MicroVal Study 2010LR32: Qualitative Method

Matrix Extension and Renewal Study Report for the ISO 16140-2:2016
Validation of the Assurance® GDS Listeria monocytogenes Tq for the
Detection of Listeria monocytogenes in a Broad Range of Foods and
Environmental Surfaces

Version 3
December 21st, 2018

Contact Details:
Q Laboratories
1400 Harrison Ave.
Cincinnati, OH 45214
Tel: 513-471-1300
www.qlaboratories.com

Report issued and authorized by:

Q Laboratories
Benjamin Bastin, Microbiology R&D Supervisor

The report is prepared in accordance with ISO 16140-1:2016 [1] and

ISO 16140-2:2016 [2] and the most recent version of the MicroVal Technical
Committee for interpretation on ISO 16140-2, version 1.0.
Page 1 of 29
Company: MilliporeSigma Bellevue

Expert Laboratory: Q Laboroatories

Method/Kit name: Assurance® GDS Listeria monocytogenes Tq

Validation standard: ISO 16140-2: 2016 Microbiology of food chain – Method

Validation – Part 2: Protocol for the validation of alternative (proprietary) methods
against a reference method.

Reference methods:

ISO 11290-1:2017 Microbiology of the food chain – Horizontal method for detection
and enumeraion of Listeria monocytogenes and Listeria spp. – Part 1: Detection
Method

Original scope of validation: Food Products and Environmental Samples

Category extension to be validated: Fresh Produce and Vegetables

Certification organization: Lloyd's Register

List of abbreviations

A(lt) Alternative method

AL Acceptability Limit
Art. Cont. artificial contamination
CFU Colony Forming Units
EL Expert Laboratory
FP False Positive
FPR False Positive Ratio
g Gram
h Hour
ILS Interlaboratory Study
LOD Level of Detection
MCS Method Comparison Study
min minute
ml millilitre
MR (MicroVal) Method Reviewer
MVTC MicroVal Technical Committee
NA Negative Agreement
na not applicable
ND Negative Deviation
neg (-) negative/no growth/no reaction/target not detected
NS Non-Suspect growth
nt not tested

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PA Positive Agreement
PD Positive Deviation
pos (+) positive/growth/target detected
PPNA Presumptive Positive Negative Argreement (False Positive result)
PPND Presumptive Positive Negative Deviation (False Positive result)
R(ef) Reference method
RLOD Relative Level of Detection
RT Relative Trueness
S Suspect growth
SE Relative Sensitivity
SP Relative Specificity
TP True Positive
PCR Polymerase Chain Reaction
HFB Half Fraser Broth
ALOA Agar Listeria Ottavani & Agosti
TSA/YE Tryptone Soya Agar with Yeast
BHI Brain Heart Infusion Broth

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Contents

1 Introduction 6

2 Method protocols 6

2.1 Reference method 6

2.2 Alternative method 7

2.3 Study design 7

3 Method comparison study 7

3.1 Sensitivity Study 7

3.1.1 Categories and sample types 7

3.1.2 Test sample preparation 8

3.1.3 Confirmation protocols 9

3.1.4 Sensitivity study results 9

3.1.5 Sensitivity study calculations 11

3.1.6 Discordant results 12

3.1.7 Conclusion sensitivity study 14

3.1.8 Enrichment broth storage 14

3.2 Relative level of detection study 15

3.2.1 Categories, sample types and strains 15

3.2.2 Test sample preparations 16

3.2.3 RLOD study results 16

3.2.4 Conclusion RLOD study 17

3.3 Inclusivity/exclusivity study Error! Bookmark not defined.

3.3.1 Protocols 17

3.3.2 Results inclusivity and exclusivity study 17

3.3.3 Conclusion inclusivity and exclusivity study 18

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4 Conclusions Method Comparsion Study 18

ANNEX A: Flow diagram of the reference method 23

ANNEX B: Flow diagram of the alternative method 23

ANNEX C: Kit insert(s) 25

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1 Introduction

This project,a MicroVal valdiation study based on ISO 16140-2 :2016, evaluated
an alternative method, the Assurance GDS Listeria monocytogenes Tq , for the
detection of Listeria monocytogenes in a category extension and a renewal
validation. The evaluation was performed by Q Laboratories as the MicroVal
Expert Laboratory.

