PCR-based tools for the detection of food-borne pathogens

PCR market PCR technology, revolution in microbiological analysis offering: Reliable results Greater efficiency and speed in obtaining the results Easy methodology Reproducible results Lower costs

PCR market In Europe, 2006 (R+D and clinical applications only): PCR reagents market: 624 M $ Thermal cycler market: 423 M $ Source: Frost & Sullivan, 2008 Areas of interest for pathogen detection: Source: adapted from Biosensors and Bioelectronics, Vol. 22, 1205-1217 (2007)

PCR market Growing interest in rapid methods based on PCR technology Source: ISI web of science 2007. Adapted from Biosensors and Bioelectronics, Vol. 22, 1205-1217 (2007)

PCR market Growing number of publications focused on pathogen detection based on PCR technology Source : ISI web of science 2007. Publications on pathogen detection methods over the last 20 years

PCR market CEN 2003 - Defines the following criteria for a standard PCR method: Analytic and diagnostic precision High detection probability Robustness Internal amplification control Low contamination risk Easy use and interpretation

Industry Problems Food Industry main problems in Quality Control are caused by Slowness in obtaining the results which causes a delay in the release of analyzed products Lack of personnel trained to perform PCR-based analyses Quality Control costs are high Due to the high number of hours of personnel required per analysis Due to long time to market product readiness Pathogens in food samples are usually hard to detect since they are Present at low levels, with accompanying bacterial flora Quiescent due to thermal, radiation, freezing treatments…

Industry Needs Food Industry requires Fast results Reliable and robust results Sensitive methods with high specificity Easy-to-perform analytical tools Access to PCR information and training

Solutions Culture & PCR: both based on exponential amplification: Culture PCR 1 2 3 4 2n 2n

Microbial Mission: Develop and produce bio-technological products to provide better, faster, easier, and cost-effective PCR analysis to industry

Microbial Solutions HIGHER SPECIFICITY AND SENSITIVITY No false negatives nor false positives Influence of accompanying bacterial flora is lower than in traditional culture or immunological systems Increased sensitivity with a standard pre-enrichment step

Microbial Solutions HIGHER THROUGHPUT Every reaction includes an Internal Amplification Control 1 reaction = 1 analysis No need of parallel control PCR reactions Different analysis in the sample plate are possible 96 well plate 94 sample analysis 1 Positive control 1 Non Template Control

Microbial Solutions EASY TO PERFORM METHODOLOGY Minimum number of steps Several pathogens analyzed in one thermocycler run SAMPLE ENRICHMENT DNA EXTRACTION REAL-TIME PCR Using Microbial DNAready kit or other systems Automation is possible A good purification of your DNA will avoid inhibitions!

Salmonella + Listeria monocytogenes Bactoplex (DNA extraction + PCR) Microbial Solutions LOWER COSTS: Price per analysis: Salmonella (ISO 6579:2002) and Listeria monocytogenes (ISO 11290:2004) versus Bactoplex (Salmonella + L. monocytogenes) Salmonella + Listeria monocytogenes Bactoplex (DNA extraction + PCR) Positive result aprox. 24 € aprox. 12,5 € Negative result aprox. 13 €

Salmonella + Listeria monocytogenes Bactoplex (DNA extraction + PCR) Microbial Solutions LOWER COSTS: Hours of a technician required per analysis: Salmonella (ISO 6579:2002) and Listeria monocytogenes (ISO 11290:2004) versus Bactoplex (Salmonella + L. monocytogenes) Salmonella + Listeria monocytogenes Bactoplex (DNA extraction + PCR) Positive result ~ 80 min ~30 min Negative result ~ 50 min

Microbial Solutions LOWER COSTS: Time to market product readiness: Culture (ISO) DNA extraction + PCR Positive result 5 days 19 hours Negative result 3 days

Microbial Products Soon available: Sample preparation Positive controls: Reference material for Real time PCR PCR Quantitation Standard Soon available:

Microbial Products Results in less than 4 hours after pre-enrichment in BPW Detection limit tested: 3ufc/l in water samples and 5ufc/25g in food With an internal amplification control to avoid false negative results 100% sensitivity and specificity 100% efficiency Validation against European Standard FOOD-PCR method in dairy products, eggs, raw and cured meat, poultry, raw and smoked fish, species, vegetables, fruits, and precooked dishes.

