PCR in Food Safety: Beyond Pathogens...

The world leader in serving science

Elisa Parrilla-Santos, Marketing Program Manager Iberia & Italy

PCR in Food Safety: Beyond Pathogens Detection

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• Introducción Thermo Fisher Scientific

• Food Safety Real-Time PCR Solutions

• Pathogens Detection• Thermo Scientific™ SureTect™ System

• Applied Biosystems™ MicroSEQ™ and RapidFinder™ Assays

• DuPont Bax® System

• Beyond Pathogens Detection:• Virulence factors: STEC

• Alicyclobacillus sp

• Speciation: meat, fish

• OGM Event Screening : Identification and Quantification

• Cost Efective Sample Preparation

Agenda

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• $20 billion in revenues• $700 million spent on R&D• 65,000 employees• 50 countries• 5 premier brands

The World Leader in Serving Science

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Analytical InstrumentsAnalytical Instruments Life Science SolutionsLife Science Solutions

Laboratory Products and Services

Laboratory Products and Services

Specialty DiagnosticsSpecialty Diagnostics

GlobalFilerPCR Amplification Kit

26%26%

18%18%

19%19%

37%37%

$20B Revenue

MassSpectrometry

Q-Exactive HF mass spectrometer

Chromatography

VanquishUltra HPLC

Chemical and Environmental Analysis

Gemini handheldanalyzer

ClinicalDiagnostics

Oral-Eze Oral Fluid Collection System Transplant

Diagnostics

NXType High Resolution Genotyping

ImmunoDiagnostics

ImmunoCAP Allergy and Asthma Tests

Microbiology

• Clinical Human & Veterinary Diagnostic

• Food Safety Solutions

QuantStudioDx R qPCR

Genetic Sciences

Human Identification

Sorvall WX+ Ultracentrifuge

Lab Equipment

Lab Consumables

E1 ClipTipPipette system

Distribution and BioPharma

Services

Chemicals

BioProduction

Cell Culture Reagents

Clinical Oncology

Next Gen Sequencing

Our Business Focus

Enterprise and Instrument Services

Biosciences

Life Science Reagents

Pathology

Rotary Microtome

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Validated pathogen detection

Detection of other pathogenic and non-pathogenic bacteria

Meat & others speciesID, screening, identification, quantification

GMO event, screening,

identification, quantification

Detection and semi-quantification of bovine

mastitis-causingorganisms

Thermo Scientific™ SureTect ™ Assays

Applied Biosystems™ TaqMan Assays

Applied Biosystems™ RapidFinder™-Imegen™ Assays

Applied Biosystems™ TaqMan™-Imegen™ Assays

Thermo Scientific™ PathoProof Assays

Applied Biosystems™ MicroSEQ™ and RapidFinder™ Assays

Thermo Scientific™ Rapid Finder™

DuPont BAX ®

System

DuPont BAX ®

System

Food Safety Real-Time PCR Solutions

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Food Safety Real-Time PCR Solutions: Pathogens Detection

Complete and Validate Solutions for Pathogens, Speciation and GMO in Food and Environmental samples

SureTect, MicroSEQ and BAX Real-Time PCR System

Assays Instruments Software

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Thermo Scientific™ PCR Systems: Main Features!!

• Reagents ready to use • Cutting edge technology in Master mix • PCR reagents liophilized in one tablet ready to

use• Multiparameter: Simultaneous analysis of

various pathogens• Internal positive control (IPC) integrated in all

PCR reaction tubes (ISO 22119)• Minimum time to manipulation• Validated Solutions

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Thermo Scientific™ SureTect™ System

Fast, convenient and user-friendly

DNA Amplification Result Interpretation

Sample Enrichment (8 – 24 hours) Cell Lysis & DNA Release (15 minutes)

80 min!

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SureTect Lysate stability

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Thermo Scientific™ SureTect™ System Assay contents:


Proteinase K

Lysis Tube Caps

PCR Tubes

Lysis Reagent 2 (Listeria kits only)

PCR Tube Caps

Pre-Filled Lysis Reagent 1

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Validations & Certifications

SureTect Assay workflow on 7500 Fast for detection of:

Meat, Dairy, Environmental, Powdered Infant Formula (25g)

Seafood and Vegetables

Meat, Dairy, Seafood, Vegetablesand Environmental samples

Meat, Dairy, Seafood, Vegetables and Environmental samples

25g Raw beef (25g, seasoned and unseasoned)

