Verification of methods

following EN ISO
16140-3; Practice

Wilma Jacobs
Member of ISO TC34/SC9/WG3:
Method validation

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Salmonella

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ISO 16140-3 Verification requirements for a
qualitative (detection) method

Performance Implementation (Food) item

Method
characteristics Verification verification

Qualitative Estimated LOD50 (eLOD50) ✓ ✓

 eLOD50: 3 available protocols to determine the eLOD50

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Implementation Verification
● Demonstrate competence 2. select 1 (food) item tested
of the user laboratory to during the validation study
perform the method that belongs within the
scope of laboratory
(obtain expected results on a application of the user
(food) item used in the laboratory, and
validation study)
3. use this 1 (food) item and
the sample size used in
the validation study to
● The user laboratory shall: perform implementation
1. review validation data for verification.
the method,

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(Food) item Verification
● Demonstrate the competence of the − select 1 challenging (food)
user laboratory to perform the item from each (food)
validated method with (food) items category listed within the
that are tested in the user
laboratory.
scope of validation, that is
also a (food) category that is
tested within the scope of
laboratory application of
the user laboratory, and
− use this 1 (food) item and
the sample size used in the
● The user laboratory shall: validation study to perform
the (food) item verification.

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 Categories: Broad range of foods and other.
CATEGORIES
Table A. 1: Classification of sample types & suggested target combinations for validation studies
Raw milk & Heat processed Raw meat & Ready-to-eat, Raw poultry & Ready-to-eat,
dairy products milk & dairy ready-to-cook ready-to-reheat ready-to-cook ready-to-reheat
products meat products meat products poultry products meat poultry
(except poultry) products

Eggs & egg Raw & ready-to- Ready-to-eat, Fresh produce & Processed fruits Dried cereals,
products cook fish & ready-to-reheat fruits & vegetables fruits, nuts,
(derivatives) seafoods fishery products seeds and
(unprocessed) vegetables
Infant formula Chocolate, Multi-component Environmental Pet food & Primary
& infant bakery products foods or meal samples (food or animal feed production
cereals & confectionary components feed production) samples

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User laboratory: RIVM-Z&O Laboratory
● Detection of Salmonella according to ISO 6579-1
● “a broad range of foods” plus the 3 “other” categories
– 1 x Implementation verification
– 8 x (Food) item verification
Verification Category Item Inoculum strain Strain source
Raw poultry & ready-to-cook
Implementation verification Chicken fillet parts Infantis Broiler
poultry products
(Food) item verification Raw milk & dairy products Raw milk Hadar Human
Raw meat & ready-to-cook
(Food) item verification Minced meat (beef/pork) STMmono Pig meat
meat products (except poultry)
Eggs & egg products
(Food) item verification Whole liquid egg Enteritidis Egg
(derivatives)
Raw & ready-to-cook fish &
(Food) item verification Mussels Agama Shellfish
seafoods (unprocessed)
(Food) item verification Fresh produce & fruits Cantaloupe Virchow Human
Environmental samples (food
(Food) item verification Spent irrigation water Environmental
or feed production
(Food) item verification Pet food & animal feed Chicken feed Senftenberg Cocoa powder
Primary production samples
(Food) item verification Chicken faeces: boot socks Typhimurium Chicken: boot swabs
(PPS)

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Verification Qualitative methods
● Estimated LOD50 (eLOD50) determination

● Enumeration of the inoculum !

– Plate counts, MPNs
● Acceptability limits

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 Protocols in ISO 16140-3

- Protocol 3: direct use of reference material (3-5 cfu/ml), 7 samples

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 Elaborated workflow at RIVM-Z&O
- Combination of Protocol 1, Protocol
2, Protocol 3 (for research reasons
only)
- Aiming at a concentration for
Dilution A at 27 cfu/ml
- Enumeration of overnight culture
(ON) of inoculation strain
- ON dilutions kept in fridge and used
to prepare Dilution A
- Actual inoculation dilutions B, C, D,
prepared from Dilution A by 1:3
steps
- Both plate counts and MPN (for
research reasons only) for
enumeration of 1 ml dilutions (A, B,
C, D)
- Testing inoculated samples (P1, P2,
P3) for presence of Salmonella

