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Rapid Pathogen Detectionusing Phage Technology
Dubai International Food Safety Conference
Workshop: Advancements in Microbiological Testing
Fabrice LESAULT – METERA Regional Business Director
February 28, 2011 – Dubai UAE
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Foodborne Pathogens risks
International regulations
International validations
Evolving solutions
Perspectives
Foodborne Pathogens risks
International regulations
International validations
Evolving solutions
Perspectives
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Microbiological risk (1/2)
Any food product is favourable to
a microorganism growth.
Sanitary risk for the consumer or the
patient
Salmonella in cooked meal No taste or smell modification
Foodborne outbreak
Risk of commercial quality deterioration by
microorganisms
Yeasts in fruit juice Turbidity, gaz, different taste
No danger for the consumer
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More ready-to-eat foods
Worldwide distribution
Mass production
Customers concentration
Risk of larger outbreaks
Microbiological risk (2/2)
Increase in the number of food poisoning cases
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Pathogens incidence worldwide
WHO data (per year and worldwide), 2006 data
2 billion of illnesses
1.8 million of deaths.
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Pathogens incidence USA
CDC data (per year and in USA), 2006 data
76 millions of foodborne illnesses,
300 000 hospitalizations,
5 000 deaths,
1 200 outbreaks.
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Pathogens incidence USA (2006)
1
10
100
1 000
10 000
100 000
1 000 000
10 000 000
nb of cases
nb of death
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Pathogens incidence Europe (2007)
1
10
100
1 000
10 000
100 000
1 000 000
nb of cases
nb of deaths
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Foodborne Pathogens risks
International regulations
International validations
Evolving solutions
Perspectives
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Due Diligence
“All food business operators are involved in food safety”
From stable To table
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North America
USA
USDA-FSIS
FDA-CFSAN
AOAC for Rapid Methods
International regulation
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Bacteriological Analytical Manual 7th Edition (1992) Provides quantitative and qualitative bacteriological
testing procedures for detecting microbiological contamination.
Chapter 4a : Diarrheagenic Escherichia coli
Chapter 5 : Salmonella
Chapter 10 : Listeria monocytogenes
Source : http://www.cfsan.fda.gov/~dms/guidance.html#proc
FDA Guidance document
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Europe
Commission regulation 2073/2005
12 articles
Based on HACCP program
Food safety criteria
Process hygiene criteria
International regulation
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n c m M1.4 Minced meat and meat preparations intended to be eaten raw
Salmonella
5 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
1.6 Minced meat and meat preparations made from other species than poultry meat intended to be eaten cooked
Salmonella
5 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
1.7 Mechanically separated Meat (MSM)
Salmonella5 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
1.8 Meat products intended to be eaten raw, excluding products where the manufacturing process or the composition of the product will eliminate the Salmonella risk
Salmonella
5 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
1.10 Gelatin and collagen Salmonella5 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
1.11 Cheeses, butter and cream made from raw milk or milk that has undergone a lower heat treatment than pasteurization
Salmonella
5 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
Absence in 25 g
Absence in 10 g
EN/ISO 6579
P roducts placed on the market during their shelf-life
Absence in 25 g (2010)
1.9 Meat products made from poultry meat intended to be eaten cooked
Absence in 10 g
Absence in 25 g
Absence in 10 g (2006)Salmonella
5 0
1.5 Minced meat and meat preparations made from poultry meat intended to be eaten cooked
Salmonella
Absence in 25 g (2010)
Food category Micro-organisms Sampling P lan Limits
5 0
Absence in 10 g
Absence in 10 g (2006)
Analytical reference method
Stage where the criterion applies
Absence in 25 g
P roducts placed on the market during their shelf-lifeEN/ISO 6579
Salmonella 1/2
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1.12 Milk powder and whey powder Salmonella5 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
1.13 Ice cream, excluding products where the manufacturing process or the composition of the product will eliminate the Salmonella risk
Salmonella
5 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
1.14 Egg products, excluding products where the manufacturing process or the composition of the product will eliminate the Salmonella risk
Salmonella
5 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
1.15 Ready-to-eat foods containing raw eggs, excluding products where the manufacturing process or the composition of the product will eliminate the Salmonella risk
Salmonella
5 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
1.16 Cooked crustaceans and molluscan shellfish
Salmonella5 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
1.17 Live bivalve molluscs and live echinoderms, tunicates and gastropods
Salmonella5 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
1.18 Sprouted seeds (ready-to-eat) Salmonella5 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
1.19 P re-cut fruit and vegetables (ready to eat)
Salmonella5 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
1.20 Unpasteurised fruit and vegetables juices (ready-to-eat)
Salmonella5 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
1.21 Dried Infant formula and dried dietary foods for special medical purposes intended for infants below 6 months of age.