This report combines the category extention to include the fresh produce and
vegetables category to the orginal claim and the renewal of the MicroVal
2010LR32 for the original 5 categories that were evaluated by ISO 16140:2003.
All data from the original evaluation was re-evaluated following ISO 16140-
2 :2016 criteria.

Below is a summary of the alternative method.

1.1. Twenty-five (25) gram samples are enriched with 225 mL of HFB and
environmental sponges are enriched in 90 mL of Half Fraser Broth. Test
portions are incubated at 30 ± 1oC for 24 ± 2 hours. Following enrichment, the
sample is concentrated using the Assurance PickPen® device and then
transferred into 0.5 mL of Half Fraser Broth. The HFB is sealed and incubated
at 30 ± 1oC for 4 to 22 hours. (Test portions will be analyzed at both 4 and 22
hours for the new food category).
1.1.1. Following incubation, the sample is concentrated using the PickPen
device, lysed and a 20 µL aliquot of lysate is analyzed using the
Assurance GDS Rotor-Gene® instrument.

Reference method: ISO 11290-1:2017

Scope of the extension validation study is: fresh fruit and vegetables

Samples were prepared in accordance with ISO 6887: parts 1, 4, and 5 and ISO
11290-1:2017.

Original Categories included (Renewal):

− Meat products
− Milk and dairy products
− Fish and seafood products
− Ready-to-eat, ready-to-reheat or ready-to-cook products
− Environmenal samples

2 Method protocols

The MCS for the original validation (MicroVal 2010LR32) was carried out using 25
gram test portions of sample material for all food matrices. For environmental
samples, testing was conducted using environmental sponges or 25 g test portions.
Data from these studies will be reanalyzed statistically to ensure acceptance criteria
for ISO 16140-2:2016 are met. In order to meet the new requirements for a broad
range of foods, one additional category will be validated (fresh fruit and vegetables).
This category will be evaluated in a paired study design using 25 g test portions.
2.1Reference method

See the flow diagram in Annex A.

Sample preparations used in the reference method and Assurance GDS Listeria
monocytogenes method will be done according to ISO 6887 – 4 and ISO 11290-
1:2017 for the Fresh Produce and Vegetables food types.

2.2Alternative method

See the flow diagram of the alternative method in Annex B.

See the Assurance GDS Listeria monocytogenes Tq kit insert in Annex C.

The alternative method principle is based on PCR.

The Assurance GDS Listeria monocytogenes is an automated nucleic acid

amplification system for the detection of Listeria monocytogenes in a broad range of
foods and environmental samples.

The food samples will be prepared for analysis following an IMS concentration
procedure as outlined in the Assurance GDS Listeria monocytogenes package insert
and previous MicroVal Validation (MicroVal 2010LR32). Results are available after
26 - 44 hours of enrichment. Appendix C presents the IFU for the method.

2.3Study design

For the 25 gram portions, the reference and the alternative method share the initial (pre)-
enrichment step, therefore the same test portion (item) was used for the two methods. All
resulting data were treated as paired data (EN-ISO 16140-2:2016) for the fresh fruits and
vegetables.

3 Method comparison study

3.1Sensitivity Study

The sensitivity study (SE) is the ability of the method selected to detect the analyte by
either the reference or the alternative method.

3.1.1 Categories and sample types

The study design of the original validation (MicroVal 2010LR32) meets the
requirements of ISO 16140-2:2016. Data from the original validation will be
reanalyzed following the new statistical parameters.

A 5th food category will be evaluated to continue the broad range of foods claim
(fresh fruit and vegetables). In accordance with ISO 16140-2:2016, a minimum of 60
individual samples, made up of at least three types with at least 20 samples
representative for each type will be tested. Each sample was tested by both the
reference method and the alternative method, aiming at a minimum of 30 positive
samples per category

The categories, the types and the number of samples analyzed are presented in Table 1.

