PCR in Food Safety: Beyond Pathogens Detection

PCR in Food Safety: Beyond Pathogens Detection Elisa Parrilla-Santos, Marketing Programme Manager, Microbiology, Iberia & Italy The world leader in serving science

Agenda 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 Speciation: meat, fish OGM Event Screening : Identification and Quantification Cost Efective Sample Preparation 2

The World Leader in Serving Science $18 billion in revenues $700 million spent on R&D 50,000 employees 50 countries 5 premier brands 3

Food Safety Real-Time PCR Solutions Validated pathogen detection Detection of other pathogenic and nonpathogenic bacteria Meat species ID, screening, identification, quantification GMO event, screening, identification, quantification Detection and semiquantification of bovine mastitis-causing organisms 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 4

Food Safety Real-Time PCR Solutions: Pathogens Detection SureTect, MicroSEQ and BAX Real-Time PCR System Assays Instruments Software Complete and Validate Solutions for Pathogens, Speciation and GMO in Food and Environmental samples 5

Thermo Scientific SureTect System Sample Enrichment (8 24 hours) Cell Lysis & DNA Release (15 minutes) DNA Amplification Result Interpretation 80 min! Fast, convenient and user-friendly 6

SureTect System: Main Features ONE enrichment step for all matrix No DNA extraction. One unique lysis step Pre alicuoted reagents. Common to all assays Minimum time to manipulation (the most simple in the market) Cutting edge technology in Master mix PCR reagents liophilized in one tablet ready to use Not centrifugation Short protocol of thermocycling - 80 minutes and common to all assays Multiparameter: Simultaneous analysis of various pathogens Internal positive control (IPC) integrated in all PCR reaction tubes (ISO 22119) Results in less than 24 hours (11 hours for Salmonella and E coli O157:H7 in raw beef) 7

8 SureTect System: Main Features

9 SureTect Lysate Stability

SureTect System SureTect System Assay contents: Lysis Reagent 2 (Listeria kits only) Proteinase K PCR Tubes Pre-Filled Lysis Reagent 1 PCR Tube Caps Lysis Tube Caps 10

Validations & Certifications SureTect Assay workflow on 7500 Fast for detection of: Meat, Dairy, Meat, Dairy, Meat, Dairy, 25g Raw beef (25g, Powdered Infant Environmental, Seafood, Vegetables Seafood, Vegetables seasoned and Formula (10g) Powdered Infant and Environmental and Environmental unseasoned) Formula (25g) samples samples Early 2017: Seafood and Vegetables Early 2017: Powdered Infant Formula (300g) 11

Validations & Certifications SureTect Assay workflow on Applied Biosystems 7500 Fast System for detection of: Raw ground beef, Pork Raw ground beef, Raw Raw ground beef, Raw Raw ground beef (1:4/1:5, frankfurters, Raw ground ground pork, Raw pork ground pork, Raw pork 375g/325g), Raw beef trim pork, 375g (1:5),Raw sausage, Pork frankfurters, sausage, Pork frankfurters, (1:4/1:5, 375g/325g), ground beef, Raw chicken Salami, Cooked slice ham, Salami, Cooked sliced ham, Bagged spinach and Apple breast, Non-fat milk powder, Raw ground turkey, Cooked Raw ground turkey, Cooked juice Pasteurized 2% milk, sliced turkey, Fresh bagged sliced turkey, Fresh bagged Cooked shrimp, Bagged spinach, Bagged lettuce, spinach, Bagged lettuce, lettuce, Cantaloupe melon, Cantaloupe, Pasteurized 2% Cantaloupe, Pasteurized 2% Bean sprouts, Stainless milk, Pasteurized brie milk, Pasteurized brie steel, Plastic surface, cheese, Processed cheese, Cheese, Processed cheese, Chilled ready-to-eat dinner, Raw cod, Smoked salmon, Ice-cream, Raw cod, Pasteurized liquid whole Cooked prawns, Ice-cream, Smoked salmon, Cooked egg, Chilled pizza dough, Stainless steel, Plastic prawns, Stainless steel Black pepper corns, Dog and Plastic food, Cat food, Chocolate 12

