PCR Based Methods for Successful Food Safety Testing from a...
Transcript of PCR Based Methods for Successful Food Safety Testing from a...
Sample & Assay Technologies
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PCR Based Methods for Successful Food Safety Testing
from a Variety of Sample Matrices and Surfaces
Matt Kramer
Director, Commercial Operations
Applied Testing North America
QIAGEN Inc. USA
Sample & Assay Technologies PCR Based Methods for Successful Food Safety Testing
Title, Location, Date 2
Introduction to QIAGEN 1
mericon Identity & Authenticity Food Safety Solutions 2
mericon Pathogen Detection Food Safety Solutions 3
Summary and Questions 4
Sample & Assay Technologies QIAGEN: Full Circle – One Core Focus
3
Wide experience of Molecular Testing in many market areas
Pharmaceutical
Industry
Molecular
Diagnostics
Biosecurity Bio-defense
Human ID Forensics
Animal Health
Academic
Research
SAMPLE
Tech.
ASSAY
Tech.
Applied Testing
Food Safety
& Quality
Sample & Assay Technologies
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1 For more information on the adjusted figures, please refer to the reconciliation tables in QIAGEN’s Q4 and fiscal year 2011 earnings release.
2 Official guidance published on January 31, 2012.
> 500 consumable products to collect, separate,
purify, stabilize, store and amplify target analytes
in samples (DNA, RNA, proteins, etc.)
Instrumentation for above consumables
Molecular diagnostics and research test kits
>1060 issued patents
>1040 pending patents
Applied Testing (food testing, animal health,
forensic human ID, and biosecurity)
Molecular Diagnostics (hospitals, labs)
Pharma (pharmaceutical & biotech companies)
Academic Research
(academia, research institutes)
~ 3,900 employees worldwide
Product Range Intellectual Property (12/11)
> 500,000 Customers Employees (as of Dec. 31st, 2011)
QIAGEN at a Glance
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
Stockach Hombrechtikon
Germantown
Frederick Gaithersburg
Venlo
Shanghai
Shenzhen
Facilities
Shenzhen
Manchester
Marseille
Hilden
Operative HQ
Shanghai
Regional HQ Asia
5
Marseille
Over 35 sites in more than 20 countries
Over 60 sales offices WW
Offices are based in Brazil and Mexico
LATAM Distributors – Costa Rica, Argentina,
Uruguay, Paraguay, Chile, Peru, Ecuador,
Colombia, Venezuela, Panama.
Sample & Assay Technologies PCR Based Methods for Successful Food Safety Testing
Title, Location, Date 6
Introduction to QIAGEN 1
mericon Identity & Authenticity Food Safety Solutions 2
mericon Pathogen Detection Food Safety Solutions 3
Summary and Questions 4
Sample & Assay Technologies
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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 of
plant and animal DNA
Screening for
allergens
Specific detection
of DNA from allergens
The food safety testing market
Sample & Assay Technologies Sample and Assay Technologies
Complex
sample
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
Sample
Technologies
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
Pure
Analyte
DNA
Assay
Technologies
Target detected
Information
1 2 3 4
SAMPLE Technologies ASSAY Technologies
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Sample & Assay Technologies
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Au
tom
ation
C
onsu
mab
les
Sample disruption DNA purification PCR analysis
QIAgility
QIAcube
QIAsymphony Rotor-Gene Q
Tissue
Lyser LT
Tissue
Lyser II
Tissue
Ruptor
mericon DNA extraction portfolio
DNeasy mericon Food Kit
QIAsymphony mericon Bacteria kit
mericon DNA Bacteria Kit
mericon DNA Bacteria Plus Kit
mericon PCR Assays portfolio
Pathogen detection assays
Ingredient authentication
GMO detection
GMO quantification
QIAGEN mericon Food Portfolio – DNA ID & Authenticity
Sample & Assay Technologies mericon Real-time PCR Portfolio
Title, Location, Date 10
DNA Identity & Authenticity
Screen 35S-pat
Screen bar
Screen CTP2-CP4EPSPS
MON 810 Corn
RR Soy
Screen 35S
Screen Nos
Quant MON 810
Quant RR Soy
Horse
Cattle
Chicken
Pig
Turkey
Ruminants
Sheep
Goat
Apricot Kernels
Corn
Soy
GMO
detection
Animal ID
detection
Plant ID
detection