Table 1 – Distribution per tested category and type

# of
Sample Positive Negative
Category Type Samples
Size Samples2 Samples
Analyzed
Fresh meat (unprocessed) 25 g 11 11 22
Cooked and cured meat
Meat 25 g 11 18 29
products
products1,3
Fermented meat products 25 g 10 14 24
Total 32 43 75
Thermisation/pasteurized
25 g 12 8 20
prodcuts
Milk and dairy Fermented/acidifed
25 g 5 13 18
products1 products
Raw milk based products 25 g 14 12 26
Total 31 33 64
Fish and shellfish 25 g 12 6 18
Fish and
Cooked fishery products 25 g 8 11 19
seafood
1 Smoked products 25 g 14 13 27
products
Total 34 30 64
Substaintial raw ingredients 25 g 11 6 17
Ready-to-eat,
Processed (cooked)
ready-to-reheat 25 g 7 9 16
products
or ready-to-
Refrigerated products 25 g 13 16 29
cook products1
Total 31 31 62
Swabs and sponges 25 g 9 13 22
Environmental Process water 25 g 11 9 20
samples1 Dusts 25 g 12 15 27
Total 32 37 69
Cut-Ready-To-Eat
25 g 11 11 22
vegetables
Fresh Produce
Produce grown in contact
and 25 g 11 11 22
with the ground
Vegetables4
Cut Ready-To-Eat fruit 25 g 11 11 22
Total 33 33 66
All Categories 160 174 334
1Original data reference: “EN ISO 16140 validation study of the GDS™ Listeria monocytogenes for the

detection of Listeria monocytogenes in food products and environmental samples” Version 4, March 24,
2014; ADRIA Developpement
2Postive by either the alternative or the reference method.

3Meat products only analyzed after 22 hours subculture.

4Fresh produce and vegetables analyzed by Q Laboratories in 2018.

3.1.2 Test sample preparation

A total of 4.5% of the samples evaluated were naturally contaminated with the target
analyte for the fresh produce and vegetables.

Artificial contaminations were done by seeding protocols. The fresh produce and
vegetables were inoculated and stored at 2-8 °C for 48 to 72 hours.

The artificial contaminations are presented in Annex D.

The Listeria monocytogenes strains used for artificial inoculation originated from food
samples. Strains used to inoculate items were used on a maximum of 5 different
items.

In total, 30 samples were artificially contaminated by seeding, using 6 different strains.

Each item seeded resulted in a positive result. Most of the spiking inoculations were
lower or equal to 5 CFU/sample.

Regardless of presumptive result, all test portions analyzed by the alternative method
were confirmed following ISO 11290-1:2017 reference method, beginning with a
direct streak of the primary enrichment. Typical colonies were confirmed according to
ISO 11290-1. See Annex A for confirmation steps of the ISO 11290-1 reference
method.

In order to improve the practicability of the method to user labs, enrichment broths for
the alternative method were also stored for 72 hours at 5 ± 3 ºC. All presumptive
positive and discrepant results were reanalyzed with the Assurance GDS Listeria
monocytogenes Tq after the 72 hour hold time. All samples were reconfirmed at 72
hours following the ISO 11290-1 reference method, and the alternative confirmation.

3.1.3 Confirmation protocols

ISO 11290-1:2017

For the fresh fruit and vegetable test portions, a 25 g sample was enriched with
225 mL of HFB. Subsequently, the enrichments were incubated at 30 ± 1°C for 25
± 1 hour.