Salmonella
30 0 EN/ISO 6579
P roducts placed on the market during their shelf-life
Absence in 25 g
Absence in 10 g
Absence in 25 g
Absence in 10 g
Absence in 25 g
Absence in 10 g
Absence in 10 g
Absence in 25 g
Absence in 10 g
Absence in 25 g
Salmonella 2/2
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n c m M
1.1 Ready-to-eat foods intended for infants and ready-to-eat foods for special medical purposes
Listeria monocytogenes
10 0 EN/ISO 11290-1
Products placed on the market during their shelf-life
5 0 EN/ISO 11290-2
Products placed on the market during their shelf-life
5 0 EN/ISO 11290-1
Before the food has left the immediate control of the food business operator, w ho has produced it
1.1 Ready-to-eat foods unable to support the grow th of L. monocytogenes , other than those intended for infants and for special medical purposes
Listeria monocytogenes
5 0 EN/ISO 11290-2
Products placed on the market during their shelf-life
100 CFU/g
100 CFU/g
Analytical reference method
Stage w here the criterionapplies
Absence in 25 g
1.1 Ready-to-eat foods able to support the grow th of L. monocytogenes , other than those intended for infants and for special medical purposes
Listeria monocytogenes
Absence in 25 g
Food category Micro-organisms Sampling Plan Limits
Listeria monocytogenes
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Conventional Methods = Reference Methods
Salmonella ISO 6579 MLG 4.04 BAM Chap.5
Listeria ISO 11290 MLG 8.06 BAM Chap.10
E. Coli O157:H7 ISO 16649 MLG 5.04 BAM Chap.4a
Conventional Methods 1/3
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Slow Results
Delays release finished products and ingredients
Delays response to data from environmental monitoring
programs
Aerobic Count = 72 hours
MPN of Coliform Bacteria = 72 hours
Listeria Neg= 4-5 days Pos= 5-7 days
Conventional Methods 2/3
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Inefficient
Laborious. Numerous supplies. High human cost.
Necessity for well-trained operators.
Very subjective results, depending on each operator’s competence. Many yield false positive and false negative results Large measurement uncertainty
Conventional Methods 3/3
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Large volume of product produced
Lack of space in warehouse
Short shelf-life
Fast sorting of raw material
Reliability and reproducibility of results
Why Rapid Methods?
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Article 5: Specific rules for testing and sampling
“The use of alternative method analytical methods is
acceptable when the methods are validated again the
reference method in Annex1 and if a proprietary method,
certified by a third party in accordance with the protocol set
out in EN/ISO standard 16140 or other internationally
accepted similar protocols, is used.”
EN ISO 16140-2003:
Food Microbiology : Protocol for validation of alternative method
Room for Alternative Methods
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Commercially available test kits
Salmonella : Any screening method under consideration for Salmonella testing must
meet or exceed the following performance characteristics: sensitivity > 97%, specificity >
90%, false negative rate < 3% and false-positive rate < 10%.
