methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety...

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ISO 16140 validated alternative methods for food safety D. Sohier 1 , M. Rannou 1 , C. Sidi 2 , C. Cordevant 2 , F. Martinez 2 1 ADRIA Développement 2 BIO-RAD

Transcript of methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety...

Page 1: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

ISO 16140 validated alternativemethods for food safety

D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2

1 ADRIA Développement2 BIO-RAD

Page 2: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

SUMMARY

• Alternative methods• ISO 16140 standard• Applications : validated methods…

üBio-Rad alternative methods!

Qualitative and quantitative

Alternative methods

Page 3: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

•European regulation for food safety (Regulation (EC) No 852/2004)specified

Alternative methods are validated against the reference method; the results are certified by a third party in accordance with the protocol set out in EN/ISO standard 16140 or other internationally accepted similar protocols

Alternative methods

Page 4: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

• The tests are validated by an expert lab according toü ISO 16140 protocol in Europe üAOAC-RI and AOAC-OMA in the US

• The results are presented and certified by a third party :üAFNOR in FranceüAOAC in the USüNORDVAL in Northern EuropeüMicroval will be the European committee?

Alternative methods

Page 5: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

SUMMARY

• Alternative methods• ISO 16140 standard• Applications : validated methods…

üBio-Rad alternative methods

Qualitative and quantitative

ISO 16140 Standard

Page 6: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

ISO 16140 : qualitative method

üPreliminary study made by the expert lab• Specificity : Inclusivity and Exclusivity• Relative detection limit• Relative, accuracy, specificity and sensibility

üCollaborative study with 10 labs at least• Reliability

ü… Practicability!

Page 7: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

ISO 16140 : quantative methodüPreliminary study made by the expert lab

• Linearity• Accuracy• Relative detection limit (intrinsic values)• Specificity : Inclusivity and Exclusivity

üCollaborative study with 8 labs at least• Reliability: repeatability and reproducibility

ü… Practicability!

Page 8: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

SUMMARY

• Alternative methods• ISO 16140 standard• Applications : validated methods…

üBio-Rad alternative methods

Qualitative and quantitative

Applications : validated methods…

Qualitative

Page 9: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

RAPID’Salmonella : ChromogeniciQ-Check Salmonella : Real-time PCR

• The regulation expects absence of Salmonella in 25 g of food

• Reference method : EN ISO 6579

Page 10: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

♦ Detection of a specific enzyme of Salmonella by a chromogenic substrate

♦Based on routine method principle, with a high flexibility (second enrichment comprised between 6h and 26h)

RAPID’Salmonellapathogen Chromogenic principle

Salmonella : Magenta coloniesMagenta colonies

Other Enterobacteria : White colonies or blue coloniesblue colonies

Page 11: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

iQ-Check SalmonellaReal time Polymerase Chain Reaction

ØSalmonella (IagA): gene involved in invasive bacterial process.

Page 12: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

Validation : Classical Methods vs Rapid Methods for Salmonella detection

Pre-enrichment(16 - 20 h)

Selectiveenrichment

(24h)

Selective plating(24h)

Confirmatory slants(24h)

Nucleic Acid-basedAssays (2 - 4h)

Selectiveenrichment

(6-24h)

Selective plating(24h)

Confirmation slantsImmuno/BiochemicalAssays (30’ – 24h)

Confirmation slantsChromogenic mediumor reference method

ISO 6570 RAPID’Salmonella iQ-Check Salmonella

Page 13: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

Accuracy and relative detection limit• To be validated for all food products, 5 food

categories (minimum) have to be tested :– Dairy products– Meat products– Sea food– Vegetables– Egg products– Animals food products– Environment

Page 14: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

RAPID’Salmonella : accuracy

Responses Positive reference

method (R+) Negative reference

method (R-) Positive alternative method

(A+) Positive agreement (A+/R+) PA = 166

Positive deviation (R-/A+) PD = 13

Negative alternative method (A-)

Negative deviation (A-/R+) ND = 12

Negative agreement (A-/R-) NA = 216

•Relative accuracy: AC = 100% (PA + NA) / N = 93.9

•Relative specificity: SP = 100% (NA / N) = 94.3

•Relative sensitivity: SE = 100% (PA / N) = 93.3

The two methods do not differ

60 samples tested / category 50% positive and 50% negative

Page 15: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

RAPID’Salmonella : Relative detection limit

The two methods do not differ

4 rates of contamination, with 6 replicatsü rate 1: 0 CFU/g or /mlü rate 2: rate necessary to obtain0 to 50% positives,ü rate 3: rate necessary to obtain 50 to 75% positives,ü rate 4: rate necessary to obtain 100% positives.

Relative level of detection (CFU / 25 g or 25 ml)

according to the Spearman-Kärber test

Couples (strain, matrix) Reference

method Alternative

method Ground beef / Salmonella infantis 14 0.8 [0.3 ; 2.4] 0.7 [0.3 ; 2.1] Raw milk / Salmonella typhimurium 305 1.8 [0.6 ; 5.6] 0.6 [0.1 ; 2.6] Filet of ling cod / Salmonella St Paul F31 0.4 [0.1 ; 2.1] 0.4 [0.1 ; 2.1] Soft egg / Salmonella enteritidis 2532 0.5 [0.1 ; 2.2] 0.8 [0.2 ; 3.4] Puff pastry in aspic/Salmonella agona A00V038

0,5 [0,1 ; 2,5] 0,5 [0,1 ; 2,5]

Page 16: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

Pure culturesØ 50 target strains Ø 30 non-target strains

RAPID’Salmonellainclusivity /exclusivity

All target strains are detected, except one collection strainAll untargeted strains tested are not detected

Ø Inclusivity = 98,1% (N=50)Ø Exclusivity = 100% (N=42)

Page 17: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

ISO 16140 : Collaborative study

Pasteurized milk samples with natural flora

ü3 different levels of pure cultures were analyzed by the reference and the alternative methods

ü6 replicats / contamination

ü10 laboratories (minimum) for each method

Page 18: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

RAPID’Salmonella and I-Q Check Salmonella collaborative studies

üRelative accuracy: AC = 99,2 %

üRelative specificity: SP = 97,7 %

üRelative sensitivity: SE = 100%

100% in agreement with the EN ISO 6579 method

alternative method … 2 positive deviations!

