ACCREDITATION
N°1-0144
PORTEE DISPONIBLE
SUR WWW.COFRAC.FR
ADRIA DEVELOPPEMENT Creac’h Gwen - F. 29196 QUIMPER Cedex - Tél. (33) 02.98.10.18.18 - Fax (33) 02.98.10.18.08
E-mail : [email protected] - Site web : http://www.adria.tm.fr
ASSOCIATION LOI DE 1901 - N° SIRET 306 964 271 00036 - N° EXISTENCE 532900006329 - N°TVA FR4530696427100036
3M
Boulevard de l’Oise
F-95029 CERGY PONTOISE CEDEX
NF VALIDATION
Validation of alternative analytical methods
Application in food microbiology
Summary report
EN ISO 16140-2:2016 validation study of
the 3MTM Molecular Detection Assay 2 -
Listeria monocytogenes for detection of Listeria monocytogenes in food products
and environmental samples
Qualitative method
This report includes 66 pages, with 5 appendixes.
Only copies including the totality of this report are authorized.
Competences of the laboratory are certified by COFRAC accreditation for the analyses
marked with symbol.
Version 0
November 14, 2016
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3M MDA 2 - Listeria monocytogenes
1 AIM OF THE STUDY ____________________________________________ 4
2 METHOD PROTOCOLS _________________________________________ 4
2.1 Reference method _______________________________________________ 4
2.2 Alternative method ______________________________________________ 4
3 METHOD COMPARISON STUDY __________________________________ 5
3.1 Sensitivity study ________________________________________________ 5
3.1.1 Number and nature of samples ______________________________________ 5
3.1.2 Artificial contamination of samples ____________________________________ 6
3.1.3 Confirmation protocols _____________________________________________ 6
3.1.4 Test results ______________________________________________________ 7
3.1.5 Calculation of relative accuracy (RT), relative sensitivity (SE) and false positive
ratio (FPR) ______________________________________________________ 9
3.1.6 Analysis of discordants ___________________________________________ 11
3.1.7 Enrichment broth storage at 2 - 8°C for 72 h ___________________________ 17
3.1.8 Confirmation ____________________________________________________ 18
3.1.9 Inhibitions ______________________________________________________ 19
3.2 Relative level of detection study __________________________________ 19
3.2.1 Experimental design ______________________________________________ 19
3.2.2 Calculation and interpretation of the RLOD ____________________________ 20
3.3 Inclusivity / exclusivity __________________________________________ 21
3.3.1 Test protocols ___________________________________________________ 21
3.3.2 Results ________________________________________________________ 21
3.4 Practicability __________________________________________________ 22
4 INTER-LABORATORY STUDY ORGANISATION AND RESULTS _______ 23
4.1 Study organisation _____________________________________________ 23
4.2 Experimental parameters controls _________________________________ 24
4.2.1 Sample stability _________________________________________________ 24
4.2.2 Logistic conditions _______________________________________________ 26
4.3 Calculation and summary of data _________________________________ 27
4.3.1 Results obtained by the expert Lab. _________________________________ 27
4.3.2 Results obtained by the collaborators ________________________________ 27
4.4 Interpretation __________________________________________________ 32
5 CONCLUSION ________________________________________________ 34
Appendix 1 – Flow diagram of the EN ISO 11290-1/A1 (2004) method: Horizontal method
for the detection and enumeration of Listeria monocytogenes - Part 1: detection of Listeria monocytogenes
in foods ___________________________________________________________________________ 36
Appendix 2 – Flow diagram of the 3MTM Molecular Detection Assay 2 - Listeria monocytogenes ______ 37
Appendix 3 – Relative level of detection study: raw data _____________________________________ 40
Appendix 4 – Inclusivity and exclusivity study: raw data _____________________________________ 46
Appendix 5 – Results obtained by the collaborators and the expert laboratory____________________ 51
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Quality Assurance documents related to this study can be consulted upon
request from 3M.
The technical protocol and the result interpretation were realised according to
the EN ISO 16140-2:2016 and the AFNOR technical rules.
Company: 3M
Boulevard de l’Oise
F-95029 CERGY PONTOISE CEDEX
Expert Laboratory: ADRIA Développement
ZA Creac’h Gwen
F-29196 QUIMPER Cedex
Studied method: 3MTM Molecular Detection Assay 2 - Listeria
monocytogenes
Validation standard : EN ISO 16140-2:2016 - Microbiology of the food
chain - Method validation
Part 1: Vocabulary
Part 2: Protocol for the validation of alternative
(proprietary) methods against a reference method
Standard method: EN ISO 11290-1/A1 (2004): Microbiology of food and
animal feeding stuffs - Horizontal method for the
detection and enumeration of Listeria
monocytogenes - Part 1: detection of Listeria
monocytogenes in foods
Scope: Food products and Environmental samples
(excluding samples from primary production)
Certification body: AFNOR Certification
Analyses performed according to the COFRAC accreditation
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1 AIM OF THE STUDY
The 3MTM Molecular Detection Assay 2 - Listeria monocytogenes for the
detection of Listeria monocytogenes was validated for human food
products and environmental samples (excluding samples from primary
production) was validated in September 2016 (Certificate number:
3M 01/15 - 09/16) according to the EN ISO 16140-2 (2016).
2 METHOD PROTOCOLS
2.1 Reference method
The reference method correspond to the ISO 11290-1/A1 (2004) standard:
Microbiology of food and animal feeding stuffs - Horizontal method for the
detection and enumeration of Listeria monocytogenes – Part 1: detection
method (See Appendix 1).
2.2 Alternative method
Principle
The 3MTM Molecular Detection Assay 2 - Listeria monocytogenes Test Kit
uses isothermal amplification of unique DNA target sequences, the amplified
sequences are detected by bioluminescence.
Protocol (see Appendix 2)
- Enrichment step in Half Fraser broth at 37°C ± 1°C:
* Specific protocol: 25 g + 225 ml Half Fraser for raw dairy, raw meat
and raw seafood products. Incubation for 22 h ± 2 h at 37°C.
Subculture in Fraser broth for 22 h ± 2 h at 37°C.
* General protocol: 25 g + 225 ml Half Fraser for all other products
(food and environmental samples). Incubation for 27 h ± 3 h at 37°C
- Lysis step
- Amplification and real-time detection
Analysis performed according to the COFRAC accreditation
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- Confirmation step by streaking 100 µl of enriched Half Fraser broth onto
O&A and Palcam plates (General protocol), and by streaking 10 µl of
Fraser broth (Specific protocol).
It is possible to store the enrichment broths for 72 h at 2 - 8°C for all the
categories and to store the lysates for 72 h at 2 - 8°C for all the categories,
except for Dairy products and environmental samples, before running the
MDA test.
The flow diagram is given in Appendix 2.
3 METHOD COMPARISON STUDY
As the enrichment procedure is different between the alternative and
the reference methods, this corresponds to an UNPAIRED DATA
STUDY DESIGN.
3.1 Sensitivity study
The sensitivity (SE) is the ability of the method to detect the analyte by either the reference or
alternative method.
3.1.1 Number and nature of samples
400 samples were analyzed.
The distribution per tested category and type is given in Table 1.
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Table 1 – Distribution per tested category and type
Categories TypesPositive
sample
Negative
samplesTotal
a Ready-to-eat 11 10 21
b Ready-to-reheat 10 10 20
c Pastries and egg products 9 12 21
Total 30 32 62
a Meat products (raw, frozen, seasoned) 17 10 27
b Ready-to-eat and processed meat products 7 14 21
c Delicatessen 10 13 23
Total 34 37 71
a Raw dairy products 13 10 23
b Pasteurized milk cheeses 9 11 20
c Ice cream, milk (pasteurized), flavoured milk
(pasteurized) 8 12 20
Total 30 33 63
a Raw vegetable products (fresh and frozen) 10 16 26
b Mapped vegetables and heat processed vegetables
14 6 20
c Vegetables based preparations, processed vegetables
7 15 22
Total 31 37 68
a Raw products (fresh, frozen) 10 10 20
b Smoked, marinated 10 10 20
c Ready-to-eat or ready-to-reheat 10 12 22
Total 30 32 62
a Process water 7 14 21
b Dusts 14 12 26
c Wipes 11 16 27
Total 32 42 74
187 213 400
SEAFOOD AND
FISHERY PRODUCTS
ENVIRONMENTAL
SAMPLES
TOTAL
COMPOSITE FOODS/
READY-TO-EAT AND
READY-TO-REHEAT
MEAT PRODUCTS
DAIRY PRODUCTS
VEGETABLES
3.1.2 Artificial contamination of samples
Artificial contaminations were done by seeding protocol.
87 samples were artificially contaminated, using 37 different strains. 62 gave
a positive result. All the samples were inoculated at level 3 CFU.
53.5 % of the samples were naturally contaminated.
3.1.3 Confirmation protocols
The positive tests were confirmed by streaking 100 µl of the enrichment broth
onto O&A. During the validation study, the typical colonies were identified by
the tests described in the reference method.
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3.1.4 Test results
The results are given in the following tables.
Table 2 – Summary of results obtained with the reference
and the alternative method: All the samples
Response Reference method
positive (R+)
Reference method
negative (R-)
Alternative method
positive (A+)
Positive agreement (A+/R+)
PA = 119
Positive deviation (R-/A+)
PD = 35
Alternative method
negative (A-)
Negative deviation (A-/R+)
ND = 33 (PPND = 1)
Negative agreement (A-/R-)
NA = 213 (PPNA = 2)
PP: positive presumptive non confirmed samples
PD = positive deviation (R-/A+) ND = negative deviation (A-/R+)
Results per category of samples
Table 3 – Summary of results obtained with the reference
and the alternative method:
composite foods / ready-to-eat and ready-to-reheat
Response Reference method
positive (R+)
Reference method
negative (R-)
Alternative method
positive (A+)
Positive agreement (A+/R+)
PA = 22
Positive deviation (R-/A+)
PD = 5
Alternative method
negative (A-)
Negative deviation (A-/R+)
ND = 3
Negative agreement (A-/R-)
NA = 32
Table 4 – Summary of results obtained with the reference
and the alternative method: Meat products
Response Reference method
positive (R+)
Reference method
negative (R-)
Alternative method
positive (A+)
Positive agreement (A+/R+)
PA = 17
Positive deviation (R-/A+)
PD = 8
Alternative method
negative (A-)
Negative deviation (A-/R+)
ND = 9
Negative agreement (A-/R-)
NA = 37
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Table 5 – Summary of results obtained with the reference
and the alternative method: Dairy products
Response Reference method
positive (R+)
Reference method
negative (R-)
Alternative method
positive (A+)
Positive agreement (A+/R+)
PA = 18
Positive deviation (R-/A+)
PD = 8
Alternative method
negative (A-)
Negative deviation (A-/R+)
ND = 4
Negative agreement (A-/R-)
NA = 33
Table 6 – Summary of results obtained with the reference
and the alternative method: Vegetables
Response Reference method
positive (R+)
Reference method
negative (R-)
Alternative method
positive (A+)
Positive agreement (A+/R+)
PA = 19
Positive deviation (R-/A+)
PD = 6
Alternative method
negative (A-)
Negative deviation (A-/R+)
ND = 6 (PPND = 1)
Negative agreement (A-/R-)
NA = 37
Table 7 – Summary of results obtained with the reference
and the alternative method: Seafood and fishery products
Response Reference method
positive (R+)
Reference method
negative (R-)
Alternative method
positive (A+)
Positive agreement (A+/R+)
PA = 22
Positive deviation (R-/A+)
PD = 4
Alternative method
negative (A-)
Negative deviation (A-/R+)
ND = 4
Negative agreement (A-/R-)
NA = 32
Table 8 – Summary of results obtained with the reference
and the alternative method: Environmental samples
Response Reference method
positive (R+)
Reference method
negative (R-)
Alternative method
positive (A+)
Positive agreement (A+/R+)
PA = 21
Positive deviation (R-/A+)
PD = 4
Alternative method
negative (A-)
Negative deviation (A-/R+)
ND = 7
Negative agreement (A-/R-)
NA = 42 (PPNA = 2)
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The summary of results per protocol is given tables 10 and 11.