E. coli O157:H7: The screening test for the detection of E. coli O157:H7/NM should meet or exceed the following performance characteristics: sensitivity > 98%, specificity >
90%, false negative rate < 2% and false-positive rate < 10%.
L. monocytogenes : Any screening method under consideration for L.
monocytogenes testing must be validated for the intended use and must be at least as
sensitive as the culture method described in this procedure
Source : http://www.fsis.usda.gov/Science/Microbiological_Lab_Guidebook/index.asp
USDA-FSIS MLG
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Pre-enrichment
24 HRS
Identification
24 HRS
Detection
on selective media
24 – 48 HRS
Selective
Enrichment
24 HRS
Genotyping
Few hrs
Automated
Identification
Few hrs
Automated
Detection
1 HR
Selective
Enrichment
(optionnal)
24 HRS
Genotyping
Few hrs
Negative samples Positive samples
Mains steps for Pathogen detection
24 HRS
Pre-enrichment
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Conventionnal Methods vs Rapid Methods
VIDAS
BPWovernight @ 37±1°C
Selective Enrichment22-26 hrs @ 41.5±1°C
heating
VIDAS
BPWovernight @ 37±1°C
Selective Enrichment22-26 hrs @ 41.5±1°C
heatingheating
X g into 10X mL BPW
16-20 hrs @ 37±1°C
XLD
1 mL into 10 mL
MKTTn broth0.1 mL into 10 mL
Rappaport Vassiliadis Soya broth
reading
21-27hrs @ 41,5 ±1 °C 21-27 hrs @ 37±1 °C
Manufacturer’s recommendations21-27 hrs @ 37±1 °C
+XLD
+Other selective media Other selective media
Pick up 1 to 5 colonies
Biochemical confirmation Serological confirmation
Manufacturer’s recommendations
Nutrient agar
21-27 hrs @ 37±1 °C
X g into 10X mL BPW
16-20 hrs @ 37±1°C
XLD
1 mL into 10 mL
MKTTn broth0.1 mL into 10 mL
Rappaport Vassiliadis Soya broth
reading
21-27hrs @ 41,5 ±1 °C 21-27 hrs @ 37±1 °C
Manufacturer’s recommendations21-27 hrs @ 37±1 °C
+XLD
+Other selective media Other selective media
Pick up 1 to 5 colonies
Biochemical confirmation Serological confirmation
Manufacturer’s recommendations
Nutrient agar
21-27 hrs @ 37±1 °C
5 days 2 days
Salmonella Testing
VIDAS
Supplemented BPW24hrs @ 37±1°C
heating
VIDAS
Supplemented BPW24hrs @ 37±1°C
heatingheating
1 day
ISO 6579 Rapid Method
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Objectives
Shorter Time to results Increase Lab Efficiency/productivity Increase Reliability by
Objective results by automation Limited steps in protocols
Alternative Methods 1/2
Characteristics
Ready to use
Fast: 1 or 2 days results Validated Could be automated
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Alternative Methods 2/2
But also… Higher risk for Interference / inhibition matrix.
Need for International Validation
Request for Internal Evaluation
Screening Method in case of Qualitative Method (necessity for confirming positive presumptive)
No Method is Perfect or Absolute !!
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Foodborne Pathogens risks
International regulations
International validations
Evolving solutions
Perspectives
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Interest for faster and/or more practical (“alternative”) methods.
Offer of important, steady-developed, alternative methods
Field evaluations are costly, require high scientific competence and a lot of time
How to choose the suitable method?
Do all of them work well?
How to choose?