Applied Biosystems MicroSEQ and RapidFinder Assays Assays Instrument Software Results 40 minutes Complete and Validate Solutions for Pathogens, Speciation and GMO in Food and Environmental samples 13

MicroSEQ Workflow with Manual Sample Preparation Using the PrepSEQ Rapid Spin Kit Sample Enrichment Sample Preparation 750uL sample Discard column & supernatant 50uL Lysis Buffer Microfuge (3 min) + Transfer to new tube for some sample types Vortex or Mix with pipette Sample Preparation cont. (30-60 min) Sample Analysis and Reporting (40 min) 250uL Water 30uL lysed sample 40 min!! Heat (10 min at 95ºC) Microfuge (1 min) 14

Preparación de muestras: Extracción de DNA Kit MicroSEQ/RapidFinder Approvals and Validations 15

MicroSEQ/RapidFinder Approvals and Validations #061602 16

Applied Biosystems TaqMan Assays Enterobacteriaceae 8-Genus (including: Citrobacter, Yokonella, Raoultella, Salmonella, Klebsiella, Escherichia, Shigella and Enterobacter) Enterococcus spp Escherichia coli spp Salmonella Heidelberg, Salmonella spp & Salmonella Enteritidis multiplex Salmonella Typhimurium Taq Man Bacillus anthracis Detection kit Taq Man Brucella spp Detection kit Taq Man Francisella tularensis Detection kit Taq Man Yersinia pestis Detection Kit 17

Food Safety Real-Time PCR Solutions DuPont BAX Real-Time PCR System Assays Instruments Software Complete and Validate Solutions for Pathogens and Hygienic indicators in Food and Environmental samples 18

DuPont Bax System 19

DuPont Bax System 20

Campylobacter Case http://ecdc.europa.eu/en/publications/publications/zoonoses-trends-sources-eu-summary-report-2014.pdf 21

Campylobacter Case 22

Campylobacter Case 23

Campylobacter Bax System 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 10 4 cfu/ml Excellent inclusivity/exclusivity Matrices: products Poultry carcass rinses, sponges, ready to eat poultry 24

Campylobacter Detection: Validated Solution DuPont BAX System 25

Virulence factors: STEC 27

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 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 EHEC which EHEC big 6 and Ecoli 0104 and which samples: on sprouts (in process and lot release), seeds for sprout production, irrigation water for production 28

Reported VTEC Cases in Humans, 2010-2014 Increase in VTEC cases reported in Europe: - >5000 in 2014 - >3000 in 2010 Source: EFSA: The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2014 29

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 30

Virulence Factors: STEC STEC strains (Shiga toxin producers) Stx1 STEC serotypes Stx2 (4 subtypes) (7 subtypes) Intimin protein (eae gene) Severe disease *Enterohemorrhagic E. coli (EHEC) Serotype O157:H7 = prototypic EHEC strain 31

Virulence Factors: STEC RapidFinder Screening Assay 32

Virulence Factors: STEC RapidFinder Confirmatory Assay 33

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 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 The result is highly specific single target PCR assays, multiplexed to provide high levels of accuracy and highthroughput screening capacity 34

Thermo Fisher Scientific PCR Solutions for STEC Detection Thermo Scientific SureTect E.coli O157:H7 Assay Applied Biosystems MicroSEQ E.coli O157:H7 Kit Applied Biosystems RapidFinder STEC Detection System E.coli O157:H7 E.coli O157:H7 Screening Assay: -stx1/2 -eae -O157:H7 indicator target Confirmation Assay: -O157:H7 confirmation target -any of O26, O45, O103, O111, O121, O145 DuPont BAX E.coli O157:H7 Assay E.coli O157:H7 DuPont BAX Assay Suite for STEC STEC Screening Assay: -stx1/2 -eae STEC Panel 1 Assay: -O26 -O111 -O121 STEC Panel 2 Assay: -O45 -O103 -O45 All our PCR Assays run on the Applied Biosystems 7500 Fast Instrument* * DuPont BAX Assays run on the DuPont BAX Q7 Real-Time PCR Instrument 35