Table 9 – Summary of results obtained with the reference
and the alternative method: General protocol
Response Reference method
positive (R+)
Reference method
negative (R-)
Alternative method
positive (A+)
Positive agreement (A+/R+)
PA = 91
Positive deviation (R-/A+)
PD = 28
Alternative method
negative (A-)
Negative deviation (A-/R+)
ND = 28 (PPND = 1)
Negative agreement (A-/R-)
NA = 183 (PPNA = 2)
Table 10 – Summary of results obtained with the reference
and the alternative method: Specific protocol
Response Reference method
positive (R+)
Reference method
negative (R-)
Alternative method
positive (A+)
Positive agreement (A+/R+)
PA = 28
Positive deviation (R-/A+)
PD = 7
Alternative method
negative (A-)
Negative deviation (A-/R+)
ND = 5
Negative agreement (A-/R-)
NA = 30
3.1.5 Calculation of relative accuracy (RT), relative sensitivity (SE) and
false positive ratio (FPR)
The calculations are presented in table 11.
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Table 11 – Calculation of the relative accuracy (RT), the relative sensitivity (SE) and the false positive ratio (FPR)
Category Type PA NA PD ND PPND PPNA SE alt % SE ref % RT % FPR %
1 Composite foods/
Ready to eat/ Ready to reheat
a Ready to eat 7 10 3 1 0 0 90.9 72.7 81.0 0.0
b Ready to reheat 7 10 2 1 0 0 90.0 80.0 85.0 0.0
c Pastries, desserts, omelettes 8 12 0 1 0 0 88.9 100.0 95.2 0.0
Total 22 32 5 3 0 0 90.0 83.3 87.1 0.0
2 Meat products
a Raw, frozen, seasoned 10 10 3 4 0 0 76.5 82.4 74.1 0.0
b Ready to eat and processed meat products 3 14 2 2 0 0 71.4 71.4 81.0 0.0
c Delicatessen 4 13 3 3 0 0 70.0 70.0 73.9 0.0
Total 17 37 8 9 0 0 73.5 76.5 76.1 0.0
3 Dairy products
a Raw dairy products 10 10 3 0 0 0 100.0 76.9 87.0 0.0
b Pasteurized milk cheese 3 11 3 3 0 0 66.7 66.7 70.0 0.0
c Ice cream, milk (pasteurized), flavored milk 5 12 2 1 0 0 87.5 75.0 85.0 0.0
Total 18 33 8 4 0 0 86.7 73.3 81.0 0.0
4 Vegetables
a Raw vegetable products (fresh, frozen) 8 16 1 1 0 0 90.0 90.0 92.3 0.0
b Mapped vegetables and heat processed vegetables 7 6 4 2 1 0 78.6 71.4 65.0 16.7
c Vegetables based preparations, processed vegetables 4 15 1 2 0 0 71.4 85.7 86.4 0.0
Total 19 37 6 5 1 0 80.6 80.6 82.4 2.7
5 Seafood and fishery products
a Raw products (fresh, frozen) 8 10 1 1 0 0 90.0 90.0 90.0 0.0
b Smoked, marinated 7 10 3 0 0 0 100.0 70.0 85.0 0.0
c Ready to eat or ready to reheat 7 12 0 3 0 0 70.0 100.0 86.4 0.0
Total 22 32 4 4 0 0 86.7 86.7 87.1 0.0
6 Environmental samples
a Process water 7 14 0 0 0 0 100.0 100.0 100.0 0.0
b Dusts 5 11 4 5 0 1 64.3 71.4 65.4 9.1
c Wipes 9 15 0 2 0 1 81.8 100.0 92.6 6.7
Total 21 40 4 7 0 2 78.1 87.5 85.1 4.8
All categories 119 211 35 32 1 2 82.4 81.3 83.0 1.4
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The following results are observed:
Table 12
Sensitivity for the alternative method
%100
)(
PDNDPA
PDPASEalt 82.4 %
Sensitivity for the reference method
%100
)(
PDNDPA
NDPASEref 81.3 %
Relative trueness %100)(
N
NAPART 83.0 %
False positive ratio for the alternative method*
FP = PPNA + PPND
%100)(
NA
FPFPR
1.4 %
* With ND = ND + PPND NA = NA + PPNA
3.1.6 Analysis of discordants
Among the 33 negative deviations (see table 13), 13 concern artificially
contaminated samples and 20 naturally contaminated samples.
The presence of Listeria monocytogenes was confirmed for 7 samples by the
tests of the alternative method (streaking onto O&A plates) and one
additional sample (No 2565) was confirmed by applying the whole protocol of
the reference method (subculture in Fraser for 48 h at 37°C and streaking
onto O&A).
For 25 samples, the negative results were probably due to sampling
heterogeneity in this unpaired data study when dealing with low
contamination levels.
Among the 35 positive deviations (see table 14), 9 concern artificially
contaminated samples and 24 concern naturally contaminated samples.
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Table 13 - Negative deviations
Category Type Sample
N° Product Inoculated strain
Inoculation level
(cfu/sample)
MDA result
Confirmation result Final result Alternative method
Reference method (Fraser 48h 37°C)
Composite foods / Ready to eat and ready to reheat
b 2761 RTRH composite food -/-/- L.monocytogenes H+ -
c 3202 Tortilla L.monocytogenes Ad1757 1.8 - / / -
a 3748 Sandwich (ham, cheese) L.monocytogenes Ad1197 0.8 - / - -
Meat products
c 1097 Rillettes - L.grayi - -
c 4451 Cooked ham - / - -
c 4664 Smoked sliced bacon - / - -
a 6178 Raw chicken meat -/-/- L.monocytogenes / -
a 6179 Beef meat -/-/- L.monocytogenes / -
a 6186 Ground poultry - / - -
a 6365 Gizzards - / - -
b 7553 Ready to reheat meal L.monocytogenes A00C052 1.4 - / - -
b 7558 Ready to reheat meal (beef) L.monocytogenes Ad266 0.8 - / - -
Dairy products
c 3189 Pasteurized half skimmed milk L.monocytogenes A00L107 0.8 - / / -
b 3405 Pasteurized cow milk cheese L.monocytogenes A00L106 1.2 -/-/- L.monocytogenes + -
b 3752 Pasteurized milk cheese L.monocytogenes Ad1201 2.4 -/-/- L.monocytogenes + -
b 7561 Pasteurized ewe milk cheese L.monocytogenes Ad1201 0.8 - / - -
Vegetables
b 4443 Heat processed vegetables - / - -
a 4445 Sprouts - / - -
b 4447 Zucchini + L.innocua - -
b 4454 Frozen broccoli - / - -
c 7372 Frozen vegetables mix - L.welshimeri/
L.innocua - -
c 7570 Processed vegetables mix L.monocytogenes 1011/1410
1.0 - / - -
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Category Type Sample
N° Product Inoculated strain
Inoculation level
(cfu/sample)
MDA result
Confirmation result Final result Alternative method
Reference method (Fraser 48h 37°C)
Seafood and
fishery products
c 1100 RTRH salmon - L.welshimeri - -
c 2565 RTRH salmon balls - / H+(L.mono) -
c 2938 Cooked shrimps i/+* L.monocytogenes / -
a 6200 Raw fish - / - -
Environmental
samples
c 4142 Swab (dairy environment) L.monocytogenes Ad629 1.6 - / - -
b 4384 Wastes (Fish industry) - L.innocua - -
b 6869 Wastes (Meat industry) - L.welshimeri - -
c 6873 Wipe (Dairy industry) -/i/+ L.monocytogenes/
L.innocua / -
b 6877 Wastes (meat) L.monocytogenes Ad1265 0.4 -/+/+ L.innocua - -
b 6878 Wastes (meat) L.monocytogenes Ad1265 0.4 -/+/+ L.welshimeri - -
b 6881 Wastes (meat) L.monocytogenes Ad1255 L.innocua Ad1251
1.4 - L.innocua - -
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Table 14 - Positive deviations
Category Type Sample
N° Product Inoculated strain
Inoculation level
(cfu/sample)
MDA result
Confirmation result Final result
Composite foods /
Ready to eat /
Ready to reheat
b 1076 Deli salad + L.monocytogenes +
a 1079 RTE + L.monocytogenes +
a 2760 Sandwich (goat cheese, vegetables) + L.monocytogenes +
a 2933 Chinese pasta + L.monocytogenes +
b 3150 RTRH composite food + L.monocytogenes +
Meat products
b 2952 Sliced cooked chicken + L.monocytogenes +
c 3158 Terrine + L.monocytogenes +
c 3165 Sausages with aromatic herbs + L.monocytogenes/
L.innocua +
c 4441 Cooked sausage +/-/- L.monocytogenes /L.innocua +
a 6180 Sheep meat + L.monocytogenes (72h) +
a 6183 Chicken leg + L.monocytogenes +
a 6190 Chicken leg + L.monocytogenes +
b 7557 Ready to reheat meal (beef) L.monocytogenes Ad266 0.8 + L.monocytogenes +
Dairy products
c 3190 Pasteurized half skimmed milk L.monocytogenes 910 1.8 + L.monocytogenes +
c 3192 Cocoa flavored milk L.monocytogenes A00L098 1.2 + L.monocytogenes +
b 3404 Pasteurized cow milk cheese L.monocytogenes A00L106 1.2 + L.monocytogenes +
b 3749 Pasteurized cow milk cheese L.monocytogenes Ad1784 1.0 + L.monocytogenes +
a 4149 Raw milk cheese L.monocytogenes Ad665 2 + L.monocytogenes +
a 4703 Raw milk cheese + L.monocytogenes +
a 6246 Raw ewe milk cheese + L.monocytogenes +
b 7563 Pasteurized ewe milk cheese L.monocytogenes Ad523 0.8 + L.monocytogenes +
Vegetables
b 3539 Frozen mushrooms + L.monocytogenes +
b 4458 Frozen precooked onions + L.monocytogenes +
b 4459 Frozen zucchini + L.monocytogenes (+72h)/L.innocua +
b 4461 Vegetables for couscous + L.monocytogenes +
a 4655 Pieces of chestnuts + L.monocytogenes +
c 7569 Processed vegetables mix L.monocytogenes 1011/1410 1.0 + L.monocytogenes +
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Category Type Sample
N° Product Inoculated strain
Inoculation level
(cfu/sample)
MDA result
Confirmation result Final result
Seafood and
fishery products
b 3528 Smoked salmon + L.monocytogenes +
b 3531 Smoked salmon + L.monocytogenes +
b 3532 Smoked salmon + L.monocytogenes +
a 6194 Raw fish + L.monocytogenes (Rapid'L.mono)
+
Environmental
samples
b 6870 Wastes (meat) + L.monocytogenes +
b 6879 Wastes (meat) L.monocytogenes Ad1255 L.innocua Ad1251
1.4 + L.monocytogenes +
b 6880 Wastes (meat) L.monocytogenes Ad1255 L.innocua Ad1251
1.4 + L.monocytogenes (Fraser 1) /
L.welshimeri +
b 7932 Dusts L.monocytogenes Ad634 1.8 + L.monocytogenes +
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The analyses of discordant results according to the EN ISO 16140-2: 2016 is
the following (See table 15):
Table 15
Category Type (ND+PPND)-
PD AL
1 Composite foods/Ready to eat/Ready to reheat
a Ready to eat -2
b Ready to reheat -1
c Pastries, desserts, omelettes 1
Total -2 3
2 Meat products
a Raw, frozen, seasoned 1
b Ready to eat and processed meat products 0
c Delicatessen 0
Total 1 3
3 Dairy products
a Raw dairy products -3
b Pasteurized milk cheese 0
c Ice cream, milk (pasteurized), flavored milk -1
Total -4 3
4 Vegetables
a Raw vegetable products (fresh, frozen) 0
b Mapped vegetables and heat processed vegetables -1
c Vegetables based preparations, processed vegetables 1
Total 0 3
5 Seafood and fishery
products
a Raw products (fresh, frozen) 0
b Smoked, marinated -3
c Ready to eat or ready to reheat 3
Total 0 3
6 Environmental samples
a Process water 0
b Dusts 1
c Wipes 2
Total 3 3
All categories -2 6
The observed values for ((ND + PPND) - PD) are below or equal to the
acceptability limit for each category and for all the categories.
