ISO 16140 validated alternativemethods for food safety

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

1 ADRIA Développement2 BIO-RAD

SUMMARY

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

üBio-Rad alternative methods!

Qualitative and quantitative

Alternative methods

•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

• 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

SUMMARY

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

üBio-Rad alternative methods

Qualitative and quantitative

ISO 16140 Standard

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!

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!

SUMMARY

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

üBio-Rad alternative methods

Qualitative and quantitative

Applications : validated methods…

Qualitative

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

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

• Reference method : EN ISO 6579

♦ 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

iQ-Check SalmonellaReal time Polymerase Chain Reaction

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

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

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

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

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]

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)

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

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!

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

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

SUMMARY

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

üBio-Rad alternative methods!

Qualitative and quantitative

Applications : validated methods…

quantitive

RAPID’Staph : culture medium

• The regulation aims at limiting coagulase positiveStaphylococci presence in foodstuff

• The reference method is EN ISO 6888 - 1

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

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)

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

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

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

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)

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

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

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

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

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

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

… Thank you!