Speciation: Meat, Fish,... 37

Quality Accreditations 38

Food Fraud 39

Food Fraud Exposed in 2013 Reputations and businesses damaged 40

The Food Fraud Iceberg Horse dressed like a beef 41

Food Fraud Occurs frequently and often no detected Potencial for significant public health harm Can cause significant financial harm when uncovered Erodes consume confidence 42

Top Foods & Ingredients for Fraud 43

Asian Catfish Counterfeits Expanding Around the Globe OCEANA Deceptive Dishes: Seafood Swaps Found Worldwide. September 7, 2016 44

Seafood Fraud was Investigated in 55 Countries OCEANA Deceptive Dishes: Seafood Swaps Found Worldwide. September 7, 2016 45

Milk and Milk-based Products - Milk from bovine cows has been added to it other types of milk from sheep, buffalo and goat. 46

Meat 47

Why Perform Species Detection Analysis? Feed analysis Food fraud Is the feed compliant with the EU Legislation? Is what is stated on the label OK (recipe)? Is my meat pure? Cleaning and control surfaces Halal and Kosher Food Is my food free of pork (halal/kosher) Is my vegan product free of animal products? 48

Workflow DNA Extraction 49

PCR Mitochondrial DNA Sensitivity: It is 100 times greater using mitochondrial DNA targets than using nuclear DNA targets 50

51 RapidFinder TM Animal Detection Kits

RapidFinder TM 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 Capreolus capreolus (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 deer, Milk, Cat, Monkey, Rat ) and also Custom Assays are possible! 52

Specificity Animal Species used during the specificity assay for the RapidFinder TM Chicken ID Kit. 53

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. 54

Legislation: e.g. Ruminant DNA Detection in Feed 55

RapidFinder TM Ruminant ID Kit Determining the presence of DNA of ruminant ( Bos taurus, Ovis aries, Capra hircus, Cervus elaphus and Capreolus capreolus ) in any feed and food. This kit can also detect the presence of DNA in Processed Ruminant Animal Protein (PAP). The most important innovation included in the design of this kits are the IPC system, needed to detect false negative result. 56

Legislation/Recommendation: Fraudulent Practices in Food 57

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 58

Other Fish Speciation Kits under IMEGEN Brand Product Name Imegen Gadus ID Kit (Gadus morhua and Gadus macrocephalus) Imegen Alaska Pollock ID Kit (Theragra chalcogramma) Imegen Pollock ID Kit (Pollachius pollachius) Imegen Haddock ID Kit (Melanogrammus aeglefinus) Imegen Basa ID Kit (Pangasius hypophthalmus) Imegen Tuna ID Kit (Thunnus) Imegen-Anchoas (Engraulis sp.) Imegen-Anchoas II (E. encrasicolus y E. japonicus) Ref. IMG-221 IMG-222 IMG-223 IMG-224 IMG-225 IMG-226 IMG-136 IMG-145 59

Unique & Reliable Detection & Quantification of Species DNA 60

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 62

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 (homogenise sample) 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. 63

TapMan GMO Screening, Detection & Quantification Kits 64

Cost Effective Sample Prep No DNA extraction SureTect SureTect 75 BAX PrepSEQ RapidSpin Bead/Retriever Legacy IMS platform promoted for food testing with culture for O157 & STEC Up to 15 samples 66

IMS Capture (Pathatrix & Dynabead/BeadRetriever/MagMax) IMS bugs of interest Y Y wash away debris Culture Molecular 67

Pathatrix Auto AFNOR Salmonella Workflow Detection Sample Preparation 68

Pathatrix is Flexible & Universal Option 1: Individual Maximize Sensitivity PCR ELISA Option 2: 5-Pool Option 3: 10-Pool Balance Maximize throughput and reduced handling Others Immuno based systems LATERAL FLOW CULTURE 69

The Future of Food Safety NOW 70

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 71