Sample & Assay Technologies GMO Detection Workflow
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DNA purification with the
DNeasy mericon Food Kit
GMO Detection with PCR
analysis using the mericon
GMO kits
Reliable and efficient
analysis of results
GMO
Positive control
Sample & Assay Technologies Ingredient Authentication Workflow
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DNA purification with the
DNeasy mericon Food Kit
Animal and Plant Ingredient
Identification with PCR
analysis using the mericon
Ingredient Authentication kits
Reliable and efficient
analysis of results
Plant or Animal
Positive control
Sample & Assay Technologies DNA Identity & Authenticity:QIAGEN’sCTAB Protocol
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”preparationDNeasy sample“toProtocolCTABtraditionalFrom
1st CHCl3 extraction
CTAB DNA precipitation
2nd CHCl3
extraction
Alcohol DNA precipitation
CTAB inhibitor precipitation CTAB inhibitor precipitation
CHCl3 extraction
Column purification
2.5 hours
(30 samples)
DNeasy mericon protocol
Sample & Assay Technologies RotorGene-Q Real-time PCR System
Rotary principle makes the difference
Samples spin continuously during a run
400 RPM (heating or cooling)
High-speed data collection
All samples read in one revolution (0.15 sec)
G-force keeps reagent at base of tube
Removes bubbles & condensation
Will not pellet components
Continuous movement means no variation
Highest well-to-well thermal and optical uniformity
400 RPM
>10 G
Sample & Assay Technologies PCR Based Methods for Successful Food Safety Testing
Title, Location, Date 15
Introduction to QIAGEN 1
mericon Identity & Authenticity Food Safety Solutions 2
mericon Pathogen Detection Food Safety Solutions 3
Summary and Questions 4
Sample & Assay Technologies
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Au
tom
ation
Consu
mab
les
Sample disruption DNA purification PCR analysis
QIAgility
QIAcube
QIAsymphony Rotor-Gene Q
Tissue
Lyser LT
Tissue
Lyser II
Tissue
Ruptor
mericon DNA extraction portfolio
DNeasy mericon Food Kit
QIAsymphony mericon Bacteria kit
mericon DNA Bacteria Kit
mericon DNA Bacteria Plus Kit
mericon PCR Assays portfolio
Pathogen detection assays
Ingredient authentication
GMO detection
GMO quantification
QIAGEN mericon Food Portfolio – Pathogen Surveillance
Sample & Assay Technologies Molecular Pathogen Testing from QIAGEN
mericon solutions provide fast, sensitive and highly specific results
Title, Location, Date 17
PCR
Methods
Immuno-
based
Methods
Traditional
Culture
Methods
Sample & Assay Technologies
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mericon DNA Bacteria Kit
mericon DNA Bacteria Plus Kit
QIAsymphony
mericon Bacteria Kit
Pathogen Detection Workflows
mericon Real-Time PCR Kits
Detection of
food-borne
pathogenic
bacteria
The mericon Pathogen Detection Workflow
mericon
Manual
workflow
Automated
workflow
QIA 36/01 – 02/13, with end of
validity: 01-Feb-2017
Sample & Assay Technologies
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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
Enriching
bacteria
Chemically aided thermal
lysis onboard at 90 °C
Inhibitor removal and
bind, wash, elute process
via magnetic beads
Lysing &
purify
bacteria
Amplification of bacterial
DNA using real-time cycler
Specific detection of
amplified DNA and
Internal Control
Detecting
bacteria
1 2
Homogenization
3
Enrichment culture
DNA extraction
4
Real-time PCR
Sample & Assay Technologies
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reagents waste eluates
PCR reagents
PCR
output
QIAsymphony: Automated Pathogen Detection
DNA extraction PCR assay setup
Sample & Assay Technologies
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Sample input
Reagent cartridges
Conductive filter-tips
TopElute Fluid
1
Sample Reagents and consumables Waste Elution
2
3
4
5 6
7
8
9 10
1
2
3
4
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Plastic consumables
Waste compartment
Cooled elution slot 1
Elution slots 2 – 4
5
6
7
8
Heated lysis station
Purification station
Robotic arm
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10
11
Sample Input
Lysis