Additional Listeria monocytogenes strains were recovered from five samples
with negative agreement results (See table 16). Three samples were
artificially contaminated; the other one were naturally contaminated samples.
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Table 16 - Strains recovered from samples with NA results
Category Sample
No Product
MDA test
MDA confirmation tests Reference method *
Type O&A Identification
Meat products 7554 Ready-to-reheat
meat - / + H+ L. monocytogenes + b
Vegetables
3146 Vegetable based
preparation - H+ L. monocytogenes + c
3538 Precooked frozen
onions - / - / + H+ L. monocytogenes / b
Dairy products 3195 Pasteurized milk
cheese - / - / - H+ L. monocytogenes + b
Environmental
samples 4144 Wastes (fish) - H+ L. monocytogenes + b
* Subculture in Fraser broth from the Half Fraser of the alternative method
3.1.7 Enrichment broth storage at 2 - 8°C for 72 h
224 samples were tested again after enrichments and lysates storage for
72 h at 2 - 8°C. The following changes are observed (See table 17).
Table 17
Sample No Before storage After storage
Enrichment Lysate
2761 ND PA ND
3150 PD PD NA
3146 NA PD PD
4441 PD PD NA
6178 ND PA PA
3527 PA PA ND
3538 NA PD NA
6179 ND ND PA
3749 PD PD NA
4382 PA PA ND
3405 ND PA PA
3752 ND PA ND
4155 PA PA ND
6248 PA PA ND
6189 PA PA ND
6201 PA PA ND
6203 PA PA ND
6873 ND PA PA
7574 NA PD NA
6881 ND PA PA
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The analysis of discordant becomes (See table 18).
Table 18
Category Enrichment storage for 72h at 2-8°C Lysate storage for 72h at 2-8°C
PD ND+PPND (ND+PPND)-PD AL PD ND+PPND (ND+PPND)-PD AL
Composite foods /Ready to eat / Ready to reheat
5 2 -3 3 4 3 -1 3
Meat products 9 8 -1 3 7 8 1 3
Dairy products 8 2 -6 3 7 4 -3 3
Vegetables 8 5 -3 3 7 6 -1 3
Seafood and fishery products
4 4 0 3 4 7 3 3
Environmental samples 4 5 1 3 4 6 2 3
Total 38 26 -12 6 33 34 1 6
The observed values for ((ND + PPND) - PD) are below or equal to the
acceptability limit for each category and for all the categories.
Based on the AFNOR technical rules, the deviations and positive samples
have to be tested for the storage protocols. Three NA were as well tested
here: the samples No 3146, 5358 and 7574. They all became positive after
storage of the broth, and one sample became positive after storage of the
lysate. Note that the inoculation level requirements were as well recently
changed in the AFNOR technical rules, corresponding now more to the limits
of detection of the methods. In that way, the probability to get positive data
from firstly screened negative samples is higher, particularly if these samples
were supposed to be naturally contaminated or inoculated.
As the AL are based on differences between ND and PD, it may be
relevant to tests as well the NA.
3.1.8 Confirmation
Confirmations of the positive MDA tests were performed by streaking the
enrichment broth (Half Fraser for General protocol and Fraser for Specific
protocol) onto O&A plates.
In 8 cases, it was necessary to apply additional confirmatory tests to confirm
the presence of Listeria monocytogenes in the enrichment broth; they are
listed in table 19.
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Table 19
Sample No Additional tests applied
4441 Subculture in Fraser
3530 Streaking onto RAPID’L.mono agar
4459 Recovered from enrichment storage for 72 h at 2 - 8°C
6203 Streaking onto RAPID’L.mono agar
6368 Streaking onto RAPID’L.mono agar
6194 Streaking onto RAPID’L.mono agar
6870 Recovered from enrichment storage for 72 h at 2 - 8°C
6880 Subculture in Fraser
3.1.9 Inhibitions
One inhibition was observed (sample No 2938); this sample gave a positive
MDA test after lysate dilution (1/10). This sample was considered negative
for interpretation, and gave a negative deviation.
3.2 Relative level of detection study
The relative level of detection is the level of detection at P = 0.50 (LOD50) of the alternative
(proprietary) method divided by the level of detection at P = 0.50 (LOD50) of the reference
method. The RLOD is defined as the ratio of the alternative and reference methods:
3.2.1 Experimental design
Six (matrix/strain) pairs were analyzed by the reference method and by the
alternative method (See Table 20):
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Table 20 - Defined (matrix/strain) pairs for the RLOD determination
Matrix and related
category number Inoculated strain Origin
Inoculation
protocol
Analysis
protocol
- Deli-salad (Piémontaise) Listeria monocytogenes Ad494 Deli salad
Seeding
protocol
with 4 °C
storage prior
analyses
General
- Beef meat Listeria monocytogenes Ad1206 Ground beef Specific
- Raw milk Listeria monocytogenes Ad618 Cheese Specific
- Bagged raw spinach Listeria monocytogenes Ad1178 Ready-to-cook General
- Cold smoked salmon Listeria monocytogenes Ad670 Smoked salmon General
- Process water Listeria monocytogenes Ad551 Environmental
sample General
3.2.2 Calculation and interpretation of the RLOD
The raw data are given in Appendix 3.
The RLOD calculations were performed using Excel spreadsheet of the
international standard (ISO 16140), as described in the EN ISO 16140-
2:2016 standard (http://standards.iso.org/iso/16140). The RLOD are given
table 21.
Table 21 – Presentation of RLOD before and after confirmation of the
alternative method results
Name RLOD RLODL RLODU b=ln
(RLOD) sd(b)
z-Test statistic
p-value
Deli-salad (Piémontaise) / L.monocytogenes Ad494 1.0 0.4 3.0 0.0 0.5 0.1 0.9
Beef meat / L.monocytogenes Ad1206 0.7 0.3 1.6 -0.4 0.4 0.9 1.6
Raw milk / L.monocytogenes Ad618 1.0 0.4 2.2 0.0 0.4 0.0 1.0
Bagged raw spinach / L.monocytogenes Ad1178 1.2 0.5 3.0 0.1 0.5 0.3 0.8
Cold smoked salmon / L.monocytogenes Ad670 1.0 0.5 2.3 0.0 0.4 0.1 0.9
Process water / L.monocytogenes Ad551 1.0 0.5 2.2 0.0 0.4 0.0 1.0
The RLOD are below the AL fixed at 2.5 for unpaired studies for all the tested
matrix/strain pairs and for the alternative method.
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3.3 Inclusivity / exclusivity
Inclusivity is the ability of the alternative method to detect the target analyte from a wide range
of strains.
Exclusivity is the lack of interference from a relevant range of non-target strains of the
alternative method.
3.3.1 Test protocols
50 targeted strains and 30 non targeted strains were tested.
Inclusivity
50 Listeria monocytogenes strains were thawed and grown in BHI broth
overnight at 37°C ± 1°C. The cultures were diluted in peptone salt in order to
inoculate between 10 to 100 cells per 225 ml of Half Fraser. The enrichment
broths were then incubated for 24 h at 37°C ± 1°C. The protocol of the
alternative method was then applied.
Exclusivity
30 non Listeria strains were thawed and grown in BHI broth overnight at
37°C ± 1°C. The cultures were diluted in order to inoculate 105 cells/ml of an
appropriate media (BPW or MRS). The broths were then incubated for 24 h
at the appropriate incubation temperature in order to have culture to test with
the alternative method.
3.3.2 Results
Raw data are given in Appendix 4.
Inclusivity
The 50 specific inclusivity strains tested were detected.
Exclusivity
Among the 30 non target strains tested, no cross reaction was observed.
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3.4 Practicability
The alternative method practicability was evaluated according to the AFNOR
criteria relative to method comparison study.
Storage conditions, shelf-
life and modalities of
utilization after first use
The storage temperature is: 2-8°C. The shelf-life is given on the package. All the
reagents shall be stored at the temperature mentioned on the package.
Time to result Negative samples
Steps Reference
method
Alternative method
General
protocol
Specific
protocol
Sampling enrichment Day 0 Day 0 Day 0
Subculture in Fraser 1 Day 1 / /
Lysate / Day 1 Day 2
MDA test / Day 1 Day 2
Streaking onto O&A plates
(O1/P1) Day 1 / /
Second streaking (O2/P2) Day 3 / /
Reading plates (O1 – P1) Day 2 - Day 3 / /
Reading plates (O2 – P2) Day 4 - ay 5 / /
Results Day 5 Day 1 Day 2
Presumptive positive or positive results
Steps Reference
method
Alternative method
General
protocol
Specific
protocol
Sub-culture of typical colonies Day 2 - Day 5 / /
Streaking onto O&A plates / Day 1 Day 2
O&A plates reading / Day 2 - Day 3 Day 3 - Day 4
Confirmatory tests Day 3 - Day 6 / /
Results Day 4 - Day 7
Day 8 - Day 11 (1) Day 2 - Day 3 Day 3 - Day 4
(1) In the case of the rhamnose and xylose, tests are realized in tubes.
Common step with the
reference method No common step
The negative results are available in one day for the general protocol, and in
two days for the specific protocol.
The positive results are available in two or three days for the general
protocol, and in three or four days for the specific protocol.
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4 INTER-LABORATORY STUDY ORGANISATION AND
RESULTS
4.1 Study organisation
Collaborators number
Samples were sent to 13 laboratories. For 2 Labs, 2 technicians were
involved in the study: Labs K and L in order to have 15 sets of data.