Following incubation, 0.1 mL of primary enrichment was transferred into 10 mL of

FB and a direct streak was also conducted to ALOA and MOX agar plates. The
FB tubes, ALOA and MOX plates were incubated at 37 ± 1°C for 48 ± 2 hours.
Following 24 and 48 hours of incubation of the FB tubes, the tubes were again
streaked to ALOA and MOX agar plates. One suspected colony from either ALOA
or MOX plates was transferred to TSA/YE and incubated at 37 ± 1°C for 18 to 24
hours. Following purification, a Gram stain was conducted and beta-hemolysis
test, and a carbohydrate test was conducted using L-rhamnose and D-xylose.

3.1.4 Sensitivity study results

All raw data for the sensitivity study (both original data and fresh produce and
vegetables) is presented in Annex E. Sample numbers in bold indicate artificial
inoculation of the sample (see Annex D for details on artificial inoculation).

Table 2 shows the summary of results of the reference method and the alternative
methods for all Categories.
Table 2 - Summary of sensitivity study results (fresh produce and vegetables) –
4 hour and 22 hour subculture analysis

4 Hour
Category PA NA1 PD ND2 PPNA3 PPND3 Total
Milk and dairy
30 34 0 0 0 0 64
products4
Fish and seafood
34 30 0 0 0 0 64
products4
Ready-to-eat, ready-
to-reheat or ready-to- 30 31 0 1 0 0 62
cook products4
Environmental
31 37 0 1 0 0 69
Samples4
Fresh Produce and
33 33 0 0 0 0 66
Vegetables5
All Categories 158 165 0 2 0 0 325
22 Hour
Category PA NA1 PD ND2 PPNA3 PPND3 Total
Meat products4 29 42 1 2 1 0 75
Milk and dairy
30 33 1 0 0 0 64
products4
Fish and seafood
34 30 0 0 0 0 64
products4
Ready-to-eat, ready-
to-reheat or ready-to- 31 31 0 0 0 0 62
cook products4
Environmental
32 37 0 0 0 0 69
Samples4
Fresh Produce and
33 33 0 0 0 0 66
Vegetables5
All Categories 189 206 2 2 1 0 400
1NA: 2ND: 3FP
Including PPNA, Including PPND, = PPNA + PPND
4Originaldata reference: “EN ISO 16140 validation study of the GDS™ Listeria monocytogenes for the
detection of Listeria monocytogenes in food products and environmental samples” Version 4, March 24,
2014; ADRIA Developpement
5Data generated by Q Laboratories in 2018.

Table 3 shows the summary of results of the reference method and the alternative
methods for all categories from the original data submitted.
Table 3 - Summary of sensitivity study results – 4 hour and 22 hour subculture
analysis1,2

Incubation Reference method Reference method

Result
Time positive (R+) negative (R-)
Alternative method Positive agreement (R+/A+) Positive deviation (R-/A+)
positive (A+) PA = 158 PD = 0
4 Hour3
Alternative method Negative deviation (R+/A-). Negative agreement (R-/A-)
negative (A-) ND = 2 NA = 165
Alternative method Positive agreement (R+/A+) Positive deviation (R-/A+)
positive (A+) PA = 189 PD = 2
22 Hour
Alternative method Negative deviation (R+/A-). Negative agreement (R-/A-)
negative (A-) ND = 2 NA = 206
1Original data reference: “EN ISO 16140 validation study of the GDS™ Listeria monocytogenes for the

detection of Listeria monocytogenes in food products and environmental samples” Version 4, March 24,
2014; ADRIA Developpement
2Q Laboratories analyzed fresh produce and vegetables.

3Meat products only analyzed after 22 hours of incubation.

3.1.5 Sensitivity study calculations

The sensitivity study parameters as specified in Table 4 were calculated for all
Categories and Types, and the overview is given in Table 5.