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Enumeration of inocula

aantal kolonies per plaat (14 cm, 1 ml)

Dil. A Dil. A Dil. B Dil. B Dil. C Dil. C
MPN table
MONSTER NR PRODUCT
Stam Stam is 20 ml Dil. A is 20 ml Dil. A is 20 ml Dil. B is 20 ml Dil. B

Dil A is 2,3 ml -6 plus

afhankelijk afhankelijk plus 40 ml PFZ plus 40 ml PFZ plus 40 ml PFZ plus 40 ml PFZ
in ISO 16140-3
57,7 ml PFZ (verwachting
27 cfu/ml) Ent S6 op NA 36 37 8 10 3 6
Dil A is 2,5 ml -6 plus
57,5 ml PFZ (verwachting
27 cfu/ml) Ent S8 op NA 47 31 12 12 6 5 0,62 [0,15 -2,6] cfu/test portion
Dil A is 2,1 ml -6 plus
57,9 ml PFZ (verwachting
27 cfu/ml)
Dil A is 1,4 ml -6 plus
Ent S10 op NA 37 35 15 10 1 2
CFU/ml Dilution D
58,6 ml PFZ (verwachting
27 cfu/ml) Ent S11 op NA 31 27 10 13 5 3
1,35 cfu/test portion

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 Combined results, per Protocol

Food item: Animal feed

Protocol 1 Dilution A Dilution B Dilution C Dilution D Blank

Strain S6 (1:3 of A) (1:3 of B) (1:3 of C)
na 1/1 4/4 3/4 0/1

Protocol 2 Dilution A Dilution B Dilution C Dilution D Blank

Strain S6 (1:3 of A) (1:3 of B) (1:3 of C)
na na 3/3 4/5 0/1

Protocol 3 Dilution A Dilution B Dilution C Dilution D Blank

Strain S6 (1:3 of A) (1:3 of B) (1:3 of C)
na na na 6/7 0/1

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 Evaluation of the results, per Protocol

Food item: Animal feed Inoculation strain S6

Published validation data Qualitative

Low Inoculation Level or assume 1 cfu/test portion Verification
Results Table 6/Table 8 (LIL) eLOD50 LOD50 4 x LOD50 Acceptability Limit
ISO 16140-3 (cfu/test portion) (cfu/test portion) (cfu/test portion) (cfu/test portion) e LOD50 ≤ 4 x LOD50

P1 1-4-3-0 = 0,5 x LIL 1,4 0,7 1 4 Accepted

1-4-3-0 = 0,5 x LIL 0,62 [0,15 - 2,6] 0,31 1 4 Accepted

P2 3-4-0 = 0,4 x LIL 1,4 0,56 1 4 Accepted

3-4-0 = 0,4 x LIL 0,62 [0,15 - 2,6] 0,25 1 4 Accepted

P3 6-0 na 1,4 ( < [3-5] ) na na minimum 6/7 Accepted

6-0 na 0,62 ( < [3-5] ) na na minimum 6/7 Accepted

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User laboratory: RIVM-Z&O Laboratory
● Detection of Salmonella according to ISO 6579-1
● “a broad range of foods” plus the 3 “other” categories
– 1 x Implementation verification
– 8 x (Food) item verification P1:
Verification Category Item Inoculum strain Strain source
Raw poultry & ready-to-cook
Implementation verification Chicken fillet parts Infantis Broiler
poultry products
(Food) item verification Raw milk & dairy products Raw milk Hadar Human
Raw meat & ready-to-cook
(Food) item verification Minced meat (beef/pork) STMmono Pig meat
meat products (except poultry)
Eggs & egg products
(Food) item verification Whole liquid egg Enteritidis Egg
(derivatives)
Raw & ready-to-cook fish &
(Food) item verification Mussels Agama Shellfish
seafoods (unprocessed)
(Food) item verification Fresh produce & fruits Cantaloupe Virchow Human
Environmental samples (food
(Food) item verification
or feed production
Spent irrigation water Environmental ~
(Food) item verification Pet food & animal feed Chicken feed Senftenberg Cocoa powder
Primary production samples
(Food) item verification Chicken faeces: boot socks Typhimurium Chicken: boot swabs
(PPS)

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 Thank you for your attention !

Any questions ?
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