Matrix and strain used
Pasteurised cheese sample (Brie: 31 % fat, 1.4 % NaCl) was inoculated with
Listeria monocytogenes 153, isolated from raw milk cheese.
Samples
Samples were prepared and inoculated on Monday 27th June 2016, as
described below:
- 24 blind coded samples for analysis of Listeria monocytogenes by the
3MTM Molecular Detection Assay 2 - Listeria monocytogenes method
(= RED LABEL)
- 24 blind coded samples for Listeria monocytogenes analysis by the
reference method (EN ISO 11290-1/A1) (= BLUE LABEL)
- 1 sample for aerobic mesophilic flora enumeration by ISO 4833-1 method,
- 1 water flask labelled “Temperature Control” with a temperature probe.
Inoculation
The targeted inoculation levels were the following:
- Level: 0 CFU/25 g,
- Level 1: 2 CFU/g, inoculation level providing as much as possible
fractional positive recovery data;
- Level 2: 8 CFU/25 g.
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Labelling and shipping
Blind coded samples were placed in isothermal boxes, which contained
cooling blocks, and express-shipped to the different laboratories.
A temperature control flask containing a sensor was added to the package in
order to register the temperature profile during the transport, the package
delivery and storage until analyses.
Samples were shipped in 24 h to 48 h to the involved laboratories. The
temperature conditions had to stay lower or equal to 8°C during transport,
and between 0°C – 8°C in the labs.
Analyses
Collaborative study laboratories and the expert laboratory carried out the
analyses on Tuesday 28th or Wednesday 29th June 2016 with the alternative
and reference methods. The analyses by the reference method and the
alternative method were performed on the same day.
Expedition conditions
The collaborative study instructions were sent on 10th June 2016.
4.2 Experimental parameters controls
4.2.1 Sample stability
Contamination levels
In order to detect the presence of Listeria monocytogenes, the reference
method was performed on five portions (25 g) before the inoculation. All the
results were negative.
The contamination levels and the confidence intervals were (See Table 22):
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Table 22
Level Samples Theoretical
target level
True level
(CFU/25 g
sample)
Low limit /
CFU/25 g sample
High limit /
CFU/25 g sample
Level 0
7 - 10 - 11 - 14 -
16 - 18 - 20 - 23 0 / / /
26 - 29 - 30 - 32 -
34 - 37 - 38 - 43
Low level
1 - 3 - 4 - 9 -
15 - 19 - 21 - 22 2 2.4 2.0 2.9
27 - 31 - 33 - 36 -
41 - 44 - 45 - 48
High level
2 - 5 - 6 -8 -
12 - 13 - 17 - 24 8 8.8 7.1 10.9
25 - 28 - 35 - 39 -
40 - 42 - 46 - 47
Strain stability during transport
Three samples inoculated at a high level (100 CFU/g) were tested for
enumeration after 24 h and 48 h storage. Three samples inoculated at a low
level were tested for detection after 24 h and 48 h storage (See Table 23).
Table 23 – Listeria monocytogenes stability in the matrix
Day of analysis Listeria monocytogenes detection Mesophilic aerobic
flora (CFU/g) CFU/g Detection/25 g
Day 0
80 +
2.0 108 110 +
100 +
Day 1
120 +
1.9 108 40 +
100 -
Day 2
110 +
3.4 108 170 +
90 +
No evolution was observed during storage for 48 h at 2 - 8°C.
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4.2.2 Logistic conditions
The temperatures measured at reception by the Labs, the temperatures
registered by the thermo-probe, and the receipt dates are given in Table 24.
Table 24 - Sample temperatures at receipt
Collaborators
Temperature
measured
at receipt (°C)
Temperature
measured
by the probe (°C)
Receipt date
and time Analysis date
A 6.0 4.0 28/06/2016 10h30 28/06/2016
B 5.8 3.5 28/06/2016 10h30 28/06/2016
C 5.5 4.5 28/06/2016 10h30 28/06/2016
D 6.4 5.5 28/06/2016 10h00 28/06/2016
E 6.9 4.5 28/06/2016 11h50 28/06/2016
F 5.5 5.0 28/06/2016 13h50 28/06/2016
G 8.0 4.5 28/06/2016 10h00 28/06/2016
H 6.8 4.0 28/06/2016 13h22 29/06/2016
I 6.5 5.0 28/06/2016 11h10 29/06/2016
J 6.5 4.5 28/06/2016 11h40 28/06/2016
K1 10.7 5.0 28/06/2016 10h10 28/06/2016
K2 10.7 4.5 28/06/2016 10h10 28/06/2016
L1 4.4 3.5 28/06/2016 11h00 29/06/2016
L 2 4.8 4.0 28/06/2016 11h00 29/06/2016
N 5.5 5.0 28/06/2016 10h50 28/06/2016
All the samples were delivered on time and in appropriated conditions.
Temperatures during shipment and at receipt were all correct.
Note that Lab K measured a temperature of 10.7°C at receipt while the
temperature measured with the probe indicated clearly a temperature of
5.0°C.
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4.3 Calculation and summary of data
The raw data are given in Appendix 7.
4.3.1 Results obtained by the expert Lab.
The results obtained by the expert Lab. are the following (See table 4).
Table 25 – Results obtained by the expert Lab.
Level Reference method Alternative method
L0 0/8 0/8
L1 8/8 7/8
L2 8/8 8/8
For Level 1, one sample (M4) gave a negative MDA test while the
confirmatory tests concluded to the presence of Listeria monocytogenes in
the enrichment broth. The lysate was tested twice again and one positive and
one negative results were observed. A second lysate was also tested and a
positive MDA result was obtained.
4.3.2 Results obtained by the collaborators
Samples were sent to 15 collaborators.
Mesophilic aerobic flora
The enumeration of the mesophilic aerobic flora varies from 6.4 107 to
4.2 109 CFU/g, which is a high level of background microflora.
Reference method
The positive results by the reference method are provided in Table 26.
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Table 26 - Positive results by the reference method (ALL the collaborators)
Collaborators Contamination level
L0 L1 L2
A 0 8 8
B 0 8 8
C 0 7 8
D 0 7 8
E 0 8 8
F 0 8 8
G 0 7 8
H 0 8 8
I 0 8 8
J 0 6 8
K1 0 8 8
K2 1 8 8
L1 0 8 8
L2 0 8 8
N 0 8 8
Total P0 = 1 P1 = 115 P2 = 120
Alternative method
The positive results of the alternative method (before and after confirmation)
are given in table 27.
Table 27 - Positive results (before and after confirmation) by the alternative
method (ALL the collaborators)
Collabo- rators
Contamination level
L0 L1 L2
Before confirmation
Confir- mation
After confirmation
Before confirmation
Confir- mation
After confirmation
Before confirmation
Confir- mation
After confirmation
A 0 0 0 8 8 8 8 8 8
B 0 0 0 5 5 5 8 8 8
C 0 0 0 8 8 8 8 8 8
D 0 0 0 8 8 8 8 8 8
E 0 0 0 8 8 8 8 8 8
F 0 4 0 8 8 8 8 8 8
G 0 0 0 6 6 6 8 8 8
H 0 0 0 8 8 8 8 8 8
I 0 0 0 8 8 8 8 8 8
J 0 0 0 7 7 7 8 8 8
K1 0 0 0 7 7 7 8 8 8
K2 0 0 0 6 6 6 8 8 8
L1 1 0 0 8 8 8 8 8 8
L2 0 0 0 7 7 7 8 8 8
N 1 3 0 7 7 7 8 8 8
Total P0 = 2 C = 7 CP0 = 0 P1 = 109 C = 109 CP1 = 109 P2 = 120 C = 120 CP2 = 120
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The following unexpected results were observed (See Appendix 7):
- Collaborator F detected the presence of Listeria monocytogenes in the
enrichment broth (O&A plates) of the alternative method for 4 unspiked
samples (F14, F16, F18 and F20) (See Appendix 7).
- Collaborator K2 found a positive result with the reference method for a
control sample (K26).
- Collaborator L obtained a positive MDA test for a control sample (L23); the
analysis was repeated twice and negative results were then observed.
- Collaborator N detected the presence of Listeria monocytogenes in the
enrichment broth of the alternative method for 3 unspiked samples (N16,
N18 and N23) (See Appendix 7)
A positive MDA test was obtained for a control sample (N14) but the
presence of Listeria monocytogenes was not confirmed in the enrichment
broth. The Collaborator proceeded to a new extraction on this sample and
a negative result was then observed.
For sample N21 (Level 1), a negative MDA test was observed while the
presence of Listeria monocytogenes was confirmed on O&A plates only.
It was asked to the Collaborators to send the strains isolated from unspiked
samples. The expert laboratory received only the plates from Collaborator N.
A fingerprinting using the Sma I restriction enzyme was run on the strains in
order to compare the profiles obtained with the profile of the inoculated strain.
The following profiles were obtained (See Figure 1).
Figure 1
These results show clearly that the strains isolated from the unspiked
samples were the same strains as that inoculated for Levels 1 and 2.
Inoculated
strain
N23
N21
N18
N16
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Two Collaborators (F and N) were not retained for interpretation due to cross
contamination at the level L0.
According to the AFNOR technical rules, it is possible to keep collaborators
with one unspiked sample providing a positive result per tested method. This
was applied in this study for Collaborators K2 and L1.
Finally, the results obtained by 13 Collaborators were kept for interpretation;
they are provided in Tables 28 and 29.
Table 28 - Positive results by the reference method
(Without Collaborators F and N)
Collaborators Contamination level
L0 L1 L2
A 0 8 8
B 0 8 8
C 0 7 8
D 0 7 8
E 0 8 8
G 0 7 8
H 0 8 8
I 0 8 8
J 0 6 8
K1 0 8 8
K2 1 8 8
L1 0 8 8
L2 0 8 8
Total P0 = 1 P1 = 99 P2 = 104
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Table 29 - Positive results (before and after confirmation)
by the alternative method (Without Collaborators F and N)
Collabo- rators
Contamination level
L0 L1 L2 Before
confirmation Confir- mation
After confirmation
Before confirmation
Confir- mation
After confirmation
Before confirmation
Confir- mation
After confirmation
A 0 0 0 8 8 8 8 8 8
B 0 0 0 5 5 5 8 8 8
C 0 0 0 8 8 8 8 8 8
D 0 0 0 8 8 8 8 8 8
E 0 0 0 8 8 8 8 8 8
G 0 0 0 6 6 6 8 8 8
H 0 0 0 8 8 8 8 8 8
I 0 0 0 8 8 8 8 8 8
J 0 0 0 7 7 7 8 8 8
K1 0 0 0 7 7 7 8 8 8
K2 0 0 0 6 6 6 8 8 8
L1 1 0 0 8 8 8 8 8 8
L2 0 0 0 7 7 7 8 8 8
Total P0 = 1 C =0 CP0 = 0 P1 = 94 C = 94 CP1 = 94 P2 = 104 C = 104 CP2 = 104
The percentage specificities (SP) of the reference method and of the
alternative method, using the data after confirmation, based on the results of
level L0 are the following:
Specificity for the reference method:
Specificity for the alternative method:
Taking into account the results obtained for Level 1 (level for which fractional
recoveries was observed), the summary is the following (See table 309).