Purification
Elution
QIAsymphony: Automated Pathogen Detection
Sample & Assay Technologies
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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
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Sample transfer Sample processing PCR assay setup
QIAsymphony: Automated Pathogen Detection
Sample & Assay Technologies
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QIAsymphony 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 Details
Sample & Assay Technologies
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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
Sample & Assay Technologies
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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
For internal use only
Protocol Design: Real-time PCR Detection
Sample & Assay Technologies
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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
Culture: 25 g food + 225 ml medium
Salmonella spp: Tested workflow parameters (AOAC approved)
External Certifications: AOAC Approved Workflow
Sample & Assay Technologies
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53 min 24 samples Batch 8
53 min 24 samples Batch 5
24 samples Batch 9
53 min 24 samples Batch 2
53 min
53 min
53 min
53 min
53 min
53 min
24 samples
24 samples
24 samples
24 samples
24 samples
Batch 4
Batch 3
Batch 1
Batch 7
Batch 6
PCR — 73 min PCR setup — 33 min
PCR — 73 min PCR setup — 33 min
PCR — 73 min PCR setup — 33 min
Sample throughput — full processing of 72 samples in 4.5 hours
Plastics refill 1
Plastics refill 2
Initial instrument setup
Results from
72 samples
in 4.5 hours
Results from
144 samples
in 7 hours
Results from
216
samples in
9.7 hours
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Pathogen Detection Workflow: Sample Throughput
Sample & Assay Technologies
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Milk Peanut butter
Minced beef Chocolate
PCR curves for detection of Salmonella spp. DNA in various matrices
Internal Control Internal Control
Internal Control Internal Control
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Pathogen detection workflow: Salmonella Data
Sample & Assay Technologies
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Pathogen detection workflow: Challenge Data
1:9
1:8
1:7
1:6
1:10
Standard 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
Increase
matrix per ml
Sample & Assay Technologies
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10
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1:6 1:7 1:8 1:9 1:10
Ct
Enrichment culture ratios [food : medium]
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1:6 1:7 1:8 1:9 1:10
Ct
Enrichment culture ratios [food : medium]
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1:6 1:7 1:8 1:9 1:10
Ct
Enrichment culture ratios [food : medium]
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1:6 1:7 1:8 1:9 1:10
Ct
Enrichment culture ratios [food : medium]
Milk Peanut butter
Minced beef Chocolate
Pathogen Detection Workflow: Salmonella Challenge Data
Sample & Assay Technologies
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Culture incubation time: 15 hours
Matrix: Hot dog juice
Cultures, each with 1, 10, 100, 1000 cfu/5 ml in 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 1475 cfu / 5ml
100 cfu / 5 ml 150 cfu / 5 ml
Part 1: Hot dog juice, 15 hours
B52
Sample & Assay Technologies
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QS mericon protocol
10
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unpooled pooled unpooled pooled unpooled pooled
30 ml 50 ml 100 ml
Culture volumes
Ct
1 cfu 10 cfu 100 cfu 1000 cfu
B52
QIAsymphony: Influence of 4-pooling of cultures with different medium volumes
General Ct level show no sensitivity problems for pooled samples
No significant titration of different inoculated cell numbers visible: cells have grown into plateau phase →
equilibrium between live and dead bacteria has been reached after 15 hours in small medium volumes
No inhibition in IC
Part 1: Hot dog juice, 15 hours
Sample & Assay Technologies