Powdered Infant Formula (10g)

Powdered Infant Formula (300g)

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Validations & Certifications

SureTect Assay workflow on 7500 Fast for detection of:

Raw ground beef, Pork frankfurters, Raw ground pork, 375g (1:5) ,Raw ground beef, Raw chicken breast, Non-fat milk powder, Pasteurized 2% milk, Cooked shrimp, Bagged lettuce, Cantaloupe melon, Bean sprouts, Stainless steel, Plastic surface, Chilled ready-to-eat dinner, Pasteurized liquid whole egg, Chilled pizza dough, Black pepper corns, Dog food, Cat food, Chocolate

Raw ground beef, Raw ground pork, Raw pork sausage, Pork frankfurters, Salami, Cooked slice ham, Raw ground turkey, Cooked sliced turkey, Fresh bagged spinach, Bagged lettuce, Cantaloupe, Pasteurized 2% milk, Pasteurized brie cheese, Processed cheese, Raw cod, Smoked salmon, Cooked prawns, Ice-cream, Stainless steel, Plastic

Raw ground beef, Raw ground pork, Raw pork sausage, Pork frankfurters, Salami, Cooked sliced ham, Raw ground turkey, Cooked sliced turkey, Fresh bagged spinach, Bagged lettuce, Cantaloupe, Pasteurized 2% milk, Pasteurized brie Cheese, Processed cheese, Ice-cream, Raw cod, Smoked salmon, Cooked prawns, Stainless steel and Plastic

Raw ground beef (1:4/1:5, 375g/325g), Raw beef trim (1:4/1:5, 375g/325g), Bagged spinach and Apple juice

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MicroSEQ™ and RapidFinder™ Assays

Assays Instrument Software

Complete and Validate Solutions for Pathogens, Speciation and GMO in Food and Environmental samples

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MicroSEQ Workflow with Manual Sample Preparation

Sample Enrichment Sample Preparation

Sample Preparation cont. (30-60 min) Sample Analysis and Reporting (40 min)

750uLsample

Microfuge (3 min)

Discardcolumn &

supernatant

50uL Lysis Buffer

Heat (10 min at 95ºC)

250uL Water

Microfuge (1 min)

30uLlysed

sample

+ Transfer to new tubefor some sample types

Vortex orMix with pipette

Using the PrepSEQ Rapid Spin Kit

40 min!!

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Preparación de muestras: Extracción de DNAKit MicroSEQ/RapidFinder approvals and validations

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MicroSEQ/RapidFinder approvals and validations

#061602

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• Taq Man Bacillus anthracis Detection kit

• Taq Man Brucella spp Detection kit

• Taq Man Francisella tularensis Detection kit

• Taq Man Yersinia pestis Detection Kit

Applied Biosystems™ - TaqMan Assays

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Food Safety Real-Time PCR Solutions

Complete and Validate Solutions for Pathogens and Hygienic indicators in Food and Environmental samples

BAX Real-Time PCR System

Assays Instruments Software

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BAX general workflow

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Bax® System

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Campylobacter case

http://ecdc.europa.eu/en/publications/publications/zoonoses-trends-sources-eu-summary-report-2014.pdf

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Campylobacter case

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Campylobacter case

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PCR quantitative

• BAX System Campylobacter – proposed protocol

25 g neck skins in 225mL BPW

2 min high speed

2 min ; 3000 g

1 ml

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PCR

• Sistema BAX Campylobacter

Add: • 20 µL sample• 200 µL lysis reagent

Incubation: •20 min 37ºC•10 min 95ºC

Cool

Transfer 30 µL to PCR tubes Load instrument

1,5 hours

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PCR• Sistema BAX Campylobacter• Software

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Technical Note

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• Detects and quantifies all three species in the same sample ( C. jejuni, C. coli and C. lari )

• Less than 90 minutes processing time

• Same-day results for highly contaminated samples

(direct protocol)

• Next day/two day results for samples requiring

enrichment.