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By working at all steps of the analysis
Enrichment: balance of selectivity and fertility : media
optimised for a full solution Proprietary media, standard media used as enrichment
broths
Screening step: A balance between the enrichment and the
detection. Sensitivity and specificity
Immuno-assay
Chromogenic media
Molecular biology
Confirmation: selective media, latex, identification
Complete validated solution
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North America
USA
AOAC Official Method
AOAC Performance tested Method
Canada
Health Canada
International Validations
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Two Phases Validation Pre-Collaborative Study
Inclusivity and Exclusivity Method Comparison
• 20 foods• USDA or FDA reference methods
Collaborative Study Method Comparison
• 20 foods• USDA or FDA reference methods
Quantitative method: 8 laboratories minimum Qualitative method: 10 laboratories minimum
Official Method of Analysis
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Adopted as…
First Action Successful Collaborative Study In accordance with AOAC specifications Recommended by General Referee Approved by Methods Committee Published in Journal of AOAC Compiled in OMA
Final Action Approved methods eligible for final action after 2
years of availability to public
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OMA Approval Process - Overview
A p provedP re co lla b ora tive
P ro to co l
P re co lla b ora tive S tu dy
A p provedC o lla bo ra tive
P ro to co l
C o lla bo ra tiveS tu dy
F in a l A c tion
F irs t A c tion
C o lla bo ra tiveF in a l
R e p o rt
P re co lla b ora tive F in a lR e p o rt
S tu d y D ire c to r G e ne ra l R e fe ree
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Monitored by the Research InstitutePTM-Approval has gained wide acceptance in the
US, Europe, and globally.
Third Party validation Independent “single” Lab Validation Certification Mark Annual Review
Performance Tested Method (PTM)
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Two-part Validation – Internal Studies and Independent Study
One Independent Laboratory required – contracted by AOAC RI
Use AOAC, FDA, USDA, ISO, AFNOR or other official reference methods
Data review by two Expert Reviewers and General Referee
Validation Time: can be less than 6 months
Performance Tested Method (PTM)
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Internal Study Inclusivity Exclusivity Method Comparison
10 foods for “Variety of Foods” choice of reference methods
Ruggedness Stability Lot-to-Lot Variation
Independent Study Method Comparison
1 laboratory 1-2 foods
Performance Tested Method (PTM)
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List of PTMSM Approved Methods
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AOAC RI Certificate
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Europe
Article 5: Specific rules for testing and sampling
“The use of alternative method analytical
methods is acceptable when the methods
are validated again the reference method
in Annex1 and if a proprietary method,
certified by a third party in accordance with
the protocol set out in EN/ISO standard
16140 or other internationally accepted
similar protocols, is used.”
International Validations
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AFNOR (French Association of Normalization) MICROVAL (European Validation Association), and other European bodies participated in the development of the first ISO international standard for the validation of alternative microbiological methods.
EN/ISO 16140 : 2003
“Microbiology of food and animal feeding stuffs -
Protocol for the validation of alternative methods”
EN ISO 16140 1/2
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Publication date: May, 2003
Objective: Protocol for the validation of alternative methods applicable to food microbiology
AFNOR applies ISO 16140 since 2004
MicroVal applies ISO 16140 since 2006
EN ISO 16140 2/2
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It depends on the organism tested:
Salmonella EN ISO 6579: Microbiology of food and animal feeding stuffs – Horizontal
Method for the detection of Salmonella spp.