Table 4 – Formula to calculate the sensitivity parameters

(PA + PD )
Sensitivity for the alternative method SEalt =  100%
( PA + ND + PD )
SEref =
(PA + ND )  100%
Sensitivity for the reference method ( PA + ND + PD )
( PA + NA)
Relative trueness RT =  100%
N
( FP )
False positive ratio for the alternative method FPR =  100%
NA
Table 5 - Overview calculated sensitivity parameters per Category and Type

4 Hours
Sample SE SE FPR
Category PA NA1 PD ND2 FP3 RT %
Size alt % ref % %
Milk and dairy
25 g 30 34 0 0 0 100.0 100.0 100.0 0.0
products

Fish and seafood

25 g 34 30 0 0 0 100.0 100.0 100.0 0.0
products
Ready-To-Eat or
Ready-To-Reheat 25 g 30 31 0 1 0 96.8 100.0 98.4 0.0
products
Environmental
N/A 31 37 0 1 0 96.9 100.0 98.6 0.0
samples

Fresh Produce and

25 g 33 33 0 0 0 100.0 100.0 100.0 0.0
Vegetables
All Categories 158 165 0 2 0 98.8 100.0 99.4 0.0
22 Hours
Sample SE SE FPR
Category PA NA1 PD ND2 FP3 RT %
Size alt % ref % %
Milk and dairy
25 g 29 42 1 2 1 93.8 96.9 94.7 2.4
products

Milk and dairy

25 g 30 33 1 0 0 100.0 96.8 98.4 0.0
products

Fish and seafood

25 g 34 30 0 0 0 100.0 100.0 100.0 0.0
products
Ready-To-Eat or
Read-To-Reheat 25 g 31 31 0 0 0 100.0 100.0 100.0 0.0
products
Environmental
N/A 32 37 0 0 0 100.0 100.0 100.0 0.0
samples

Fresh Produce and

25 g 33 33 0 0 0 100.0 100.0 100.0 0.0
Vegetables
All Categories 189 206 2 2 1 99.0 99.0 98.8 0.5
1 2 3
NA: Including PPNA, ND: Including PPND, FP = PPNA + PPND
4Originaldata reference: “EN ISO 16140 validation study of the GDS™ Listeria monocytogenes for the
detection of Listeria monocytogenes in food products and environmental samples” Version 4, March
24, 2014; ADRIA Developpement
5Data analyzed by Q Laboratories in 2018.

3.1.6 Discordant results

There were no deviations observed during the sensitivity evaluation of the fresh fruit
and vegetables.

The devidations from the original sensitivity evaluation are displayed below in Table
6.
Table 6 – Discordant Results (Original Data)1

4 Hour
Alternative
Sample Sample Confirmatory Inoculating Organism
Category Type Size method
no test results (CFU/Sample)
results
Ready-To-
Eat, Ready-
To-Reheat or Spinach 25 g 4624 - + Natural Contamination
ready to cook
products
Environmental L. monocytogenes
Wipe na 6834 - +
samples Ad 548 (3.8)
22 Hour
Alternative
Sample Sample Confirmatory Inoculating Organism
Category Type Size method
no test results (CFU/Sample)
results
Meat products Merguez 25 g 3344 - + Natural Contamination
Sliced
L monocytogenes
Meat products dehydrated 25 g 5242 - +
Ad 267 (2.8)
sausage
Milk and dairy L monocytogenes
Cream 25 g 4771 + -
products 18312 (5.6)
Dehydrated L monocytogenes
Meat products 25 g 5244 + -
Sausage Ad 267 (2.8)
1Originaldata reference: “EN ISO 16140 validation study of the GDS™ Listeria monocytogenes for the
detection of Listeria monocytogenes in food products and environmental samples” Version 4, March 24,
2014; ADRIA Developpement

Six positive deviations were observed. Two of the deviations were associated with
naturally contaminated samples, and four of the deviations were observed with
artificial contaminated samples.