• Results for enriched samples as soon as next day

• Validated on poultry carcass rinses and ready-to-eat products

• Level of detection down to 104 cfu/mL

• Excellent inclusivity/exclusivity

• Matrices : Poultry carcass rinses, sponges, ready to eat poultry products

Campylobacter Bax® System

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Campylobacter Detection: Validated Solution DuPont BAX® System

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Bax® System

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Agenda

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Virulence factors: STEC

Shiga toxin -producing E. coli

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Regulatory Landscape for STEC/VTEC in Europe (focus sprouts)• EU defines products at risk for STEC (CE 2073/2005):

• beef and food based on beef

• raw milk and products based on raw milk

• sprouted seeds; fruit juice and non pasteurized vegetables

�It ”aimed at reducing the faecal contamination along the food chain“ by using E. coli as indicator

• EFSA opinion in 2007 that O26, O111, O103, O145 and O157 are the strains of significance in Europe (90% cases)

• Outbreak in 2011 caused by E.coli 0104:H4 and has created lot of discussions at regulatory level

• EFSA added O104 to the list in May 2011

• ISO/TS 13136:2012

• EU released the EC209/2013 (amending CE 2073/2005) on the microbiological criteria for sprouted seeds

• sprouted seeds, producers and importers, need to test on EHECwhich EHEC big 6 and Ecoli 0104 and which samples: on sprouts (in process and lot release), seeds for sprout production, irrigation water for production

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Reported VTEC Cases in Humans, 2010-2014

Source: EFSA: The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2014

Increase in VTEC cases reported in Europe:- >5000 in 2014 - >3000 in 2010

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Scenario

http://www.foodqualitynews.com/Food-Outbreaks/Authorities-suspect-contaminated-frozen-items-for-E.-coli-cases

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Scenario

https://www.efsa.europa.eu/en/supporting/pub/1017e

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Scenario

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Scenario

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STEC strains(Shiga toxin producers)

Stx1 Stx2

(4 subtypes) (7 subtypes) STEC serotypes

Intimin protein(eae gene)

Severe disease*Enterohemorrhagic E. coli (EHEC)

Serotype O157:H7 = prototypic EHEC strain

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Collaboration with the USDA ARS

Thermo Fisher Scientific collaborates with the USDA Agricultural Research Service (ARS) to confirm assay designs against an extensive panel of pathogenic and non-pathogenic STEC of various O-group and origin, as well as non-E. coli organisms

The result is highly specific single target PCR ass ays, multiplexed to provide high levels of accuracy and high-

throughput screening capacity

Assay Inclusion Exclusion

O45 11/11 0/150

O121 10/10 0/151

O103 13/13 0/148

O145 10/10 0/151

O111 14/14 0/147

O26 17/17 0/144

stx and eae assays are designed to be inclusive

of all known allelic variants including

stx 2g and 2f

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Thermo Scientific™ SureTect™ E.coliO157:H7 Assay

Applied Biosystems™ MicroSEQ E.coli

O157:H7 Kit

Applied Biosystems™ RapidFinder STEC Detection System

DuPont BAX® E.coliO157:H7 Assay

DuPont BAX® Assay Suite for STEC

E.coli O157:H7 E.coli O157:H7 Screening Assay:-stx1/2-eae-O157:H7 indicator target

Confirmation Assays:-O157:H7 confirmation target- O45, O121- O26, O103, O145- O111, O104

E.coli O157:H7 STEC Screening Assay:-stx1/2-eae

STEC Panel 1 Assay:-O26-O111-O121

STEC Panel 2 Assay:-O45-O103-O145

Thermo Fisher Scientific PCR Solutions for STEC Detection

All our PCR Assays run on the Applied Biosystems™ 75 00 Fast Instrument*

* DuPont BAX Assays run on the DuPont BAX Q7 Real-Time PCR Instrument

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MicroSEQ Workflow Manual

Sample Enrichment Sample Preparation

Preparación de muestra (30-60 min) Análisis de muestra y resultados (40 min)

750uLmuestra

Microfuge (3 min)

Descartar columna &

sobrernadante

50uL Lisis Buffer

calentar(10 min a 95ºC)

250uL Water

Microfuga (1 min)

30uLmuestralisada

Vortex o Mezclar con pipeta

Utilizando el kit PrepSEQ Rapid Spin

40 min!!

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Dynabeads for Immunomagnetic Separation (IMS)• Original and most trusted name in IMS technology can

make your detection method simpler and more consistent

• Applied Biosystems™ Dynabeads™ reagents for E.coliO26, O103, O111, O45, O121 and O145 are listed in the USDA MLG 5B.01

• Dynabeads E.coli O157:H7 reagent is listed as an optimal method in numerous regulatory confirmation methods such as FDA-BAM, USDA MLG, ISO , Health Canada and Japanese Ministry of Health ( as well STEC Dynabeads)

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Agenda

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Alicyclobacillus: a Food Spoiling Organism

• Alicyclobacillus has been implicated in food spoilage• Fruit juice industry has acknowledged Alicyclobacillus spp. as a major quality

control target microorganism

• A. acidoterrestris: fruit juice industry• pear, orange, peach, mango, apple

and white grape juice, fruit juice blends

• A. acidocaldarius: tomato products

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Alicyclobacillus testing FROM today on !

