1 rapid pathogen detection using phage technology dubai international food safety conference...

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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




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

4

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

5

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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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


1.7 Mechanically separated Meat (MSM)

Salmonella5 0 EN/ISO 6579


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


1.10 Gelatin and collagen Salmonella5 0 EN/ISO 6579


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


Absence in 25 g

Absence in 10 g

EN/ISO 6579


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


Food category Micro-organisms Sampling P lan Limits

5 0

Absence in 10 g


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


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


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


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


1.16 Cooked crustaceans and molluscan shellfish



1.17 Live bivalve molluscs and live echinoderms, tunicates and gastropods



1.18 Sprouted seeds (ready-to-eat) Salmonella5 0 EN/ISO 6579


1.19 P re-cut fruit and vegetables (ready to eat)



1.20 Unpasteurised fruit and vegetables juices (ready-to-eat)



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


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


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


5 0 EN/ISO 11290-2


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


Absence in 25 g

Food category Micro-organisms Sampling Plan Limits


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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


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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


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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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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


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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


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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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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


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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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

1 rapid pathogen detection using phage technology dubai international food safety conference...

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