Page 19: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

Practicability Reference RAPID’

Salmonella iQ-Check Salmonella

TTiimmee ttoo rreessuullttss PPrreesseennccee//aabbss

With confirmation Salmonella

3 days 5 days

2 days 3-5 days

1 day 3-5 days

Workflows

negative sample positive sample (with confirmation)

7 minutes 14 minutes

5 minutes 7 minutes

5 minutes 8 minutes

Page 20: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

Performances assessment according to the ISO 16140 standard

Rapid’Salmonella

ü is sensitive and specific

ü gives comparable results to the stantard method

ü shows a very high practicability… I-Q Check Salmonella

… Rapid’L.mono and I-Q Check L. monocytogenes

Page 21: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

SUMMARY

• Alternative methods• ISO 16140 standard• Applications : validated methods…

üBio-Rad alternative methods!

Qualitative and quantitative

Applications : validated methods…

quantitive

Page 22: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

RAPID’Staph : culture medium

• The regulation aims at limiting coagulase positiveStaphylococci presence in foodstuff

• The reference method is EN ISO 6888 - 1

Page 23: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

RAPID’StaphBased on routine method principle

Enumeration of coagulase positive Staphylococci

Coagulase postive Satphylococci : Black colonies Black colonies withwith a a specificspecific halohalo

Other bacteria : White coloniesWhite colonies

Fast and easy confirmation : ØPASTOREX STAPH PLUS or BP+RPF

Page 24: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

Validation : Classical Method vs Rapid Method for coagulase positive Staphylococci

Suspension 1/10 of the sampleAnd decimal dilutions

PlatingOn BPA plates

Incubation(24h +/- 2h)

Enumeration of characteristic and non characteristic colonies

PlatingOn RAPID’Staph

plates

Confirmation slants : PASTOREX STAPH PLUS (2’)

Or BP + RPF

ISO 6888 RAPID’Staph

Enumeration of characteristic colonies

Coagulase confirmation (48h)

Page 25: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

ISO 16140 : Relative linearity and accuracy

• To be validated for all food products, 5 food categories (minimum) have to be tested :- Dairy products- Meat products- Sea food- Vegetables- Egg products- Animals food products- Environment

Page 26: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

RAPID’Staph relative linearity5 contamination levels, 4 log range1 matrix per category, x 5 categories

Regression to compare both methods (alternative and standard)

Ø Lack of fit test

Correlation coefficients > 0.98

OLS1 Regression

0,001,002,003,004,005,006,00

0,00 2,00 4,00 6,00

Standard

Alt

ern

ativ

e

Page 27: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

RAPID’Staph relative accuracy

5 categories of food products

Min 10 samples tested per category

Ø Slope = 1

Ø Ordinate = 0

All products

Accepted for each categories tested

OLS1 Regression OLS 1

0,00

2,00

4,00

6,00

8,00

0,00 2,00 4,00 6,00 8,00

Reference

Alt

ern

ativ

e

Page 28: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

Pure culturesØ 30 target strains Ø 30 non-target strains

ISO 16140 : Sensitivity and specificity

RAPID’Staph

All target strains are detectedAll untargeted strains tested are not detected

ØInclusivity = 100% (N=30)ØExclusivity = 100% (N=20)

Page 29: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

ISO 16140 : collaborative study

• Pasteurized milk samples with natural flora • 4 different levels of pure cultures were analyzed

by the reference and the alternative methods• 8 laboratories (minimum) for each method

Page 30: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

RAPID’Staph : Collaborative study

Limit of reproducibility Reference method

Alternative method

0,352 0,881 0,264 0,338 0,117 0,323

Limit of repeatability Contaminations

levels Reference method

Alternative method

1,18 0,365 0,881 2,70 0,268 0,377 3,86 0,280 0,461

Depending on the contamination levels tested, Rapid’Staph shows similar orhigher limit of repeatability and limit of reproducibility values comparing to

the standard plate count method

Page 31: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

Bias between the reference and alternative methods

- 0.03 < bias < 0.13, perfect equivalence

RAPID’Staph : Collaborative study

Dispersion between laboratories Contaminations

levels Reference method

Alternative method

1,18 1,15 0,78 2,70 1,06 1,50 3,86 10,35 3,08

Rapid’Staph is characterized by a better laboratories results dispersion

Page 32: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

Practicability !

DDeessccrriippttiioonn Reference RAPID’Staph TTiimmee ttoo rreessuullttss ((CCoonnffiirrmmaattiioonn))

4 days

1-2 days

WWoorrffllooww Timing required for 1 sample analysis

32’5

11’8

Page 33: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

Performances assessment according to the ISO 16140 standard

Rapid’Staphüis sensitive and specific

ügives comparable results to the stantard methods

üshows a very high practicability

… Rapid’E. coli 2

Page 34: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

Validation according to the ISO 16140 standard and the AFNOR technical rules :

AAssessmentssessment of performancesof performances… + + + !

Alternative methods

Ø represent valuable methods for food testing

Ø offer an important economic savings by

- standardizing the analysis

- minimizing the training time and the workflow

Page 35: methods for food safety - · PDF fileISO 16140 validated alternative methods for food safety D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA Développement 2

… Thank you!