The analysis of discordant results according to ISO 16140-2:2016 for a paired study is
given in Table 7.
Table 7 – Interpretation of the sensitivity study results

4 Hour
Negative Positive
ND1- Acceptability Acceptability
Category Deviations deviations ND1+PD
PD Limit (AL) Limit (AL)
(ND1) (PD)

Milk and Dairy Products2 0 0 0 3 0 6

Fish and Seafood

Products2 0 0 0 3 0 6
Read-to-Eat, Ready-To-
Reheat or Ready-To-Cook 1 0 1 3 1 6
Products2

Environrmental Samples2 1 0 1 3 1 6

Fresh Produce and

Vegetables3 0 0 0 3 0 6

Total 2 0 2 5 2 14
22 Hour
Meat Products2 2 1 1 3 3 6
Milk and Dairy Products2 0 1 -1 3 1 6
Fish and Seafood
Products2 0 0 0 3 0 6
Ready-To-Eat, Ready-To-
Reheat or Ready-To-Cook 0 0 0 3 0 6
Products2
Environrmental Samples2 0 0 0 3 0 6
Fresh Produce and
Vegetables3 0 0 0 3 0 6
Total 2 2 0 6 4 16
1 ND: including PPND
2Originaldata reference: “EN ISO 16140 validation study of the GDS™ Listeria monocytogenes for the
detection of Listeria monocytogenes in food products and environmental samples” Version 4, March 24,
2014; ADRIA Developpement
3Data generated by Q Laboratories in 2018.

3.1.7 Conclusion sensitivity study

Paired Testing: The observed values for ND-PD and ND+PD for the individual categories
and for all categories meet the acceptability limits (observed values ≤ AL).

3.1.8 Enrichment broth storage

Enrichment broth HFB storage was done at 2-8 °C for 72 h.

All positive samples were tested again after enrichment broth storage. Zero changes
were observed for the fresh produce and vegetables. But there were a few changes
observed for the original data, which is presented below in Table 8.
Table 8 – Difference in Results After Storage (Original Data)1

Before storage After storage

Sample n°
4h 22 h 4h 22 h
4624 ND PA PA PA
4765 PA PA ND PA
4766 PA PA ND PA
5248 / PA / ND
6844 NA NA PD NA
1Originaldata reference: “EN ISO 16140 validation study of the GDS™ Listeria monocytogenes for the
detection of Listeria monocytogenes in food products and environmental samples” Version 4, March 24,
2014; ADRIA Developpement

Table 9 – Interpretation of the sensitivity study results

4 Hour
Negative Positive
ND1- Acceptability Acceptability
Category Deviations deviations ND1+PD
PD Limit (AL) Limit (AL)
(ND1) (PD)
Total 4 1 3 5 5 14
22 Hour
Total 3 2 1 6 5 16
1 ND: including PPND

The observed values for ND-PD and ND+PD for the individual categories and for all
categories meet the acceptability limits (observed values ≤ AL) after storage of the pre-
enrichment broth for 72 hours at 2-8 °C.

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 method divided by the level of detection at P = 0.50 (LOD50) of the reference
method.

3.2.1 Categories, sample types and strains

One sample type and one relevant target micro-organism for this sample type was
chosen for each of the categories in this validation study, as shown in Table 10.
Table 10- List of selected types and strains per category, as tested within the
relative level of detection study.

Storage
Analysis
Sample Study Inoculated Strain conditions
Type Time
size design strain origin before
Points
analysis
L. monocytogenes Seeding
Sausage 25 g Paired 22 Hour Meat Product
Ad 669 48 h at 5 ± 3°C

Goat 4 Hour L. monocytogenes Seeding

25 g Paired Cheese
Cheese 22 Hour Ad 618 48 h at 5 ± 3°C

Smoked 4 Hour L. monocytogenes Smoked Seeding

25 g Paired
Salmon 22 Hour Ad 670 Salmon 48 h at 5 ± 3°C

4 Hour Vegetable
L. monocytogenes Seeding
Potatoes 25 g Paired based, Ready
Ad 279 48 h at 5 ± 3°C
22 Hour to Eat Food

4 Hour
Process L. monocytogenes Dairy Seeding
25 g Paired
Water Ad 631 Environrment 48 h at 5 ± 3°C
22 Hour

Pre-Cut 4 Hour
L. monocytogenes Seeding
Bagged 25 g Paired Dairy Product
CWD 15554 48 h at 5 ± 3°C
Spinach 22 Hour