Table 30 - Summary of the obtained results with the reference method
and the alternative method for Level 1
Response Reference method
positive (R+)
Reference method
negative (R-)
Alternative method
positive (A+)
Positive agreement (A+/R+)
PA = 90
Positive deviation (R-/A+)
PD = 4
Alternative method
negative (A-)
Negative deviation (A-/R+)
ND = 9
Negative agreement (A-/R-)
NA = 1
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Based on the data summarised in Table 30, the values of sensitivity of the
alternative and reference methods, as well as the relative trueness and false
positive ratio for the alternative method taking account the confirmations, are
the following (See Table 31):
Table 31
Sensitivity for the
alternative method: SEalt =
91.3 %
Sensitivity for the
reference method: SEref =
96.1 %
Relative trueness AC RT =
87.5 %
False positive ratio for
the alternative method FPR =
0 %
4.4 Interpretation
For Level 1, the negative deviations are listed in Table 32 and the positive
deviations in Table 33.
Table 32 - Negative deviations
Collaborator Sample No MDA 2 test Confirmation
B
B15 - -
B21 - -
B22 - -
G G9 - -
G15 - -
K
K3 - -
K27 - -
K31 - -
L L36 - -
Note that for all the samples, the presence of Listeria monocytogenes was
not confirmed in the enrichment broths of the alternative method.
Table 33 - Positive deviations
Collaborator Sample No
C C19
D D21
G G19
J J19
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For an unpaired study, the difference between (ND – PD) is calculated for
the level(s) where fractional recovery is obtained (so L1 and possibly L2). The
observed value found for (ND – PD) shall not be higher than the AL. The AL
is defined as [(ND – PD)max] and calculated per level where fractional
recovery is obtained as described below using the following three
parameters:
refx
x
Pp
N
where
Px = number of samples with a positive result obtained with the reference method
at level x (L1 or L2) for all the laboratories
Nx = number of samples tested at level x (L1 or L2) with the reference method by
all the laboratories.
altx
x
CPp
N
where
CPx = number of samples with a confirmed positive result obtained with the
alternative method at level x (L1 or L2) for all the laboratories;
Nx = number of samples tested at level x (L1 or L2) with the alternative method by
all the laboratories.
max ref alt ref altND-PD 3 2xN p p p p
where
Nx = the total number of samples tested for level x (L1 or L2) with the
reference method by all the laboratories.
The AL is not met when the observed value is higher than the AL.
In this study, fractional recovery was observed at Level 1. According to the
EN ISO 16140-2:2016, the calculations are the following (See table 34).
Table 34
NX 104
(p+)ref 1.0
(p+)alt 0.9
AL = (ND - PD) max 6.49
ND - PD 5
Conclusion ND - PD < AL
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The EN ISO 16140-2:2016 requirements are fulfilled as (ND - PD) is lower
than the AL for Level 1.
The RLOD calculations were performed using Excel spreadsheet of the
international standard (ISO 16140), as described in the EN ISO 16140-
2:2016 standard (http://standards.iso.org/iso/16140). The results are used
only for information (see table 35).
Table 35
RLOD RLODL RLODV b=ln(RLOD) sd(b) z Test
statistic p.value
1.3 0.9 1.9 0.3 0.2 1.4 0.2
5 CONCLUSION
The method comparison study conclusions are:
The method comparison study scheme corresponds to an UNPAIRED
STUDY design as the alternative and reference methods do have split
enrichment procedures.
In the sensitivity study, 6 categories were tested: 5 food categories
and the environmental samples. The protocol of the alternative
method shows 35 positive deviations (PD) and 33 negative deviations
(ND) for the overall categories. The values for ((ND+ PPND) - PD) are
below the acceptability limits (AL) whatever the categories, and as well
for the 6 tested categories.
The Relative Levels of Detection (RLOD) are all below the AL fixed at
2.5 for the unpaired data study whatever the matrix/strain pairs.
The inclusivity and exclusivity testing did give the expected results for
the 50 target strains and the 30 non target strains.
It is possible to store the primary enrichment broth for all the
categories and the lysates for 72 h at 2-8°C for all the categories,
except dairy products and environmental samples.
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The alternative method allows a one-day screening of the negative
samples for the general protocol, and two-days for the specific
protocol.
The alternative method fulfils all the EN ISO 16140-2:2016 and
AFNOR technical rules requirements.
The inter-laboratory study conclusions are:
The data and interpretations comply with the EN ISO 16140-2:2016
requirements. The 3MTM Molecular Detection Assay 2 -
Listeria monocytogenes is considered equivalent to the ISO
standard.
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Appendix 1 – Flow diagram of the EN ISO 11290-1/A1 (2004) method:
Horizontal method for the detection and enumeration of Listeria
monocytogenes - Part 1: detection of Listeria monocytogenes in foods
25 g or ml of the sample
1/10 dilution in ½ Fraser enrichment broth
Incubation 30°C 1°C, 24 h 3 h
0,1 ml ½ Fraser broth in 10 ml
Fraser 1 enrichment broth
Streaking on Palcam and O&A
Incubation 37°C 1°C,
48 3h
Streaking on Palcam
and OAA
Incubation 37°C 1°C, 24 h 3 h
Presence of characteristic colonies
of Listeria spp.
Yes No
Confirmation test Incubation 18 - 24 h added
37°C 1°C
Presence of characteristic
colonies of Listeria spp.
L. monocytogenes Other than
L. monocytogenes
Yes
Confirmation test
No
Presence of
L. monocytogenes
x g or ml
Absence of
L. monocytogenes
x g or ml
L. monocytogenes Other than
L. monocytogenes
Absence of
L. monocytogenes
x g or ml
Presence of
L. monocytogenes
x g or ml
Absence of
L. monocytogenes
x g or ml
Confirmation tests: Gram, Catalase, Haemolysis, Camp Test, API Listeria Gallery
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Appendix 2 – Flow diagram of the 3MTM Molecular Detection Assay 2 -
Listeria monocytogenes
Specific protocol General protocol
Raw dairy products
Raw Seafood and
Raw meat products
Food products
(excluding raw meats and raw
seafood and raw milk based
products)
Environmental samples
25 g + 225 ml
Half Fraser
25 g + 225 ml
Half Fraser
Incubation for
22 h ± 2 h
at 37°C
Subculture in
Fraser broth
Incubation for
22 h ± 2 h
at 37°C
Incubation for
27 h ± 3 h
at 37°C ± 1°C
Possibility
to store 72 h
at 2-8°C
Lysis
Possibility
to store the
lysates for
72 h at 2-8°C
except for dairy
products and
environmental
samples
Amplification and detection by the Listeria monocytogenes kit
Confirmation:
- by streaking 100 µl of Half Fraser broth onto Palcam and O&A plates
(General protocol)
- by streaking 10 µl of Fraser broth onto Palcam and O&A plates
(Specific protocol)
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Specific protocol 1 Raw dairy, Raw meat, Raw seafood
products
25 g + 225 ml Half Fraser (d 1/10)
Incubation for 22 h 2 h
at 37°C 1°C
Lysis on 10 µl enrichment broth
MDA 2 test on 20 µl lysate (Listeria monocytogenes kit)
-
Confirmatory tests: streak 10 µl Fraser
onto O&A and Palcam
Storage for 72 h at 2-8°C
Lysis
MDA 2 test
Confirmatory tests:
streak 10 µl Fraser onto O&A and Palcam
STOP
* This is done during the validation study in order to confirm that the typical colonies observed on the plates are really Listeria strains.
+
- +
Subculture in Fraser broth
Incubation for 22 h 2 h
at 37°C 1°C
3M
ADRIA Développement 39/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
General protocol
Food products
(excluding raw meats and raw seafood and
raw milk based products)
Environmental samples
25 g + 225 ml Half Fraser (d 1/10)
Incubation for 27 h 3 h
at 37°C 1°C
Lysis on 20 µl enrichment broth
MDA 2 test on 20 µl lysate (Listeria monocytogenes kit)
-
Confirmatory tests: streak 100 µl Half Fraser
onto O&A and Palcam
Storage for 72 h at 2-8°C
Storage for 72 h at 2-8°C
(except for Dairy products and environmental
samples)
Lysis
MDA 2 test
Confirmatory tests:
streak 100 µl Half Fraser
onto O&A and Palcam
STOP
+
- +
3M
ADRIA Développement 40/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Appendix 3 – Relative level of detection study: raw data
Matrix : Deli-salad (Piémontaise) Strain : Listeria monocytogenes Ad494 Aerobic mesophilic flora :210 CFU/g
N° sample Level Contamination level- (cfu/sample)-MPN
determination
Reference method: ISO 11290-1/A1 Number positive samples/Total
Alternative method: 3M MDA 2 - Listeria monocytogenes Number positive
samples/Total Half Fraser Fraser Final result O&A Palcam O&A Palcam MDA test
Confirmation result
Final result
3369
0 /
st - - - -
0/5
- - -
0/5 3370 st - - - - - - - 3371 st - - - - - - - 3372 st st - - - - - - 3373 st - - - - - - - 3374
Low 0,6
st - - - -
4 /20
- - -
6/20
3375 st - - - - + + + 3376 st st st st - + + + 3377 H+ + / / + + + + 3378 st - - - - - - - 3379 st - - - - + + + 3380 st - - - - - - - 3381 st st - - - + + + 3382 st st st st - - - - 3383 st st - - - - - - 3384 st - - - - - - - 3385 st - - - - - - - 3386 H+ + / / + - - - 3387 st st - - - - - - 3388 st - - - - - - - 3389 H+ + / / + - - - 3390 H+ + / / + - - - 3391 st - - - - - - - 3392 st - - - - + + + 3393 st st - - - - - - 3394
