ISO 16140 validated alternative methods for food safety. D. Sohier1, M. Rannou1,
C. Sidi2, C. Cordevant2, F. Martinez2. 1 ADRIA Développement. 2 BIO-RAD ...
ISO 16140 validated alternative methods for food safety
D. Sohier1, M. Rannou1, C. Sidi2, C. Cordevant2, F. Martinez2 1 ADRIA
Développement
2 BIO-RAD
SUMMARY • Alternative Alternative methods 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 to ü ISO 16140 protocol in Europe ü AOAC-RI and AOAC-OMA in the US
• The results are presented and certified by a third party : ü AFNOR in France ü AOAC in the US ü NORDVAL in Northern Europe ü Microval will be the European committee?
SUMMARY • Alternative methods ISO 16140 16140 standard Standard • ISO • Applications : validated methods… üBio-Rad alternative methods Qualitative and quantitative
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 method üPreliminary 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 Applications :: validated validated methods… methods… üBio-Rad alternative methods Qualitative and quantitative
RAPID’Salmonella : Chromogenic iQ-Check Salmonella : Real-time PCR • The regulation expects absence of Salmonella in 25 g of food • Reference method : EN ISO 6579
RAPID’Salmonella pathogen Chromogenic principle ♦ 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) Salmonella : Magenta colonies Other Enterobacteria : White colonies or blue colonies
iQ-Check Salmonella Real 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) ISO 6570 Selective enrichment (24h) Selective plating (24h) Confirmatory slants (24h)
RAPID’Salmonella Selective enrichment (6-24h)
iQ-Check Salmonella
Selective plating (24h)
Nucleic Acid-based Assays (2 - 4h)
Confirmation slants Immuno/Biochemical Assays (30’– 24h)
Confirmation slants Chromogenic medium or reference method
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 60 samples tested / category 50% positive and 50% negative Positive reference method (R+) Positive alternative method Positive agreement (A+/R+) (A+) PA = 166 Negative alternative method Negative deviation (A-/R+) (A-) ND = 12 Responses
Negative reference method (R-) Positive deviation (R-/A+) PD = 13 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
RAPID’Salmonella : Relative detection limit Relative level of detection (CFU / 25 g or 25 ml) according to the Spearman-Kärber 4 rates of contamination, with 6 replicats test ü rate 1: 0 CFU/g or /ml Reference Alternative Couples (strain, matrix) ü rate 2: rate necessary to obtainmethod method Ground beef0/ Salmonella infantis 14 0.8 [0.3 ; 2.4] 0.7 [0.3 ; 2.1] to 50% positives, Raw milk / Salmonella typhimurium 305 1.8 [0.6 ; 5.6] 0.6 [0.1 ; 2.6] ü rate 3: rate necessary to obtain Filet of ling cod / Salmonella St Paul F31 0.4 [0.1 ; 2.1] 0.4 [0.1 ; 2.1] 50 to 75% positives, Soft egg / Salmonella enteritidis 2532 0.5 [0.1 ; 2.2] 0.8 [0.2 ; 3.4]
Puff pastry ü rate in aspic/Salmonella 4: rate necessary agona to obtain 100% positives. 0,5 [0,1 ; 2,5] 0,5 [0,1 ; 2,5] A00V038
The two methods do not differ
RAPID’Salmonella inclusivity /exclusivity Pure cultures Ø 50 target strains Ø 30 non-target strains All target strains are detected, except one collection strain All 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% alternative method … 2 positive deviations! 100% in agreement with the EN ISO 6579 method
Practicability Reference
RAPID’ Salmonella
iQ-Check Salmonella
3 days
2 days
1 day
5 days
3-5 days
3-5 days
negative sample
7 minutes
5 minutes
5 minutes
positive sample (with confirmation)
14 minutes
7 minutes
8 minutes
Ti me to resul ts Presence/abs With confirmation Salmonella Workflows
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 Applications :: validated validated methods… methods… üBio-Rad alternative methods! Qualitative and quantitative quantitive
RAPID’Staph : culture medium
• The regulation aims at limiting coagulase positive Staphylococci presence in foodstuff • The reference method is EN ISO 6888 - 1
RAPID’Staph Based on routine method principle Enumeration of coagulase positive Staphylococci Coagulase postive Satphylococci : Black colonies with a specific halo Other bacteria : White 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 sample And decimal dilutions RAPID’Staph ISO 6888 Plating Plating On RAPID’Staph On BPA plates plates Incubation(24h +/- 2h) Enumeration of characteristic and non characteristic colonies
Coagulase confirmation (48h)
Enumeration of characteristic colonies Confirmation slants : PASTOREX STAPH PLUS (2’) Or BP + RPF
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 linearity 5 contamination levels, 4 log range 1 matrix per category, x 5 categories Regression to compare both methods (alternative and standard) Ø Lack of fit test
Correlation coefficients > 0.98
Alternative
OLS1 Regression 6,00 5,00 4,00 3,00 2,00 1,00 0,00 0,00
2,00
4,00
Standard
6,00
RAPID’Staph relative accuracy Min 10 samples tested per category 5 categories of food products
All products OLS1 Regression OLS 1 8,00
Ø Slope = 1 Ø Ordinate = 0
Alternative
6,00
4,00
2,00
0,00 0,00
2,00
4,00
6,00
8,00
Reference
Accepted for each categories tested
ISO 16140 : Sensitivity and specificity Pure cultures Ø 30 target strains Ø 30 non-target strains
RAPID’Staph All target strains are detected All 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
Contaminations levels
1,18 2,70 3,86
Limit of repeatability Reference Alternative method method 0,365 0,881 0,268 0,377 0,280 0,461
Limit of reproducibility Reference Alternative method method 0,352 0,881 0,264 0,338 0,117 0,323
Depending on the contamination levels tested, Rapid’Staph shows similar or higher limit of repeatability and limit of reproducibility values comparing to the standard plate count method
RAPID’Staph : Collaborative study Dispersion between laboratories Contaminations Reference Alternative levels method 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 Bias between the reference and alternative methods - 0.03 < bias < 0.13, perfect equivalence
Practicability ! Description Time to results
Reference
RAPID’Staph
4 days
1-2 days
32’5
11’8
(Confirmation)
Worflow Timing required for 1 sample analysis
Performances assessment according to the ISO 16140 standard Rapid’Staph üis 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 :
Assessment of 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!