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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
Part 2: non-fat dry milk, 15 hours
B54
Sample & Assay Technologies
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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
Part 2: non-fat dry milk, 15 hours
B54
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
Sample & Assay Technologies
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PCR curves for detection of Listeria monocytogenes DNA in various matrices
Internal Control
Smoked salmon
Internal Control
Cheese
Milk
Internal Control
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Pathogen Detection Workflow: Listeria Data
Sprouts
Internal Control
Sample & Assay Technologies
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Smoked salmon
Sprouts
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14
18
22
26
30
34
38
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1:6 1:7 1:8 1:9 1:10
Ct
Enrichment culture ratios [food - medium]
10
14
18
22
26
30
34
38
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1:6 1:7 1:8 1:9 1:10
Ct
Enrichment culture ratios [food - medium]
10
14
18
22
26
30
34
38
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1:6 1:7 1:8 1:9 1:10
Ct
Enrichment culture ratios [food - medium]
10
14
18
22
26
30
34
38
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1:6 1:7 1:8 1:9 1:10
Ct
Enrichment culture ratios [food - medium]
Gouda cheese
Whole milk
Pathogen Detection Workflow: Listeria Data
Sample & Assay Technologies Why Should I Perform QIAsymphony Extraction?
Presumed positive but confirmed negative (relative specificity)
Title, Location, Date 38
Automated method has fewer PPNA (false positives) than the manual method
Better specificity due to removal of inhibitors with automated protocol
Sample & Assay Technologies PCR Based Methods for Successful Food Safety Testing
Title, Location, Date 39
Introduction to QIAGEN 1
mericon Identity & Authenticity Food Safety Solutions 2
mericon Pathogen Detection Food Safety Solutions 3
Summary and Questions 4
Sample & Assay Technologies
40
External Certifications: AOAC & AFNOR
Successfully finished studies
AOAC-RI: Salmonella spp (manual + automated)
AFNOR: Salmonella spp workflow (manual + automated)
Studies just started
AOAC-RI: Listeria spp workflow (manual + automated)
AOAC-RI: Listeria monocytogenes workflow (manual + automated)
AFNOR: Listeria spp workflow (manual + automated
AFNOR: Listeria monocytogenes workflow (manual + automated
AFNOR: Legionella spp and Legionella pneumophila (quantitative assays)
Planned extension of certification portfolio
Campylobacter
Certification roadmap
QIA 36/01 – 02/13, with end of validity:
01-Feb-2017
Sample & Assay Technologies mericon Automated Solutions - Benefits
Automated PCR-based solutions for pathogen screening in food safety testing 41
Increased lab productivity, efficiencies, process safety
Staff can multitask due to automation
No sample mix-ups and switches, bar code reading
One system for all pathogens, samples, medias
Rapid enrichment
QIAGEN Tech staff to support your internal validation
More sensitive and robust results
Removal of PCR inhibitors by DNA purification
No cross reactivity
Neutralizing buffers do not inhibit PCR
Cheaper
42% cost savings on traditional methods, VIDAS, etc.
75% cost savings on standard PCR methods
Sample & Assay Technologies mericon Automated Solutions - Benefits
Automated PCR-based solutions for pathogen screening in food safety testing 42
Enhance your ISO 17025 capabilities
Sample tracking with bar code reader
Output files and reports
Low cost to start
Reagent Rental
Capital purchases
Fast and Ease of use
Continuous, closed system
PCR Run time
Universal PCR cycling protocols
Standardization
Uniform extraction protocol for all pathogens
Harmonized PCR protocols
Automated data analysis
Sample & Assay Technologies PCR Based Methods for Successful Food Safety Testing
43
Thank you
Gracias
Matt Kramer
Director, Commercial Operations
Applied Testing North America
QIAGEN Inc. USA
Contact:
Cell: +1 703-623-0564
Email: [email protected]
Resource: