24
From traditional CTAB Protocol to “DNeasy sample preparation”
1st CHCl3 extraction
CTAB DNA precipitation
2nd CHCl3extraction
Alcohol DNA precipitation
CTAB inhibitor precipitation CTAB inhibitor precipitation
CHCl3 extraction
Column purification
2.5 hours(30 samples)
DNeasy mericon protocol
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
mericon DNA Bacteria Kitmericon DNA Bacteria Plus Kit
QIAsymphony mericon Bacteria Kit
Pathogen Detection WorkflowsPathogen Detection WorkflowsPathogen Detection WorkflowsPathogen Detection Workflows
mericon Real-Time PCR Kits
Detection offood-borne pathogenic bacteria
The mericon Pathogen Detection Workflow
mericonmericonmericonmericon
Manual Manual Manual Manual workflowworkflowworkflowworkflow
Automated Automated Automated Automated workflowworkflowworkflowworkflow
QIAGEN - McGill Food Safety & Quality Program 25
Sample & Assay Technologies
Standardized, automated workflow
mericon pathogen detection workflow: automated solutions
26
One protocol & one workflow for all pathogens
Minimal hands on time; true walk-away capability
QIAGEN - McGill Food Safety & Quality Program 26
Sample & Assay Technologies Automated mericon pathogen detection workflows
� 25 g of food in 225 ml
of enrichment medium
� Homogenization in lab
pedal blender (stomacher)
Releasing bacteria
� Incubation of mixture at
raised temperature
� Loading of unprocessed
culture onto QIAsymphony
Enrichingbacteria
� Chemically aided thermal
lysis onboard at 90 °C
� Inhibitor removal and
bind, wash, elute process
via magnetic beads
Lysingbacteria
� Amplification of bacterial
DNA using real-time cycler
� Specific detection of
amplified DNA and
Internal Control
Detectingbacteria
1 2Homogenization
3
Enrichment culture
DNA extraction4
Real-time PCR
QIAGEN - McGill Food Safety & Quality Program 27
Sample & Assay Technologies
One system for sample preparation and PCR assay setup
QIAsymphony SP QIAsymphony AS
DNA extraction PCR assay setup
QIAsymphony: Automated pathogen detection
QIAGEN - McGill Food Safety & Quality Program 28
Sample & Assay Technologies
reagents waste eluatesPCR reagents
PCRoutput
QIAsymphony: Automated pathogen detection
DNA extraction PCR assay setup
QIAGEN - McGill Food Safety & Quality Program 29
Sample & Assay Technologies
Sample input
Reagent cartridges
Conductive filter-tips
TopElute Fluid
1111
Sample Reagents and consumables Waste Elution
2222
3333
4444
5555 6666
7777
8888
9999 10101010
1111
2222
3333
4444
11111111
Plastic consumables
Waste compartment
Cooled elution slot 1
Elution slots 2 – 4
5555
6666
7777
8888
Heated lysis station
Purification station
Robotic arm
9999
10101010
11111111
Sample Input
LysisPurification
Elution
QIAsymphony: Automated pathogen detection
QIAGEN - McGill Food Safety & Quality Program 30
Sample & Assay Technologies
� Continuous loading
� Various formats
� Bar code reading
� Liquid-level detection
� Conductive tips
� Transfer of 4 samples
� Thermal bacteria lysis
� Chemically supported lysis
� Magnetic bead purification
� Automated transfer
� Highly accurate
� Flexible formats
Sample loading
31
Sample transfer Sample processing PCR assay setup
QIAsymphony: Automated pathogen detection
QIAGEN - McGill Food Safety & Quality Program 31
Sample & Assay Technologies
Standardized workflow and process safety
From Sample to Result
High efficiency due to continuous sample loading - processing of 72 samples in 4,5 hours
Standardized workflows and process safety
One for all protocol
Minimal hands on time
32QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
DNA extraction protocols (QS SP Module)
mericon Bacteria 400
� Recommended default extraction protocol
� One-for-all protocol
� For Gram-negative & Gram-positive bacteria
� Tested with Salmonella and Listeria