3.2.2 Test sample preparations

For the original RLOD (sausage, goat cheese, smoked salmon, potatoes and process
water), six replicates were performed. The contamination levels were:

- 0 CFU/g or ml
- Level required to get 0-50% positive samples
- Level required to get 50-75% positive samples
- Level required to get 75-100% positive samples

Three levels of artificial contamination were prepared for the pre-cut bagged spinach:

- Negative control level: One uninoculated in order to get 5 test portions,

- Low level: One inoculated between 0.2 and 2.0 CFU/sample in order to get 20 test
portions providing fractional recovery (5-15 positive results out of 20),
- High level: One inoculated between 2.0 and 5.0 CFU/sample in order to get 5 test
portions contaminated at a higher level.

A bulk lot of the matrix was inoculated at each level, homogenized and stored as
described in Table 10.

3.2.3 RLOD study results

The tabulated raw data on the RLOD study are given in Annex G.

The RLOD calculations were performed using the Excel spread sheet (version 06-07-
2015) of the international standard as described in ISO 16140-2: 2016.
The RLOD per Category is given in Table 11.

Table 11 – Presentation of RLOD before and after confirmation of the alternative

method results

RLOD using the alternative RLOD using the confirmed

Item
method results alternative method results
Sausage 1.000 1.000
Goat Cheese 1.000 1.000
Smoked Salmon 1.000 1.000
Potatoes 1.000 1.000
Process Water 1.000 1.000
Pre-cut Bagged Spinach 1.000 1.000
Combined 1.000 1.000

3.2.4 Conclusion RLOD study

The RLOD values (using the confirmed alternative method results) meet the acceptability
limit, 1.5 for paired test portions for all categories tested.

Inclusivity/exclusivity study

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.

4.2.1 Protocols

Inclusivity: Fifty (50) Listeria monocytogenes strains were freshly cultured in BHI
medium at 37 ± 1°C. Dilutions were made in order to inoculate 10 CFU/ 225 ml HFB. The
alternative method protocol was then performed. For 2 strains, ultra high temperature
milk was added to the enrichment broth (25 ml) in order to mimic the real conditions of
analysis.

Exclusivity: Thirty (30) strains were freshly cultured in BHI medium at 37 ± 1°C.
Dilutions were made in order to inoculate about 105 CFU/ ml BHI. The alternative method
was then performed.

4.2.2 Results inclusivity and exclusivity study

All raw data on inclusivity and exclusivity are given in Annex F.

A total of 50 strains were tested for inclusivity. Forty-nine (49) of these strains showed
the expected positive result, with Listeria monocytogenes Ad 253 producing a negative
result following the alternative method but a positive result following the reference
method.

A total of 30 strains were tested for exclusivity. Thirty (30) of these strains showed the
expected negative result. Zero (0) strains showed a positive result.
4.2.3 Conclusion inclusivity and exclusivity study

The alternative Assurance® GDS Listeria monocytogenes Tq detection method is both

selective and specific for the detection of Listeria monocytogenes.

4 Conclusions Method Comparison Study

Overall, the conclusions for the Method Comparison Study are:

The observed values for ND-PD and ND+PD for the individual categories and for all
categories meet the acceptability limits (observed values ≤ AL).

The observed values for ND-PD for the individual categories and for all categories meet
the acceptability limits (observed values ≤ AL).

The RLOD values (using the confirmed alternative method results) meet the acceptability
limit, which is 1.5 for paired studies for all categories tested.

The alternative Assurance® GDS Listeria monocytogenes Tq detection method is

selective and specific.

5 Interlaboratory Study

5.1General Overview

In this collaborative study, one food type was evaluated (cheese). The matrix was
artificially contaminated with Listeria monocytogenes Ad153 at two inoculation levels:
a high inoculation level of approximately 5-50 colony-forming units (CFU)/test portion
and a low inoculation level of approximately 1-10 CFU/test portion. A set of
uninoculated control test portions (0 CFU/test portion) were also included.