Thermo Fisher Scientific offers a rapid method:

• Extraction: PrepSEQ™ Rapid Spin (Prot K)

• Detection: TaqMan™ real-time PCR basedcustom assays:

• A. acidoterrestris assay

• A. multiplex assay: • simultaneous detection of A. acidoterrestris and A. acidocaldarius

• workflow

• Fast detection, means in process testing and faster release of

• products, creating less economic loss

Classicalenrichment

PrepSEQ RapidSpin Extraction

(Prot K)

TaqMan® real-time PCR

detecton

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PrepSEQ™ Rapid Spin Kit protocol

Total time < 30 min

Current enrichmentin BAT 45°C

PrepSEQ Rapid Spin Extraction (Prot K)

TaqMan™ A. multiplex assay

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Agenda

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Speciation: meat, fish,...

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QualityAccreditations

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Food Fraud

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Food fraud exposed in 2013

Reputations and businesses damaged

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The food fraud iceberg

Horse dressed like a beef…

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Food Fraud

• Occurs frequently and often no detected

• Potencial for significant public health harm

• Can cause significant financial harm when uncovered

• Erodes consume confidence

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Top Foods & Ingredients for Fraud

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Asian Catfish Counterfeits Expanding Around the Globe

OCEANA | Deceptive Dishes: Seafood Swaps Found Worldwide. September 7, 2016

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Seafood fraud was investigated in 55 countries

OCEANA | Deceptive Dishes: Seafood Swaps Found Worldwide. September 7, 2016

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Milk and Milk-based Products

- Milk from bovine cows has been added to it other types of milk from sheep, buffalo and goat.

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Meat

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Why to perform species detection analysis?

Food fraud

Feed analysis

Cleaning and control surfaces

Halal and Kosher Food

Is my meat pure?

Is what is stated on the label OK (recipe)?

Is my food free of pork (halal/kosher)

Is the feed compliant with the EU Legislation?

Is my vegan product free of animal products?

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Workflow DNA extraction

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PCR Mitochondrial DNA

Sensitivity: It is 100 times greater using mitochondrial DNA targets than using nuclear DNA targets

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RapidFinderTM Animal Detection kits

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RapidFinderTM Animal Detection kits

Product name Part no. Volume Species name

GMO Extraction kit A15570 48 rxn Generic for Speciation & GMO

RapidFinder™ Beef ID kit A24391 48 rxn Bos taurus

RapidFinder™ Equine ID kit A15570 48 rxn Equus caballus

RapidFinder™ Pork ID kit A24392 48 rxn Sus scrofa

RapidFinder™ Chicken ID kit A24393 48 rxn Gallus gallus

RapidFinder™ Turkey ID kit A24394 48 rxn Meleagris gallopavo

RapidFinder™ Sheep ID kit A24395 48 rxn Ovis aries

RapidFinder™ Poultry ID kit A24397 48 rxn Several avian species, including Gallus gallus (chicken), Meleagris gallopavo (turkey), Anas platyrhynchos(duck), Struthio camelus (ostrich), and Anser (goose)

RapidFinder™ Ruminant ID kit A24396 48 rxn Bos taurus (beef), Ovis aries (sheep), Capra hircus(goat), Cervus elaphus (red deer), and Capreoluscapreolus (roe deer)

RapidFinder™ Fish ID kit A24398 48 rxn Atlantic Salmon, Angler, Cod, Hake, Halibut, Seabass, Anchovy, Sole, Dab, Trout, Tiger Shark, European Eel, Red Bandfish

RapidFinder™ Quant Multi Meat Set A24399 48 rxn

Optimized for Applied Biosystems® 7500 FAST Real-Time PCR Instrument

More predesigned assays available (Goat, Fallow dee r, Milk, Cat, Monkey, Rat…) and also Custom Assays are possible!

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Specificity

Animal Species used during the specificity assay for the RapidFinder TM Chicken ID Kit .

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Limit of Detection

Results corresponding to the PCR amplification of samples, which contain different percentages of chicken DNA

• Adding a greater amount of DNA (200ng/µl is recommended)

• Increase the PCR cycles at 38-40 and increase the Ct cut-off.