Listeria monocytogenes EN ISO 11290-1: Microbiology of food and animal feeding stuffs – Horizontal
Method for the detection of Listeria monocytogenes EN ISO 11290-2: Microbiology of food and animal feeding stuffs – Horizontal
Method for the enumeration of Listeria monocytogenes
E. coli O157 ISO 16654:2001: Microbiology of food and animal feeding stuffs – Horizontal
Method for the detection of Escherichia coli O157
Most Frequent Reference Methods used
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Preliminary Study Inclusivity and Exclusivity LOD50 (Relative detection limit)
Method Comparison Ease of Use
Collaborative Study Method Comparison
• 1 food, 8 replicates• 1 strain at 3 levels
10 laboratories minimum
Two Phases Validation
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Study Protocol Methods
Inclusivity study 50 pure positive strains Alternative method
Exclusivity study 30 pure negative strains Alternative method
Relative detection level
5 food products
5 positive strains
4 level of contamination
6 replicates/level
Alternative method
+
Reference Method
Comparative study
5 food categories
60 products per category
~ 50% of positive results
Alternative method
+
Reference Method
Interlaboratory study
10 labs without outliers
1 food products
1 positive strain
3 levels of contamination
8 replicates per level
Alternative method
+
Reference Method
Qualitative methods
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6 months
Expertise and comparison of method study + report
C
Analysis inter-lab study + report
2-3 months C5 weeks
Protocol Study
Expert Lab - Organizer
C
General Committee
Validation Process
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ISO 16140
All key performance criteria are specified
Renewed every 4 years Available on the AFNOR
website
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ISO 16140
All key detailled performance criteria are specified
Renewed once there is a change in the protocol
Available on the AFNOR website
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USA regulation Meat, Poultry, Eggs = USDA
Traditional Methods = MLG Rapid Methods = AOAC RI or OMA (comparing with MLG)
Others = FDA Traditional Methods = BAM Rapid Methods = AOAC RI or OMA (comparing with BAM)
Ease of Use
EU regulation Food Micro criteria = 2073/2005
Traditional Methods = ISO Methods Rapid Methods = ISO 16140
Summary
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Foodborne Pathogens risks
International regulations
International validations
Evolving solutions
Perspectives
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LSXNextDay
LMO2 SET2
Fab FragmentELFA
1992 1996
ImmunoConcentration
2002 2003 2004 2006 20091993
ECO LDUOListeria ICS+ SLM
SLMXLMX
2008
ECPT
Recombinant phage protein
2007
VIDASHeat & Go
to improve the reliability and the TTR of the solution
VIDAS…always evolving
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VIDAS Principle
SANDWICH TEST
A second antibody conjugated with an enzyme binds to specific antigens
DETECTION
The intensity of the reaction is interpreted by the system
370 nm 450 nm
CAPTURE OF ANTIGENS
The antibody captures
the target pathogens
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Portfolio
Salmonella
Listeria spp
Listeria monocytogenes
Escherichia coli 0157 (including H7)
Campylobacter
Staphyloccocal enterotoxins
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1992 1996 2002 2003 2004 2006
Listeria
20091993
ECO
20082007
Automated ELFA
An objective result with Ready to Use reagents
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1992 1996 2002 2003 2004 2006
ICS+ SLM
Listeria
20091993
ECO
20082007
Immuno Concentration
A faster result within 24 hours
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LMO2 SET2
Fab Fragment
1992 1996 2002 2003 2004 2006
ICS+ SLM
Listeria
20091993
ECO
20082007
Improved performanes
Antibodies fragment
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LMO2 LSXNextDay
SET2
1992 1996 2002 2003 2004 2006
LDUOICS+ SLM
Listeria
20091993
ECO
2008
ECPT
Recombinant phage protein
2007
VIDASHeat & Go
Most advanced technology for high performances
A new technology on VIDAS
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What’s a bacteriophage ?
Virus: A virus that only infects bacteria.
Very common: The most abundant form of life on earth.
Optimized by nature: Need for a specific host for its reproduction
From phage to VIDAS
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A new technology on VIDAS
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Raw beef25 gr.
in BPW
7-24 hrs
41.5 +/- 1 °C
VIDAS UP E. coli O157 VIDAS UP E. coli O157 including H7including H7
Heat & Go 5 min
A simple and rapid protocol…
Raw beef375 g
in BPW +/- vancomycin
8-24 hrs
41.5 +/- 1 °C
VIDAS UP E. coli O157 VIDAS UP E. coli O157 including H7including H7
Heat in boiling bath 5 min
50 min. 50 min.
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Internationally recognized and validated…
ISO 16140/AFNORISO 16140/AFNOR Certification in May 2009 for raw ground beef.
ISO 16140/AFNORISO 16140/AFNOR Certification in December 2009 for all food and environmental samples
AOAC RIAOAC RI validated in July 2009 for raw ground beef, beef trim, produce and irrigation water.