High 1,7
H+ + / / +
3/5
- - -
1/5 3395 H+ + / / + - - - 3396 H+ + / / + + + + 3397 st - - - - - - - 3398 st - - - - - - -
Analyses performed according to the COFRAC accreditation
3M
ADRIA Développement 41/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Matrix : Beef meat Strain : Listeria monocytogenes Ad1206
Aerobic mesophilic flora : 5,0.105 CFU/g
N° sample Level Contamination level- (cfu/sample)-MPN
determination
Reference method: ISO 11290-1/A1 Number positive samples/Total
Alternative method: 3M MDA 2 - Listeria monocytogenes
Number positive samples/Total Half Fraser Fraser
Final result O&A Palcam O&A Palcam MDA test Confirmation result Final result
7950
0 /
st st st st -
0/5
- - -
0/5 7951 st st st st - - - - 7952 st st st - - - - - 7953 st st st - - - - - 7954 H- (L.welshimeri) + H- + - - - - 8026
Low 0,7
H- + H- + -
8/20
- - -
8/20
8027 H+/H- + H+/H- + + - - - 8028 H+/H- + H+/H- + + - - - 8029 H+/H- + H+/H- + + - - - 8030 H- + H- + - + + + 8031 H- + H- + - - - - 8032 H+/H- + H+/H- + + - - - 8033 H- + H- + - - - - 8034 H+/H- + H+/H- + + + + + 8035 H- + H- + - - - - 8036 H- + H- + - + + + 8037 H- + H- + - + + + 8038 H+/H- + H+/H- + + - - - 8039 H- + H- + - + + + 8040 H+/H- + H+/H- + + - - - 8041 H+/H- + H+/H- + + - - - 8042 H- + H- + - + + + 8043 H- + H- + - + + + 8044 H- + H- + - - - - 8045 H- + H- + - + + + 8046
High 2
H+/H- + H+/H- + +
3/5
+ + +
5/5 8047 H+/H- + H+/H- + + + + + 8048 H+/H- + H+/H- + + + + + 8049 H- + H- + - + + + 8050 H- + H- + - + + +
Analyses performed according to the COFRAC accreditation
3M
ADRIA Développement 42/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Matrix : Raw milk
Strain : Listeria monocytogenes Ad618 Aerobic mesophilic flora : 6,4.105 CFU/g
N° sample Level Contamination level- (cfu/sample)-MPN
determination
Reference method: ISO 11290-1/A1 Number positive samples/Total
Alternative method: 3M MDA 2 - Listeria monocytogenes Number positive
samples/Total Half Fraser Fraser Final result O&A Palcam O&A Palcam MDA test
Confirmation result
Final result
5481
0 /
St St - - -
0/5
- - -
0/5 5482 St St St - - - - - 5483 St - St - - - - - 5484 St - - - - - - - 5485 St St - - - - - - 5486
Low 0,5
St St - - -
11/20
- - -
11/20
5487 - - - - - - - - 5488 H+ + H+ + + - - - 5489 H+ + H+ + + -/+/+ + - 5490 H+ + H+ + + - - - 5491 H+ + H+ + + + + + 5492 - - St St - + + + 5493 H+ + H+ + + + + + 5494 H+ + H+ + + + + + 5495 H+ + H+ + + + + + 5496 - - St - - - - - 5497 St St - - - + + + 5498 St - - - - + + + 5499 H+ + H+ + + - - - 5500 St - St - - - - - 5501 - - - - - + + + 5502 H+ + H+ + + + + + 5503 H+ + H+ + + + + + 5504 St - - - - + + + 5505 St - H+ + + - - - 5506
High 1,7
H+ + H+ + +
5/5
+ + +
5/5 5507 H+ + H+ + + + + + 5508 H+ + H+ + + + + + 5509 H+ + H+ + + + + + 5510 H+ + H+ + + + + +
Analyses performed according to the COFRAC accreditation
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Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Matrix : Bagged raw spinach
Strain : Listeria monocytogenes Ad1178 Aerobic mesophilic flora : 8.0 106 CFU/g
N° sample Level Contamination level- (cfu/sample)-MPN
determination
Reference method: ISO 11290-1/A1 Number positive samples/Total
Alternative method: 3M MDA 2 - Listeria monocytogenes Number positive
samples/Total Half Fraser Fraser Final result O&A Palcam O&A Palcam MDA test
Confirmation result
Final result
2369
0 /
- - - - -
0/5
- - -
0/5 2370 - - - - - - - - 2371 - - - - - - - - 2372 - - - - - - - - 2373 - - st st - - - - 2374
Low 1,2
H+ + H+ + +
10 /20
+ - + (subculture in Fraser)
9/20
2375 - - - - - - + - 2376 - - - - - + + + 2377 - - - - - + - + (subculture in Fraser) 2378 H+ + H+ + + - - - 2379 - - st st - - - - 2380 H+ + H+ + + + + 2381 H+ + H+ + + - - - 2382 - - - - - + + + 2383 - - - - - + + + 2384 - - - - - - - - 2385 H+ + H+ + + + + + 2386 - - - - - - - - 2387 H+ + H+ + + + + + 2388 H+ + H+ + + - - - 2389 H+ + H+ + + - - - 2390 H+ + H+ + + - - - 2391 H+ + H+ + + + + + 2392 - - st st - - - - 2393 - - - - - - - - 2394
High 2,9
H+ + H+ + +
5/5
+ + +
5/5 2395 H+ + H+ + + + + + 2396 H+ + H+ + + + + + 2397 H+ + H+ + + + + + 2398 H+ + H+ + + + + +
Analyses performed according to the COFRAC accreditation
3M
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Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Matrix : Cold smoked salmon
Strain : Listeria monocytogenes Ad670 Aerobic mesophilic flora : 400 CFU/g
N° sample Level Contamination level-
(cfu/sample)- MPN determination
Reference method: ISO 11290-1/A1 Number positive samples/Total
Alternative method: 3M MDA 2 - Listeria monocytogenes Number positive
samples/Total Half Fraser Fraser Final result O&A Palcam O&A Palcam MDA test
Confirmation result
Final result
1999
0 /
- - st st -
0/5
- - -
/5 2000 st st st st - - - - 2001 - - st st - - - - 2002 st st st st - - - - 2003 st st st st - - - - 2004
Low 0,4
H+ + / / +
9/20
- - -
10 /20
2005 H+ + / / + + + + 2006 st st st st - - - - 2007 st st st st - + + + 2008 st st st st - - - - 2009 H+ + / / + - - - 2010 H- + H- + - + + + 2011 st st st st - - - - 2012 H+ + / / + - - - 2013 H+ + / / + - - - 2014 st st st st - - - - 2015 H+ + / / + + + + 2016 H- + H- + - + + + 2017 H+ + / / + + + + 2018 st - - st - + + + 2019 H+ + / / + - - - 2020 st st st st - + + + 2021 H+ + / / + - - - 2022 st st st st - + + + 2023 st st st st - + + + 2024
High 2,7
st st st st -
4/5
+ + +
3/5 2025 H+ + / / + - - - 2026 H+ + / / + - - - 2027 H+ + / / + + + + 2028 H+ + / / + + + +
Analyses performed according to the COFRAC accreditation
3M
ADRIA Développement 45/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Matrix : Process water
Strain : Listeria monocytogenes Ad551 Aerobic mesophilic flora : 9,7.103CFU/g
N° sample Level Contamination level-
(cfu/sample)- MPN determination
Reference method: ISO 11290-1/A1 Number positive samples/Total
Alternative method: 3M MDA 2 - Listeria monocytogenes Number positive
samples/Total Half Fraser Fraser Final result O&A Palcam O&A Palcam MDA test
Confirmation result
Final result
4135
0 /
st st - - -
0/5
- - -
0/5 4136 st st st st - - - - 4137 st st st - - - - - 4138 st st st st - - - - 4139 st st st st - - - - 4353
Low 0,6
st st st st -
11 /20
- - -
11/20
4354 H+ + / / + - - - 4355 st st st st - - - - 4356 st st st st - + + + 4357 H+ + / / + + + + 4358 H+ + / / + - - - 4359 H+ + / / + - - - 4360 st st st st - - - - 4361 H+ + / / + + + + 4362 st st st st - + + + 4363 H+ + / / + + + + 4364 H+ + / / + + + + 4365 st st st st - + + + 4366 st st st st - + + + 4367 H+ + / / + + + + 4368 H+ + / / + + + + 4369 H+ + / / + - - - 4370 st st st st - + + + 4371 H+ + / / + - - - 4372 st st st st - - - - 4373
High 1,7
st st st st -
2/5
+ + +
2/5 4374 st st st st - - - - 4375 st st st st - - - - 4376 H+ + / / + - - - 4377 H+ + / / + + + +
Analyses performed according to the COFRAC accreditation
3M
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Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Appendix 4 – Inclusivity and exclusivity study: raw data
INCLUSIVITY
Strain Species Reference Origin Inoculation level
(cfu/225ml)
3M Molecular Detection Assay 2 Half Fraser broth 24h at 37°C
MDA results Confirmation 100 µl
MDA Listeria monocytogenes ALOA Palcam
1 Listeria monocytogenes 153 Soft cheese (Munster) 39 + H+ +
2 Listeria monocytogenes 1011/1410 Frozen broccoli 28 + H+ +
3 Listeria monocytogenes 1972/2399 Puff pastry with mushrooms 38 + H+ +
4 Listeria monocytogenes 1973/2400 Puff pastry egg and ham (Quiche-lorraine) 36 + H+ +
5 Listeria monocytogenes 2407/3139 Tripes with tomatoes 31 + H+ +
6 Listeria monocytogenes 2760/3145 Raw bacon 44 + H+ +
7 Listeria monocytogenes 32.183 Croque-Monsieur 31 + H+ +
8 Listeria monocytogenes 38/181 Toulouse sausages 33 + H+ +
9 Listeria monocytogenes 5721/6179 Smoked bacon 49 + H+ +
10 Listeria monocytogenes 7111/7516 Pâté (Rillettes) 64 + H+ +
11 Listeria monocytogenes 850/109 RTE food (deli salad with seafood) 34 + H+ +
12 Listeria monocytogenes 877/113 Environmental sample (pastry) 28 + H+ +
13 Listeria monocytogenes 913/1048 Black pudding 39 + H+ +
14 Listeria monocytogenes A00C014 Sausage 37 + H+ +
15 Listeria monocytogenes A00C022 Merguez 33 + H+ +
16 Listeria monocytogenes A00C024 Sausage 21 + H+ +
17 Listeria monocytogenes A00C036 Poultry (guinea) 41 + H+ +
18 Listeria monocytogenes A00C039 Sausages 20 + H+ +
19 Listeria monocytogenes A00C040 Cooked delicatessen (Museau) 36 + H+ +
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Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
INCLUSIVITY
Strain Species Reference Origin Inoculation level
(cfu/225ml)
3M Molecular Detection Assay 2 Half Fraser broth 24h at 37°C
MDA results Confirmation 100 µl
MDA Listeria monocytogenes ALOA Palcam
20 Listeria monocytogenes A00C041 Sausage 38 + H+ +
21 Listeria monocytogenes A00C042 Raw sausage 32 + H+ +
22 Listeria monocytogenes A00C043 Smoked Bacon 50 + H+ +
23 Listeria monocytogenes A00C044 Poultry (duck) 34 + H+ +
24 Listeria monocytogenes A00C052 RTE food (Osso bucco with turkey) 34 + H+ +
25 Listeria monocytogenes A00C053 Gizzards 30 + H+ +
26 Listeria monocytogenes A00C054 Beef hart 30 + H+ +