Listeria monocytogenes Microbial Products Determination of Listeria monocytogenes Results in less than 4 hours after pre-enrichment in Fraser Detection limit tested: 3-4 ufc/reaction With an internal amplification control to avoid false negative results 100% sensitivity and specificity 100% efficiency Validation in dairy products, eggs, raw and cured meat, poultry, raw fish, species, and precooked dishes.

Microbial Products Results in less than 4 hours after pre-enrichments in BPW and Fraser Detection limit tested: 3-6 ufc/25g for Salmonella, and 3-4 ufc/reaction for Listeria monocytogenes With an internal amplification control to avoid false negative results 100% sensitivity and specificity 100% efficiency Validation in dairy products, eggs, raw meat, poultry, raw vegetables, raw and smoked fish, and precooked dishes

Microbial Products Results in less than 4 hours Detection limit: 3-4 ufc/reaction With an internal amplification control to avoid false negative results 100% sensitivity and specificity (detects all 15 serogroups L. pneumophila) 100% efficiency Validation against ISO 11731 in water sample from showers, refrigeration towers, environmental outbreaks, and water supply systems.

Microbial Products Results in less than 4 hours Quantification limit: 1 ufc/reaction With an internal amplification control to avoid false negative results 100% sensitivity and specificity (detects all Legionella species) 100% efficiency SYBRGreen based detection, easy interpretation of the results by dissociation curves.

Microbial Products Results in less than 4 hours Detection limit: 1 ufc/reaction With an internal amplification control to avoid false negative results 100% sensitivity and specificity (detects all thermotolerant Campylobacter) 100% efficiency Validation against ISO 10272 in raw meat, poultry, fruits, vegetables, dairy products, raw fish, and others.

Microbial Products Results in less than 4 hours Detection limit: 1 ufc/reaction With an internal amplification control to avoid false negative results 100% sensitivity and specificity (detects all Cronobacter species) 100% efficiency Validation against ISO 22964 in powdered infant formula.

Microbial Products Results in less than 4 hours Detection limit: 1 ufc/reaction With an internal amplification control to avoid false negative results 100% sensitivity and specificity (detects the presence of all Salmonella and Cronobacter species) 100% efficiency Validation against ISO 6579:2002 and ISO 22964 in powdered infant formula

Microbial Products Fast DNA extraction Minimum number of steps Tested on dairy products, raw vegetables, eggs, fruits, fruit juices, precooked dishes, raw and cured meat, poultry, raw and smoked fish, ice- creams

Microbial technical product benefits OPEN KITS: Microbial products work with most of the thermocyclers in the market. LOWER COSTS: cheaper analysis, less technician’s hours, shorter time to market. AVOID RISKS: protect your brand with our carefully chosen oligonucleotides using scientific criteria: Highest stability / lowest variability Highest inclusivity and specificity Avoid false negative results Microbial Kits 27

Microbial support to implement PCR TURN-KEY SOLUTION We study your needs depending on the type and number of samples analyzed We propose the best solution to your needs We come to your lab and set-up your real-time PCR-based system We perform the first analysis with you in your lab We provide courses and training on PCR analysis We offer full support for any technical problem you may find

Microbial Microbial efforts are exclusively directed to research, development, and production of new tools for the detection of pathogenic bacteria by PCR Years of experience help in identifying the best genetic targets, and in the design of the appropriate molecular tools to detect them Quality is our main goal Our analysis systems are based on years of rigorous investigations, and on the application of the characteristics and conditions that a good molecular target must accomplish. This has lead us to present a product which goes a step forward in the detection of food-borne pathogens.

Microbial Official Supporters CDTi CIDEM PCiT de Girona AGAUR Universitat de Girona (UdG) Generalitat de Catalunya (Catalonia Government) Ministerio de Educación y Ciencia (Spanish Government) Ministerio de Industria, Turismo y Comercio (Spanish Government) 30

Microbial Certifications All our products have been validated against International Standard Procedures UNE EN ISO 9001:2008 (ENAC, UKAS) AFNOR certification (In progress) 31

Parc Científic i Tecnològic de Girona 32 Parc Científic i Tecnològic de Girona Pic de Peguera, 15. Edifici Jaume Casademont, E E-17003 Girona Spain Tel. +34 972 183236 Fax. +34 972 183236 www.microbial-systems.com info@microbial-systems.com