Sample input volume 500 µl
Protocol run time (24 samples) 53 min
Elution volume 100 µl / 200 µl / 400 µl
Preferred input format 2 ml micro tube
Preferred output format EMTR
Software version 3.5 / 4.0
Protocol design: DNA extraction
QIAGEN - McGill Food Safety & Quality Program 33
Sample & Assay Technologies
PCR protocols on the QS AS Module
Currently featured targets Featured PCR output formats
� Salmonella spp
� Listeria monocytogenes
� Listeria spp
� Campylobacter triple (jejuni, lari, coli)
� Campylobacter spp
� Cronobacter spp
� Rotor-Disc (for 72 Rotor-Disc rotor)
� Rotor Strips (for 72 Rotor-Strips rotor)
� 96 well plate (for the ABI 7500 cycler)
Applicable PCR assay mericon PCR Detection Assays
Run times for maximal
sample number
� 72 Rotor Disc: 33 min
� 72 Rotor Strips: 27 min
� 96 Well Plate: 26 min
Software version 3.5 / 4.0
Protocol design: real-time PCR setup
QIAGEN - McGill Food Safety & Quality Program 34
Sample & Assay Technologies
PCR reaction step Temperature Time
Activation of the HotStar Taq Plus DNA polymerase
95 °C 5 min
Denaturation 95 °C 15 s
Annealing 60 °C 15 s
Extension 72 °C 10 s
PCR reaction step Temperature Time
Activation of the HotStar Taq Plus DNA polymerase
95 °C 5 min
Denaturation 95 °C 15 s
Annealing 60 °C 23 s
Extension 72 °C 10 s
� Harmonized cycling protocol
throughout the mericon portfolio
� Open system
� Number of cycles
� Pathogen detection: 40
� Ingredient authentication: 45
� Applied fluorescent probes
� Target
– FAM
– 495 / 520 nm (green)
� Internal Amplification Control
– MAX
– 524 / 557 (yellow)
– Equivalent to VIC / Hex
Cycling protocol for other real-time block cycler
Cycling protocol for the Rotor-Gene Q
Protocol design: real-time PCR detection
QIAGEN - McGill Food Safety & Quality Program 35
Sample & Assay Technologies Why validate?
� Are fit for purpose� Perform in a manner equivalent to standard methods� Have been developed and tested in the methods
developers labs� AND tested completely independently by an outside
expert laboratory� Methods may be microbiological, chemical,
analytical,,,,,� ����Methods stand up in court
� Lab adopting new method must perform internal lab VERIFICATION
36
To ensure that newanalytical methods
Caveat:
Because trust is the most important ingredient
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies Comparison of AOAC and AFNOR processes
37
AOAC AFNOR
Expert lab studies "Independent" "Preliminary"
inclusivity x x
exclusivity x x
ruggedness x x
lot to lot/stability x x
Matrix study design 5 - 10 foods 5 or more foods/ group
design neg, low and high levels contamination 30 neg, 30 pos per group
contamination artificial 15% natural, rest artificial
Matrices studied Chocolate dairy products
Ground beef 30% fat meat products
peanut butter seafood and vegetable products
spinach
various products (eggs, pastries, RTE
meals
chicken carcass rinses environmental samples
shell eggs animal feed
Milk, 3% fat
Non-fat, dry milk
Project presentation PTM certification Permission to go to ring trial
Ring trial (OMA certification) AFNOR certification
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies NF Validation: Relative Accuracy
Results - Automated Protocol
38QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies “False” Results
False negatives � negative test when pathogen actually present
False negative rates� Salmonella 5%� Campylobacter 9%
Failure to grow� Incubation time, temperature� Growth medium
� Campy higher false negative, harder to cultivate
Low level pathogen in sample � Pathogens below LOD for PCR testing� Heterogeneous distribution
Solutions:� Internal control to detect inhibition� Careful attention to growth conditions
False positives � positive test when pathogen absent
Generally found to be around 2 – 5%