27 Listeria monocytogenes A00C055 Raw sausages 43 + H+ +
28 Listeria monocytogenes A00E008 Environmental sample 27 + H+ +
29 Listeria monocytogenes A00E049 Environmental sample (smoked salmon) 41 + H+ +
30 Listeria monocytogenes A00E082 Environmental sample (smoked salmon) 33 + H+ +
31 Listeria monocytogenes A00L097 Milk 60 + H+ +
32 Listeria monocytogenes A00M009 Smoked salmon 40 + H+ +
33 Listeria monocytogenes A00M032 Smoked salmon 11 + H+ +
34 Listeria monocytogenes A00M045 Smoked salmon 62 + H+ +
35 Listeria monocytogenes A00M088 Smoked salmon 79 + H+ +
36 Listeria monocytogenes Ad235 Poultry 45 + H+ +
37 Listeria monocytogenes Ad253 Hard cheese 58 + H+ +
38 Listeria monocytogenes Ad260 Semi hard cheese 27 + H+ +
39 Listeria monocytogenes Ad265 Tong 62 + H+ +
40 Listeria monocytogenes Ad266 Poultry 26 + H+ +
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Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
INCLUSIVITY
Strain Species Reference Origin Inoculation level
(cfu/225ml)
3M Molecular Detection Assay 2 Half Fraser broth 24h at 37°C
MDA results Confirmation 100 µl
MDA Listeria monocytogenes ALOA Palcam
41 Listeria monocytogenes Ad267 Dry sausage 26 + H+ +
42 Listeria monocytogenes Ad268 Cured ham 35 + H+ +
43 Listeria monocytogenes Ad270 Fermented sausage 42 + H+ +
44 Listeria monocytogenes Ad272 Fermented sausage 56 + H+ +
45 Listeria monocytogenes Ad273 Cured delicatessen 27 + H+ +
46 Listeria monocytogenes Ad274 Ready-to-eat food (Asiatic meal) 40 + H+ +
47 Listeria monocytogenes Ad534 Fruits 54 + H+ +
48 Listeria monocytogenes Ad544 Onion 43 + H+ +
49 Listeria monocytogenes Ad546 Flour 83 + H+ +
50 Listeria monocytogenes Ad623 Bread crumbs 50 + H+ +
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3M MDA 2 - Listeria monocytogenes
EXCLUSIVITY
Strain Species Reference Origin Inoculation level
(cfu/ml)
3M Molecular Detection Assay 2
MDA results
MDA Listeria monocytogenes MC
1 Listeria grayi Ad 1198 Smoked salmon 3.2 105 - +
2 Listeria grayi Ad 1443 Pork meat sausages 4.9 105 - +
3 Listeria innocua 1 Smoked salmon 4.9 105 - +
4 Listeria innocua Ad 658 Gorgonzola 5.9 105 - +
5 Listeria ivanovii Ad 466 Raw veal meat 3.3 105 - +
6 Listeria ivanovii Ad 662 Environment (dairy industry) 3.6 105 - +
7 Listeria seeligeri Ad 649 Cheese 5.4 105 - +
8 Listeria seeligeri BR1 Trout 5.1 105 - +
9 Listeria welshimeri Ad1276 Environment (Slaughterhouse) 5.2 105 - +
10 Listeria welshimeri Ad1175 Ready-to-eat-food 6.4 105 - +
11 Bacillus cereus Ad 465 Salmon Terrine 7.2 104 - +
12 Bacillus circulans Ad 760 Vegetables 2.0 104 - +
13 Bacillus coagulans Ad 731 Dairy product <2.0 103 (opacity +) - +
14 Bacillus licheniformis Ad 978 Dairy product 1.6 104 - +
15 Bacillus pumilus Ad 284 Ready-to-eat 1.7 105 - +
16 Brochrotrix campestris CIP 102920T
Environment <2.0 103 (opacity +) - +
17 Carnobacterium piscicola Ad 369 Raw milk <2.0 103 (opacity +) - +
18 Enterococcus durans Ad 149 Ham <2.0 103 (opacity +) - +
19 Enterococcus faecalis 89L326 Soft cheese (Vacherin) 1.6 105 - +
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3M MDA 2 - Listeria monocytogenes
EXCLUSIVITY
Strain Species Reference Origin Inoculation level
(cfu/ml)
3M Molecular Detection Assay 2
MDA results
MDA Listeria monocytogenes MC
20 Lactobacillus brevis 86L126 Ham 1.4 105 - +
21 Lactobacillus curvatus Ad 380 Delicatessen <2.0 103 (opacity +) - +
22 Lactobacillus sakei Ad 473 Ham <2.0 103 (opacity +) - +
23 Leuconostoc carnosum Ad 411 Ham <2.0 103 (opacity +) - +
24 Leuconostoc citreum Ad 396 Ham 4.9 105 - +
25 Micrococcus luteus Ad 432 Cocktail <2.0 103 (opacity +) - +
26 Staphylococcus aureus Ad 165 Smoked delicatessen <2.0 103 (opacity +) - +
27 Staphylococcus epidermis Ad 931 Fruits 2.0 103 - +
28 Staphylococcus haemoliticus Ad 989 Dairy product 2.8 104 - +
29 Streptococcus bovis 92L622 Cheese 2.0 103 - +
30 Streptococcus salivarus Ad 441 Dairy product <2.0 103 (opacity +) - +
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3M MDA 2 - Listeria monocytogenes
Appendix 5 - Results obtained by the collaborators and the expert laboratory
Collaborator A Aerobic mesophilic flora: 2,0.109/g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement
Half Fraser Fraser Final result MDA test result O&A Palcam Final result
O&A Palcam O&A Palcam
A7 - - - - - - - - - NA
A10 - - - - - - - - - NA
A11 - - - - - - - - - NA
A14 - - - - - - - - - NA
A16 - - - - - - - - - NA
A18 - - - - - - - - - NA
A20 - - - - - - - - - NA
A23 - - - - - - - - - NA
A1 + + + + + + + + + PA
A3 + + + + + + + + + PA
A4 + + + + + + + + + PA
A9 + + + + + + + + + PA
A15 + + + + + + + + + PA
A19 + + + + + + + + + PA
A21 + + + + + + + + + PA
A22 + + + + + + + + + PA
A2 + + + + + + + + + PA
A5 + + + + + + + + + PA
A6 + + + + + + + + + PA
A8 + + + + + + + + + PA
A12 + + + + + + + + + PA
A13 + + + + + + + + + PA
A17 + + + + + + + + + PA
A24 + + + + + + + + + PA
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3M MDA 2 - Listeria monocytogenes
Collaborator B Aerobic mesophilic flora: 9,6 108 /g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement
Half Fraser Fraser Final result MDA test result O&A Palcam Final result
O&A Palcam O&A Palcam
B7 - - - - - - - - - NA
B10 - - - - - - - - - NA
B11 - - - - - - - - - NA
B14 - - - - - - - - - NA
B16 - - - - - - - - - NA
B18 - - - - - - - - - NA
B20 - - - - - - - - - NA
B23 - - - - - - - - - NA
B1 + + + + + + + + + PA
B3 + + + + + + + + + PA
B4 + + + + + + + + + PA
B9 + + + + + + + + + PA
B15 + + + + + - - - - ND
B19 + + + + + + + + + PA
B21 + + + + + - - - - ND
B22 + + + + + - - - - ND
B2 + + + + + + + + + PA
B5 + + + + + + + + + PA
B6 + + + + + + + + + PA
B8 + + + + + + + + + PA
B12 + + + + + + + + + PA
B13 + + + + + + + + + PA
B17 + + + + + + + + + PA
B24 + + + + + + + + + PA
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3M MDA 2 - Listeria monocytogenes
Collaborator C Aerobic mesophilic flora: 4,2 109/g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement
Half Fraser Fraser Final result MDA test result O&A Palcam Final result
O&A Palcam O&A Palcam
C7 - - - - - - - - - NA
C10 - - - - - - - - - NA
C11 - - - - - - - - - NA
C14 - - - - - - - - - NA
C16 - - - - - - - - - NA
C18 - - - - - - - - - NA
C20 - - - - - - - - - NA
C23 - - - - - - - - - NA
C1 + + + + + + + + + PA
C3 + + + + + + + + + PA
C4 + + + + + + + + + PA
C9 + + + + + + + + + PA
C15 + + + + + + + + + PA
C19 - - - - - + + + + PD
C21 + + + + + + + + + PA
C22 + + + + + + + + + PA
C2 + + + + + + + + + PA
C5 + + + + + + + + + PA
C6 + + + + + + + + + PA
C8 + + + + + + + + + PA
C12 + + + + + + + + + PA
C13 + + + + + + + + + PA
C17 + + + + + + + + + PA
C24 + + + + + + + + + PA
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3M MDA 2 - Listeria monocytogenes
Collaborator D Aerobic mesophilic flora: 2,0 109/g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement
Half Fraser Fraser Final result MDA test result O&A Palcam Final result
O&A Palcam O&A Palcam
D7 - - - - - - - - - NA
D10 - - - - - - - - - NA
D11 - - - - - - - - - NA
D14 - - - - - - - - - NA
D16 - - - - - - - - - NA
D18 - - - - - - - - - NA
D20 - - - - - - - - - NA
D23 - - - - - - - - - NA
D1 + + + + + + + + + PA
D3 + + + + + + + + + PA
D4 + + + + + + + + + PA
D9 + + + + + + + + + PA
D15 + + + + + + + + + PA
D19 + + + + + + + + + PA
D21 - - - - - + + + + PD
D22 + + + + + + + + + PA
D2 + + + + + + + + + PA
D5 + + + + + + + + + PA
D6 + + + + + + + + + PA
D8 + + + + + + + + + PA
D12 + + + + + + + + + PA
D13 + + + + + + + + + PA
D17 + + + + + + + + + PA
D24 + + + + + + + + + PA
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3M MDA 2 - Listeria monocytogenes
Collaborator E
Aerobic mesophilic flora: 1,2 109/g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement Half Fraser Fraser
Final result MDA test result O&A Palcam Final result O&A Palcam O&A Palcam
E7 - - - - - - - - - NA
E10 - - - - - - - - - NA
E11 - - - - - - - - - NA
E14 - - - - - - - - - NA
E16 - - - - - - - - - NA
E18 - - - - - - - - - NA
E20 - - - - - - - - - NA
E23 - - - - - - - - - NA
E1 + + + + + + + + + PA
E3 + + + + + + + + + PA
E4 + + + + + + + + + PA
E9 + + + + + + + + + PA
E15 + + + + + + + + + PA
E19 + + + + + + + + + PA
E21 + + + + + + + + + PA
E22 + + + + + + + + + PA
E2 + + + + + + + + + PA
E5 + + + + + + + + + PA
E6 + + + + + + + + + PA
E8 + + + + + + + + + PA
E12 + + + + + + + + + PA
E13 + + + + + + + + + PA
E17 + + + + + + + + + PA
E24 + + + + + + + + + PA
3M
ADRIA Développement 56/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Collaborator F Aerobic mesophilic flora: 4,2 108/g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement
Half Fraser Fraser Final result MDA test result O&A Palcam Final result
O&A Palcam O&A Palcam
F7 - - - - - - - - - NA
F10 - - - - - - - - - NA
F11 - - - - - - - - - NA
F14 - - - - - - + - - NA
F16 - - - - - - + - - NA
F18 - - - - - - + - - NA
F20 - - - - - - + - - NA
F23 - - - - - - - - - NA
F1 + + + + + + + + + PA
F3 + + + + + + + + + PA
F4 + + + + + + + + + PA
F9 + + + + + + + + + PA
F15 + + + + + + + + + PA
F19 + + + + + + + + + PA
F21 + + + + + + + + + PA
F22 + + + + + + + + + PA
F2 + + + + + + + + + PA
F5 + + + + + + + + + PA
F6 + + + + + + + + + PA
F8 + + + + + + + + + PA
F12 + + + + + + + + + PA
F13 + + + + + + + + + PA
