PCR Based Methods for Successful Food Safety Testing from a...

Sample & Assay Technologies

1

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


4

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.


7

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


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

8


9

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


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

11

DNA purification with the


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

12

DNA purification with the


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

13

”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


16

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


QIAsymphony mericon Bacteria 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


PCR

Methods

Immuno-

based

Methods

Traditional

Culture

Methods


18



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


19

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


amplified DNA and

Internal Control

Detecting

bacteria

1 2

Homogenization

3

Enrichment culture

DNA extraction

4

Real-time PCR


20

reagents waste eluates

PCR reagents

PCR

output

QIAsymphony: Automated Pathogen Detection

DNA extraction PCR assay setup


21

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

11

Plastic consumables

Waste compartment

Cooled elution slot 1

Elution slots 2 – 4

5

6

7

8

Heated lysis station

Purification station

Robotic arm

9

10

11

Sample Input

Lysis

Purification

Elution


http://commons.wikimedia.org/wiki/Image:DIN_4844-2_Warnung_vor_heisser_Oberflaeche_D-W026.svg

http://commons.wikimedia.org/wiki/Image:DIN_4844-2_Warnung_vor_Kaelte_D-W017.svg


22

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

22

Sample transfer Sample processing PCR assay setup



23

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


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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


25

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


26

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


27

53 min 24 samples Batch 8


24 samples Batch 9


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



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

27

Pathogen Detection Workflow: Sample Throughput


29

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

29

Pathogen detection workflow: Salmonella Data


30

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





Increase

matrix per ml


31

10

12

14

16

18

20

22

24

26

28

30

32

34

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

Ct

Enrichment culture ratios [food : medium]

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


Milk Peanut butter

Minced beef Chocolate

Pathogen Detection Workflow: Salmonella Challenge Data


32

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



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


33

QS mericon protocol

10

12

14

16

18

20

22

24

26

28

30

32

34

36

38

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


34

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




Expected cell number Plate count

1000 cfu / 5 ml 1250 cfu / 5ml

Part 2: non-fat dry milk, 15 hours

B54


35

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


36

PCR curves for detection of Listeria monocytogenes DNA in various matrices

Internal Control

Smoked salmon

Internal Control

Cheese

Milk

Internal Control

36

Pathogen Detection Workflow: Listeria Data

Sprouts

Internal Control


37

Smoked salmon

Sprouts

10

14

18

22

26

30

34

38

42

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

Ct

Enrichment culture ratios [food - medium]

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

Pathogen Detection Workflow: Listeria Data

Sample & Assay Technologies Why Should I Perform QIAsymphony Extraction?

Presumed positive but confirmed negative (relative specificity)‏


Automated method has fewer PPNA (false positives) than the manual method

Better specificity due to removal of inhibitors with automated protocol


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


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]

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