Result in additional investigations� Confirmation of positive result� Discard /divert potentially contaminated food� Time consuming � cultures can take a week
� Food vs environmental positives
� Causes� Possibly from dead, non-viable/ non-
culturable cells� Poor primer/probe design? Cross reactivity� Presumptive positive (not confirmed) vs
false positive (carried to cultural confirmation)
39QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies QIAGEN workflow solutions for pathogen detection
Pathogen detection workflow
Detection of food-borne pathogenic
bacteria
Automated workflow
Manual workflow
mericon DNA Bacteria Kitmericon DNA Bacteria Plus Kit
QIAsymphony mericon Bacteria Kit
mericon PCR Detection Kits
40QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies External certifications: AOAC PTM & NF ISO 16140
� Successfully finished studies
� AOAC-RI: Salmonella spp (manual + automated)� AOAC-RI: Environmental surfaces extension� AFNOR: Salmonella spp workflow (manual + automated) All foods, animal
feed and environmental surfaces (except primary production)
� Studies finishing Q1 2014
� AOAC-RI: Listeria spp workflow (manual + automated)� AOAC-RI: Listeria monocytogenes workflow (manual + automated)
� Studies finishing 2014
� AFNOR: Listeria spp workflow (manual + automated)� AFNOR: Listeria monocytogenes workflow (manual + automated)� AFNOR : Legionella spp and L.pneumophila Quant
� Planned extension of certification portfolio
� Campylobacter� E. coli Screening O157 Plus� E. coli “Big Six”� Additional matrices for approvals already in hand
General certification roadmap
41QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
Peanut butter
Spinach
Eggs
Non-fat dry milk
Whole milk
Chicken carcass rinses
Milk chocolate
Ground beef (30% fat)
� Certified: Automated pathogen detection
QIAsymphony RG-Q + one-for-all protocol
� Certified: Manual pathogen detection
� Enrichment broth: Buffered Peptone Water
� Enrichment time: 18 ± 2 hours @ 37 ± 1oC
� Culture: 25 g food + 225 ml medium
Salmonella spp: Tested workflow parameters (AOAC approved)
External certifications: AOAC approved workflow
42QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies Salmonella spp: Tested workflow parameters
AOAC extension to environmental surfaces
Stainless steel
Sealed concrete
Plastic
Ceramic Tile
� Certified: Automated pathogen detection
QIAsymphony RG-Q + one-for-all protocol
� Certified: Manual pathogen detection
� Enrichment broth: Buffered Peptone Water
� Enrichment time: 18 ± 2 hours @ 37 ± 1oC
Culture: Swab in100 ml medium
Sponge in 250 ml medium
43QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
Smoked salmon
Sliced Turkey
Bean Sprouts
Gouda Cheese
Hot Dogs
Milk
�Automated pathogen detection
QIAsymphony RG-Q + one-for-all protocol
�Manual pathogen detection
� Enrichment broth: ONE Broth + supplement
� Enrichment time: 22 ± 2 hours @ 30 ± 1oC
� Culture: 25 g food + 225 ml medium
Listeria spp and L. monocytogenes: Tested workflow parameters
External certifications: AOAC validation studies
44QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies External certifications: AOAC validation studies
Listeria spp and L. monocytogenes – environmental surfaces
Stainless steel
Sealed concrete
Plastic
Ceramic Tile
� Certified: Automated pathogen detection
QIAsymphony RG-Q + one-for-all protocol
� Certified: Manual pathogen detection
� Enrichment broth: ONE Broth + supplement
� Enrichment time: 22 ± 2 hours @ 30 ± 1oC
Culture: Swab in100 ml medium
Sponge in 250 ml medium
45QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies AOAC-RI Performance TestedSM Status
46QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies NF Validation certification
47QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies Why validate?