F17 + + + + + + + + + PA
F24 + + + + + + + + + PA
3M
ADRIA Développement 57/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Collaborator G Aerobic mesophilic flora: 1,6 109/g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement
Half Fraser Fraser Final result MDA test result O&A Palcam Final result
O&A Palcam O&A Palcam
G7 - - - - - - - - - NA
G10 - - - - - - - - - NA
G11 - - - - - - - - - NA
G14 - - - - - - - - - NA
G16 - - - - - - - - - NA
G18 - - - - - - - - - NA
G20 - - - - - - - - - NA
G23 - - - - - - - - - NA
G1 + + + + + + + + + PA
G3 + + + + + + + + + PA
G4 + + + + + + + + + PA
G9 + + + + + - - - - ND
G15 + + + + + - - - - ND
G19 - - - - - + + + + PD
G21 + + + + + + + + + PA
G22 + + + + + + + + + PA
G2 + + + + + + + + + PA
G5 + + + + + + + + + PA
G6 + + + + + + + + + PA
G8 + + + + + + + + + PA
G12 + + + + + + + + + PA
G13 + + + + + + + + + PA
G17 + + + + + + + + + PA
G24 + + + + + + + + + PA
3M
ADRIA Développement 58/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Collaborator H Aerobic mesophilic flora: 1,7 109/g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement
Half Fraser Fraser Final result MDA test result O&A Palcam Final result
O&A Palcam O&A Palcam
H7 - - - - - - - - - NA
H10 - - - - - - - - - NA
H11 - - - - - - - - - NA
H14 - - - - - - - - - NA
H16 - - - - - - - - - NA
H18 - - - - - - - - - NA
H20 - - - - - - - - - NA
H23 - - - - - - - - - NA
H1 + + + + + + + + + PA
H3 + + + + + + + + + PA
H4 + + + + + + + + + PA
H9 + + + + + + + + + PA
H15 + + + + + + + + + PA
H19 + + + + + + + + + PA
H21 + + + + + + + + + PA
H22 + + + + + + + + + PA
H2 + + + + + + + + + PA
H5 + + + + + + + + + PA
H6 + + + + + + + + + PA
H8 + + + + + + + + + PA
H12 + + + + + + + + + PA
H13 + + + + + + + + + PA
H17 + + + + + + + + + PA
H24 + + + + + + + + + PA
3M
ADRIA Développement 59/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Collaborator I
Aerobic mesophilic flora: 2,4 109/g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement
Half Fraser Fraser Final result MDA test result O&A Palcam Final result
O&A Palcam O&A Palcam
I7 - - - - - - - - - NA
I10 - - - - - - - - - NA
I11 - - - - - - - - - NA
I14 - - - - - - - - - NA
I16 - - - - - - - - - NA
I18 - - - - - - - - - NA
I20 - - - - - - - - - NA
I23 - - - - - - - - - NA
I1 + + + + + + + + + PA
I3 + + + + + + + + + PA
I4 + + + + + + + + + PA
I9 + + + + + + + + + PA
I15 + + + + + + + + + PA
I19 + + + + + + + + + PA
I21 + + + + + + + + + PA
I22 + + + + + + + + + PA
I2 + + + + + + + + + PA
I5 + + + + + + + + + PA
I6 + + + + + + + + + PA
I8 + + + + + + + + + PA
I12 + + + + + + + + + PA
I13 + + + + + + + + + PA
I17 + + + + + + + + + PA
I24 + + + + + + + + + PA
3M
ADRIA Développement 60/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Collaborator J Aerobic mesophilic flora: 9,2 108/g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement
Half Fraser Fraser Final result MDA test result O&A Palcam Final result
O&A Palcam O&A Palcam
J7 - - - - - - - - - NA
J10 - - - - - - - - - NA
J11 - - - - - - - - - NA
J14 - - - - - - - - - NA
J16 - - - - - - - - - NA
J18 - - - - - - - - - NA
J20 - - - - - - - - - NA
J23 - - - - - - - - - NA
J1 + + + + + + + + + PA
J3 + + + + + + + + + PA
J4 + + + + + + + + + PA
J9 + + + + + + + + + PA
J15 + + + + + + + + + PA
J19 - - - - - + + + + PD
J21 - - - - - - - - - NA
J22 + + + + + + + + + PA
J2 + + + + + + + + + PA
J5 + + + + + + + + + PA
J6 + + + + + + + + + PA
J8 + + + + + + + + + PA
J12 + + + + + + + + + PA
J13 + + + + + + + + + PA
J17 + + + + + + + + + PA
J24 + + + + + + + + + PA
3M
ADRIA Développement 61/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Collaborator K1
TECHNICIAN 1 Aerobic mesophilic flora: 2,1 109/g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement
Half Fraser Fraser Final result MDA test result O&A Palcam Final result
O&A Palcam O&A Palcam
K7 - - - - - - - - - NA
K10 - - - - - - - - - NA
K11 - - - - - - - - - NA
K14 - - - - - - - - - NA
K16 - - - - - - - - - NA
K18 - - - - - - - - - NA
K20 - - - - - - - - - NA
K23 - - - - - - - - - NA
K1 + + + + + + + + + PA
K3 + + + + + - - - - ND
K4 + + + + + + + + + PA
K9 + + + + + + + + + PA
K15 + + + + + + + + + PA
K19 + + + + + + + + + PA
K21 + + + + + + + + + PA
K22 + + + + + + + + + PA
K2 + + + + + + + + + PA
K5 + + + + + + + + + PA
K6 + + + + + + + + + PA
K8 + + + + + + + + + PA
K12 + + + + + + + + + PA
K13 + + + + + + + + + PA
K17 + + + + + + + + + PA
K24 + + + + + + + + + PA
3M
ADRIA Développement 62/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Collaborator K2
TECHNICIAN 2 Aerobic mesophilic flora: 3,4.109 /g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement
Half Fraser Fraser Final result MDA test result O&A Palcam Final result
O&A Palcam O&A Palcam
K26 + + - - + - - - - ND
K29 - - - - - - - - - NA
K30 - - - - - - - - - NA
K32 - - - - - - - - - NA
K34 - - - - - - - - - NA
K37 - - - - - - - - - NA
K38 - - - - - - - - - NA
K43 - - - - - - - - - NA
K27 + + + + + - - - - ND
K31 + + + + + - - - - ND
K33 + + + + + + + + + PA
K36 + + + + + + + + + PA
K41 + + + + + + + + + PA
K44 + + + + + + + + + PA
K45 + + + + + + + + + PA
K48 + + + + + + + + + PA
K25 + + + + + + + + + PA
K28 + + + + + + + + + PA
K35 + + + + + + + + + PA
K39 + + + + + + + + + PA
K40 + + + + + + + + + PA
K42 + + + + + + + + + PA
K46 + + + + + + + + + PA
K47 + + + + + + + + + PA
3M
ADRIA Développement 63/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Collaborator L1
TECHNICIAN 1 Aerobic mesophilic flora: 6,4 107/g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement
Half Fraser Fraser Final result MDA test result O&A Palcam Final result
O&A Palcam O&A Palcam
L7 - - - - - - - - - NA
L10 - - - - - - - - - NA
L11 - - - - - - - - - NA
L14 - - - - - - - - - NA
L16 - - - - - - - - - NA
L18 - - - - - - - - - NA
L20 - - - - - - - - - NA
L23 - - - - - +/-/-(1) - - - PPNA
L1 + + + + + + + + + PA
L3 + + + + + + + + + PA
L4 + + + + + + + + + PA
L9 + + + + + + + + + PA
L15 + + + + + + + + + PA
L19 + + + + + + + + + PA
L21 + + + + + + + + + PA
L22 + + + + + + + + + PA
L2 + + + + + + + + + PA
L5 + + + + + + + + + PA
L6 + + + + + + + + + PA
L8 + + + + + + + + + PA
L12 + + + + + + + + + PA
L13 + + + + + + + + + PA
L17 + + + + + + + + + PA
L24 + + + + + + + + + PA
(1): MDA tests run on the 5 July /2016
3M
ADRIA Développement 64/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Collaborator L2
TECHNICIAN 2 Aerobic mesophilic flora: 7,0 108 /g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement
Half Fraser Fraser Final result MDA test result O&A Palcam Final result
O&A Palcam O&A Palcam
L26 - - - - - - - - - NA
L29 - - - - - - - - - NA
L30 - - - - - - - - - NA
L32 - - - - - - - - - NA
L34 - - - - - - - - - NA
L37 - - - - - - - - - NA
L38 - - - - - - - - - NA
L43 - - - - - - - - - NA
L27 + + + + + + + + + PA
L31 + + + + + + + + + PA
L33 + + + + + + + + + PA
L36 + + + + + - - - - ND
L41 + + + + + + + + + PA
L44 + + + + + + + + + PA
L45 + + + + + + + + + PA
L48 + + + + + + + + + PA
L25 + + + + + + + + + PA
L28 + + + + + + + + + PA
L35 + + + + + + + + + PA
L39 + + + + + + + + + PA
L40 + + + + + + + + + PA
L42 + + + + + + + + + PA
L46 + + + + + + + + + PA
L47 + + + + + + + + + PA
3M
ADRIA Développement 65/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Collaborator N
Aerobic mesophilic flora: 1,4 109/g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement Half Fraser Fraser
Final result MDA test result O&A Palcam Final result O&A Palcam O&A Palcam
N7 - - - - - - - - - NA
N10 - - - - - - - - - NA
N11 - - - - - - - - - NA
N14 - - - - - +/+ /-* -/- * -/-* - PPNA
N16 - - - - - - + - - NA
N18 - - - - - - + - - NA
N20 - - - - - - - - - NA
N23 - - - - - - + + - NA
N1 + + + + + + + + + PA
N3 + + + + + + + + + PA
N4 + + + + + + + + + PA
N9 + + + + + + + + + PA
N15 + + + + + + + + + PA
N19 + + + + + + + + + PA
N21 + + + + + - + - - ND
N22 + + + + + + + + + PA
N2 + + + + + + + + + PA
N5 + + + + + + + + + PA
N6 + + + + + + + + + PA
N8 + + + + + + + + + PA
N12 + + + + + + + + + PA
N13 + + + + + + + + + PA
N17 + + + + + + + + + PA
N24 + + + + + + + + + PA
* new extraction
3M
ADRIA Développement 66/66 November 14, 2016
Summary Report (Version 0)
3M MDA 2 - Listeria monocytogenes
Expert Laboratory M (ADRIA) Aerobic mesophilic flora: 3,4.108/g
N°Sample
Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria
for detection of Listeria monocytogenes Agreement
Half Fraser Fraser Final result MDA test result O&A Palcam Final result
O&A Palcam O&A Palcam
M7 st st st st - - st - - NA
M10 st st st st - - st - - NA
M11 st st st st - - st - - NA
M14 st st st st - - st - - NA
M16 st st st st - - st - - NA
M18 st st st st - - st - - NA
M20 st st st st - - st - - NA
M23 st st - st - - st - - NA
M1 + + + + + + H+ + + PA
M3 + + + + + + H+ + + PA
M4 + + + + + -/+(1)/-(1)/+ (2) H+ + - ND
M9 + + + + + + H+ + + PA
M15 + + + + + + H+ + + PA
M19 + + + + + + H+ + + PA
M21 + + + + + + H+ + + PA
M22 + + + + + + H+ + + PA
M2 + + + + + + H+ + + PA
M5 + + + + + + H+ + + PA
M6 + + + + + + H+ + + PA
M8 + + + + + + H+ + + PA
M12 + + + + + + H+ + + PA
M13 + + + + + + H+ + + PA
M17 + + + + + + H+ + + PA
M24 + + + + + + H+ + + PA (1): same lysate (2): second extraction
Analyses performed according to the COFRAC accreditation