Sample & Assay Technologies - McGill University€¦ · (3) As of January 1, 2014, QIAGEN has...

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


3

Food Supply Chain from Farm to Fork

QIAGEN - McGill Food Safety & Quality Program


� 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



� Regulation… Because they HAVE to…

1 Follow international and national guidances…


Regulatory agencies



use the correct methods… based on official methods, like ISO or BAM, or based on alternative methods, such as EN ISO 16140 or AOAC




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?


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


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


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


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


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)


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


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


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


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



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


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


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


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


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



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



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


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



One system for sample preparation and PCR assay setup

QIAsymphony SP QIAsymphony AS

DNA extraction PCR assay setup

QIAsymphony: Automated pathogen detection



reagents waste eluatesPCR reagents

PCRoutput


DNA extraction PCR assay setup



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




� 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




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



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



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



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


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


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


Sample & Assay Technologies NF Validation: Relative Accuracy

Results - Automated Protocol


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)


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


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



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


Sample & Assay Technologies Salmonella spp: Tested workflow parameters

AOAC extension to environmental surfaces

Stainless steel

Sealed concrete

Plastic

Ceramic Tile




� Enrichment broth: Buffered Peptone Water


Culture: Swab in100 ml medium

Sponge in 250 ml medium



Smoked salmon

Sliced Turkey

Bean Sprouts

Gouda Cheese

Hot Dogs

Milk

�Automated pathogen detection


�Manual pathogen detection

� Enrichment broth: ONE Broth + supplement


� Culture: 25 g food + 225 ml medium

Listeria spp and L. monocytogenes: Tested workflow parameters

External certifications: AOAC validation studies


Sample & Assay Technologies External certifications: AOAC validation studies

Listeria spp and L. monocytogenes – environmental surfaces

Stainless steel

Sealed concrete

Plastic

Ceramic Tile




� Enrichment broth: ONE Broth + supplement


Culture: Swab in100 ml medium

Sponge in 250 ml medium


Sample & Assay Technologies AOAC-RI Performance TestedSM Status


Sample & Assay Technologies NF Validation certification


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


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


Sample & Assay Technologies NF Validation: Relative Accuracy

Results - Automated Protocol


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)


Sample & Assay Technologies Blu-V Viability PMA Kit & Blu-V System



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.


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.



One-for-all Protocol development


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





Increasematrix per ml



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



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12

14

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20

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30

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34

1:6 1:7 1:8 1:9 1:10

Enrichment culture ratios [food : medium]

Ct

10

12

14

16

18

20

22

24

26

28

30

32

34

1:6 1:7 1:8 1:9 1:10


Ct

10

12

14

16

18

20

22

24

26

28

30

32

34

1:6 1:7 1:8 1:9 1:10


Ct

10

12

14

16

18

20

22

24

26

28

30

32

34

1:6 1:7 1:8 1:9 1:10


Ct

MilkMilkMilkMilk Peanut butterPeanut butterPeanut butterPeanut butter

Minced beefMinced beefMinced beefMinced beef ChocolateChocolateChocolateChocolate

Pathogen detection workflow: Salmonella data



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



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


Ct

10

14

18

22

26

30

34

38

42

1:6 1:7 1:8 1:9 1:10


Ct

10

14

18

22

26

30

34

38

42

1:6 1:7 1:8 1:9 1:10


Ct

Gouda cheese

Whole milk



Pooling studies: Feasibility



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



Expected cell number Plate count

1000 cfu / 5 ml 1250 cfu / 5ml

Salmonella Culture Pooling : non-fat dry milk, 15 hours



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


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



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


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



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


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)


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



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: [email protected]


Sample & Assay Technologies - McGill University€¦ · (3) As of January 1, 2014, QIAGEN has...

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Transcript of Sample & Assay Technologies - McGill University€¦ · (3) As of January 1, 2014, QIAGEN has...