� Are fit for purpose� Perform in a manner equivalent to standard methods� Have been developed and tested in the methods
developers labs� AND tested completely independently by an outside
expert laboratory� Methods may be microbiological, chemical,
analytical,,,,,� ����Methods stand up in court
� Lab adopting new method must perform internal lab VERIFICATION
48
To ensure that newanalytical methods
Caveat:
Because trust is the most important ingredient
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies Comparison of AOAC and AFNOR processes
49
AOAC AFNOR
Expert lab studies "Independent" "Preliminary"
inclusivity x x
exclusivity x x
ruggedness x x
lot to lot/stability x x
Matrix study design 5 - 10 foods 5 or more foods/ group
design neg, low and high levels contamination 30 neg, 30 pos per group
contamination artificial 15% natural, rest artificial
Matrices studied Chocolate dairy products
Ground beef 30% fat meat products
peanut butter seafood and vegetable products
spinach
various products (eggs, pastries, RTE
meals
chicken carcass rinses environmental samples
shell eggs animal feed
Milk, 3% fat
Non-fat, dry milk
Project presentation PTM certification Permission to go to ring trial
Ring trial (OMA certification) AFNOR certification
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies NF Validation: Relative Accuracy
Results - Automated Protocol
50QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies “False” Results
False negatives � negative test when pathogen actually present
False negative rates� Salmonella 5%� Campylobacter 9%
Failure to grow� Incubation time, temperature� Growth medium
� Campy higher false negative, harder to cultivate
Low level pathogen in sample � Pathogens below LOD for PCR testing� Heterogeneous distribution
Solutions:� Internal control to detect inhibition� Careful attention to growth conditions
False positives � positive test when pathogen absent
Generally found to be around 2 – 5%
Result in additional investigations� Confirmation of positive result� Discard /divert potentially contaminated food� Time consuming � cultures can take a week
� Food vs environmental positives
� Causes� Possibly from dead, non-viable/ non-
culturable cells� Poor primer/probe design? Cross reactivity� Presumptive positive (not confirmed) vs
false positive (carried to cultural confirmation)
51QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies Blu-V Viability PMA Kit & Blu-V System
52QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
Excess PMA in solution
++
+
+
+
+
Dead cell
Live cell +
+
+ + +
+
LIGHT
activation
+ + +
+ + +
Inactivated excess PMA
DNA with intercalated PMA
PMA-masked dead cell DNA
Propidium Monoazide: Mode of Action
Blu-V System
PMA cannot permeate intact membranes of living microorganisms and so downstream PCR is unaffected. Conversely, PMA is capable of permeating the membranes of dead microorganisms, followed by modification of DNA and inhibition of downstream PCR amplification.
53QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies Performance Data
PMA inhibits amplification of dead cell DNA.
� Various concentrations of dead bacteria were prepared. There is a distinct signal shift to higher CT values for the cells with PMA treatment compared to those without PMA treatment, with an average ∆ CT value of ~11.4.
54QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
One-for-all Protocol development
55QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies Pathogen detection workflow: Data
1:9
1:8
1:7
1:6
1:10Standard enrichment culture:
25 g food + 225 ml culture medium
25 g food + 200 ml culture medium
25 g food + 175 ml culture medium
25 g food + 150 ml culture medium
25 g food + 125 ml culture medium
Increasematrix per ml
QIAGEN - McGill Food Safety & Quality Program 56
Sample & Assay Technologies
MilkMilkMilkMilk Peanut butterPeanut butterPeanut butterPeanut butter
Minced beefMinced beefMinced beefMinced beef ChocolateChocolateChocolateChocolate
PCR curves for detection of Salmonella spp. DNA in various matrices
Internal ControlInternal ControlInternal ControlInternal Control Internal ControlInternal ControlInternal ControlInternal Control
Internal ControlInternal ControlInternal ControlInternal Control Internal ControlInternal ControlInternal ControlInternal Control
57
Pathogen detection workflow: Salmonella data
QIAGEN - McGill Food Safety & Quality Program 57
Sample & Assay Technologies
10
12
14
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18
20
22
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26
28
30
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34
1:6 1:7 1:8 1:9 1:10
Enrichment culture ratios [food : medium]
Ct
10
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14
16
18
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22
24
26
28
30
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34
1:6 1:7 1:8 1:9 1:10
Enrichment culture ratios [food : medium]
Ct
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1:6 1:7 1:8 1:9 1:10
Enrichment culture ratios [food : medium]
Ct
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1:6 1:7 1:8 1:9 1:10
Enrichment culture ratios [food : medium]
Ct
MilkMilkMilkMilk Peanut butterPeanut butterPeanut butterPeanut butter
Minced beefMinced beefMinced beefMinced beef ChocolateChocolateChocolateChocolate
Pathogen detection workflow: Salmonella data
QIAGEN - McGill Food Safety & Quality Program 58
Sample & Assay Technologies
PCR curves for detection of Listeria monocytogenes DNA in various matrices
Internal Control
Smoked salmon
Internal Control
Cheese
Milk
Internal Control
59
Pathogen detection workflow: Listeria data
Sprouts
Internal ControlInternal ControlInternal ControlInternal Control
QIAGEN - McGill Food Safety & Quality Program 59
Sample & Assay Technologies
Smoked salmon
Sprouts
Pathogen detection workflow: Listeria data
10
14
18
22
26
30
34
38
42
1:6 1:7 1:8 1:9 1:10
Enrichment culture ratios [food - medium]
Ct
10
14
18
22
26
30
34
38
42
1:6 1:7 1:8 1:9 1:10
Enrichment culture ratios [food - medium]
Ct
10
14
18
22
26
30
34
38
42
1:6 1:7 1:8 1:9 1:10
Enrichment culture ratios [food - medium]
Ct
10
14
18
22
26
30
34
38
42
1:6 1:7 1:8 1:9 1:10
Enrichment culture ratios [food - medium]
Ct
Gouda cheese
Whole milk
QIAGEN - McGill Food Safety & Quality Program 60
Sample & Assay Technologies
Pooling studies: Feasibility
61QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
Culture incubation time: 15 hours
Matrix: 10 % solution of non-fat dry milk
Cultures, each with 1, 10 100 1000 cfu/5 ml of test matrix
� 30 ml 15 ml BPW + 10 ml DE Buffer + 5 ml Hot Dog juice inoculum
� 50 ml 35 ml BPW + 10 ml DE Buffer + 5 ml Hot Dog juice inoculum
� 100 ml 85 ml BPW + 10 ml DE Buffer + 5 ml Hot Dog juice inoculum
Expected cell number Plate count
1000 cfu / 5 ml 1250 cfu / 5ml
Salmonella Culture Pooling : non-fat dry milk, 15 hours
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Sample & Assay Technologies
QS mericon protocol
10
12
14
16
18
20
22
24
26
28
unpooled pooled unpooled pooled unpooled pooled
30 ml 50 ml 100 ml
Culture volumes
Ct
1 cfu 10 cfu 100 cfu 1000 cfu
Salmonella Culture Pooling : non-fat dry milk, 15 hours
QIAsymphony: Influence of 4-pooling of cultures with different medium volumes
� General Ct level show no sensitivity problems for pooled samples
� Here titration of different inoculated cell numbers is visible: cells have not yet completely grown into
plateau phase
� No inhibition in IC channel
QIAGEN - McGill Food Safety & Quality Program 63
Sample & Assay Technologies
Manual unpooled - pooled Ct differences
0,00
0,50
1,00
1,50
2,00
2,50
3,00
3,50
4,00
4,50
5,00
30 ml 50 ml 100 ml
Ct
1 cfu 10 cfu 100 cfu 1000 cfu
QIAsymphony unpooled - pooled Ct differences
0,00
0,50
1,00
1,50
2,00
2,50
3,00
3,50
4,00
4,50
5,00
30 ml 50 ml 100 ml
Ct
1 cfu 10 cfu 100 cfu 1000 cfu
Salmonella Culture Pooling : non-fat dry milk, 15 hours
Ct differences between unpooled and
pooled samples
� Expected Ct difference:
2 Cts due to a 4-pooling
� QS results
� Very stable and homogeneous
� Only 30 ml culture has a little
variance
� Manual results:
� Less stable and more data
variance
� 100 ml culture seems most stable
QIAGEN - McGill Food Safety & Quality Program 64
Sample & Assay Technologies Food projects --
QIAxcel – Automated gel analysis � pathogen strain detection, endpoint multiplex PCR detection.
New protocols for DNA purification from food (for GMO, authenticity testing)
65QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies Values of QIAGEN solutions for food safety testing
66
The mericon sample and assay portfolio
� Complete Workflow solutions from sample management through sample analysis
� Broad coverage� Pathogens� GMOs� Food Authentication
� Harmonized cycling protocol within the mericon assay products for all segments – one workflow for all targets
� Sensitive RotorGene-Q real-time PCR system as backbone of performance
� Tailored automated solutions across the workflow� QIAsymphony RGQ� QIAcube, QIAgility
� External method validation: AOAC approval received for Salmonella workflow / AFNOR approval in process
QIAGEN - McGill Food Safety & Quality Program
Sample & Assay Technologies
Thank youThank youThank youThank you
Dr. Marcia ArmstrongDr. Marcia ArmstrongDr. Marcia ArmstrongDr. Marcia ArmstrongScientific Affairs ManagerFood Safety TestingGlobal Applied SciencesQIAGEN GmbH
Contact:Contact:Contact:Contact:Cell: +1 617 852 8131Email: marcia.armstrong@qiagen.com
QIAGEN - McGill Food Safety & Quality Program 67
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