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Legislation: e.g. Rumiant DNA detection in feed

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RapidFinderTM Ruminant ID Kit

Determining the presence of DNA of ruminant ( Bos taurus, Ovis aries, Capra hircus, Cervus elaphus and Capreoluscapreolus ) in any feed and food.

The most important innovation included in the design of this kits are the IPC system, needed to detect false

negative result.

This kit can also detect the presence of DNA in Processed Ruminant Animal Protein (PAP).

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Legislation/Recommendation: Fraudulent practices in food

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Quantification of meat species DNA in food and feed samples

RapidFinder™ Quant Multi-Meat

• PCR detection of a highly conserved

mitochondrial genomic region from animal

species

• Multi-Meat Standard, a plasmid

• Relative quantification of 0.05% of specific

animal species DNA with respect to total animal

DNA in a sample

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Other Fish speciation Kits under IMEGEN Brand

Product Name Ref.

Imegen Gadus ID Kit (Gadus morhua and Gadus macrocephalus)IMG-221

Imegen Alaska Pollock ID Kit (Theragra chalcogramma) IMG-222

Imegen Pollock ID Kit (Pollachius pollachius) IMG-223

Imegen Haddock ID Kit (Melanogrammus aeglefinus) IMG-224

Imegen Basa ID Kit (Pangasius hypophthalmus) IMG-225

Imegen Tuna ID Kit (Thunnus) IMG-226

Imegen-Anchoas (Engraulis sp.) IMG-136

Imegen-Anchoas II (E. encrasicolus y E. japonicus) IMG-145

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Unique & reliable detection & quantification of species DNA

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• OGM Event Screening : Identification and Quantifica tion


Agenda

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Genetically Modified Organisms

• GMOs : “plants whose genome has been stably modified by genetic engineering in order to introduce one gene or one group of genes”

Usual Reasons:•Resistance to insects•Virus resistance•Delayed maturation•Nutritional features alteration

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• Detection of P35S promotor and TNOS terminator use in a large numberof GMO crops

• Relative quantification of 0,01% of P35S respect to total maize (MSS target)

Taq Man GMO eg Maize Quantification Kit

Promoter Coding region Terminator

TNOSP35

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GMO Extraction Kits (common for Speciation)

GMO Extraction Kit, 50 isolations• easy silica-based DNA purification method applicable on

DNA from a wide variety of food samples , like raw materials, processed food and feed – 2 hrs workflow

• High sample input: up to 20g of sample (homogenisesample)

• Safe: no toxic reagents

• Reliable: inhibitors removal, isolation of high pure DNA (A260/A280 > 1.8)

• Easy to use: minimal handling steps

• Automated or manual: adaptable to the needs of your lab and the matrices analysed

• Validated: extensive validation carried out on many matrices.

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• Extraction of DNA

• Creation layout of test samples

• Run on RT PCR

Process for Quantify GMO Maize: generic protocol

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Results

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Results

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Results

% P35= (copiesP35 /copies Maize )X100

%= 0,006

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TapManR GMO Screening, Detection & Quantification Kits

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GMO Testing – ThermoFisher Scientific Offering

GM events covered by ThermoFisher Scientific’s GMO S creening Kit

MON15985LLCotton25MON88913

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Agenda

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No

DN

A e

xtra

ctio

nCost Effective Sample Prep

SureTect

BAXSureTect 75

Bead/Retriever

•Legacy IMS platform promoted for food testing with culture for O157 & STEC

•Up to 15 samples

PrepSEQRapidSpin

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IMS capture (Pathatrix & Dynabead/BeadRetriever/MagMax)

Y Y

Culture

Molecular

wash away debrisIMS bugs of interest

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Pathatrix Auto – AFNOR Salmonella WorkflowS

ampl

e P

repa

ratio

nD

etec

tion

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Pathatrix is Flexible & Universal

Option 1:Individual

Option 2:5-Pool

Option 3:10-Pool

MaximizeSensitivity

Maximizethroughput

and reduced handling

Balance

PCR

ELISA

CULTURE

LATERALFLOW

Others Immunobased systems

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The future of Food Safety NOW!

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Let Us Help You Find a Better Solution

Work with your Thermo Fisher Scientific Food Safety Specialist to improve your workflow and expand your real-time PCR testing capability

http://www.thermofisher.com/foodmicrosolutions

PCR in Food Safety: Beyond Pathogens...

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