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LMO2 LSXNextDay
SET2
1992 1996 2002 2003 2004 2006
LDUOICS+ SLM
Listeria
2009
SLMXLMX
1993
ECO
2008
ECPT
2007
VIDASHeat & Go
One step protocol for Next Day results
…always evolving
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Raw beef and veal
Pasteurized eggs (liquid, powder…)
225 ml of BPW
Pre-warmed at 41.5 °C
16-24 hr
41.5°C +/- 1°C
45 min.
Heating Step 5 min*Except for pasteurized eggs
VIDAS SLMX protocol
Validated according to ISO 16 140 on raw beef & veal and pasteurized egg products. (BIO 12/26-07/09)
VIDAS SLMX
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VIDAS SLMX is certified by AFNOR Validation according to the ISO 16 140 norm on raw beef & veal and pasteurized egg products. (BIO 12/26-07/09)
Results from ISO 16 140 preliminary study
Reference Method
(ISO 6579)
Positive Negative
VIDAS SLMX
Positive 63 0
Negative 0 61
VIDAS SLMX performances
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225 ml LMX broth
+ 0.5 ml of LMX supplement
26-30 h
37°C +/- 1°C
5 min
VIDAS Listeria monocytogenes Xpress
Validated according to ISO 16 140 on human food and production environmental samples.
(BIO 12/27-02/10)
VIDAS LMX80 min.
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ALPALP
ALPALP
ALPALP
VIDAS LMX PRINCIPLE
AcM
SS
Streptavidin-PAL
biotin
Fab’
Specificity Sensitivity+
Next Day Results=
P60 protein
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Foodborne Pathogens risks
International regulations
International validations
Evolving solutions
Perspectives
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VIDAS UP Salmonella
VIDAS UP Listeria
Keep extending Phage technology
Simple and Rapid
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SimplifiedSimplified protocol for Next DayNext Day detection of Salmonella
Detection of both motile and non motile strains
VIDAS UP SALMONELLA
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41.5 +/- 1 °C
18-24 hrs
Heat & Go 5 min
Food & environment.
BPW + supplement
VIDAS UP SALMONELLA
VIDAS UP Salmonella UP Salmonella
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757 food products : Meat, poultry, vegetables, seafood, dairy products, egg products, confectionary, environmental samples from production area, feeds and pet foods.
657 negative products to evaluate the specificity of the method
100 positive products, 85 naturally contaminated and 15 artificially contaminated with stressed Salmonella (less than 10 cells/25g)
ISO 6579:2002 as the reference method
External study
VIDAS UP SALMONELLA
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ISO 6579
+ -
VIDAS SPT
+ 100 0
- 0 657 *
* 6 presumptive positive samples by the alternative method were negative after confirmation
Specificity of 98.8% Sensitivity 100%
VIDAS UP SALMONELLA
External study
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SimplifiedSimplified protocol for Next DayNext Day detection of Listeria species
Listeria
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SimplifiedSimplified protocol for Next DayNext Day detection of Listeria species
Detection protocols for Food samples and environmental samples
VIDAS UP LISTERIA SPP
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24-26 hrs
30 +/- 1 °C
Environmental samples
Heat & Go 5 min
Listeria broth + supplement
26-30 hrs
30 +/- 1 °C
Food
Heat & Go 5 min
Listeria broth + supplement
VIDAS UP LISTERIA SPP
VIDAS UP ListeriaVIDAS UP ListeriaVIDAS UP ListeriaVIDAS UP Listeria
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TAKE AVWAY
Reminder International regulations keep room for Rapid Method
International validations have strong process to assess rapid methods
Time to result can be shortened to less than 24 hours for Salmonella
Beyond TTR, Rapid Methods bring Ease of use w/ limited steps
Automation limits risk of error
Rapid Methods are evolving with state of the art technology
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THANK YOU
FOR YOUR ATTENTION