The Assurance GDS Listeria monocytogenes Tq and ISO 11290-1:2017 share the
same pre-enrichment for this food type, therefore the study was designed using
paired samples. A total of 24 samples were evaluated per collaborator, Within each
sample set were 8 replicate test portions from each of the two inoculation levels and
one unioculated level. A total of 14 collaborators particpated. Collaborators were
also sent a test portion for determining the total background microflora following the
ISO 4833 reference method and a water flask labelled “Temperature Control” with a
termpature probe on the day samples were received.

5.2 Test Portion Distribution

All samples were labeled with a randomized and blind-coded. All test portions were
sent via overnight delivery according to the Category B Dangerous Goods shipment
regulations set forth by the International Air Transport Association (IATA). Test
portions were set up and analysis was started on Wednesday May 15th, 2013. All
test portions were sent via express-shipping according to the Category B Dangerous
Goods shipment regulations set forth by the International Air Transport Association
(IATA).

Collaborators received a data logger. Data loggers were programmed to monitor the
temperature of the shipment to ensure remained cool. Participants were instructed to
send the data file from the logger to the Expert Lab upon receipt of the samples.
Results from the temperature probes from each collaborator are presented in Table
12.

Table 12: Temperature Records for Collaborator Shipments

Temperature Temperature
Temperature Temperature
Measure at Measure at
Collaborator Measured by Collaborator Measured by
Receipt Receipt
Sensor (oC) Sensor (oC)
(oC) (oC)
1 Lost 3.9 8 1.0 4.3
2 2.0 4.8 9 2.5 5.3
3 10 2.1 10 0.5 2.5
4 3.5 4.4 11 Lost 3.0
5 2.0 3.2 12 1.0 2.5
6 3.0 3.0-4.0 13 Out of Order 4.0
7 Lost 3.0-4.0 14 2.0 5.0

5.3 Stability of Test Portions

Prior to shipping test portions, the stability of the inoculum in the matrix had been
verified by the Expert Lab by confirming the viability of ten replicates of a bulk lot of
the inoculated food type at 0, 1 and 2 days.

5.4 Inoculum Levels

Table 13 presents the inoculum results for the ILS evaluation

Table 13: Inoculum Levels

High Limit
Theoretical Low Limit per
Level True Level per 25 g
target level 25 g sample
sample
Uninoculated 0 / / /
Low 2 1.9 1.5 2.3
High 25 25.0 22.1 28.4

5.5 Results

Table 14 summarizes the collaborative study results for participant.each matrix. For
each level (L0, L1, L2) 8 unpaired test portions were evaluted by each collaborator.
Results for the raw data can be found in ANNEX H. The average aerobic mesophilic
flora result obtained by the collaborators varied between 1.7 x 10 6 CFU/g to 6.0 x 106
CFU/g.
 Table 14: Summary of ILS Results
Alternative Method Alternative Method
Reference Method
Collaborator Presumptive Result Confirmed Result
L0 L1 L2 L0 L1 L2 L0 L1 L2
1 0 8 8 0 8 8 0 8 8
2 0 7 8 0 7 8 0 7 8
3 0 8 8 0 8 8 0 8 8
4 0 6 8 0 6 8 0 6 8
5 0 7 8 0 7 8 2 8 8
6 0 8 8 0 8 8 0 8 8
7 0 8 8 0 8 8 0 8 8
8 0 7 7 0 7 7 0 7 7
9 0 4 8 0 4 8 0 7 8
10 0 8 8 0 8 8 0 8 8
11 1 8 8 0 8 8 0 8 8
12 0 6 8 0 6 8 0 6 8
13 0 3 8 0 3 8 0 4 8
14 0 7 8 0 7 8 0 7 8
Total 1 88 103 0 88 103 2 92 103
Note: Collaborator 5 was excluded from the interpretation due to cross contamination on control
samples.

Table 15: Summary of agreements and deviations for the ILS

Reference Method Reference Method

Positive (R+) Negative (R-)
Positive Agreement Positive Deviation
Alternative Method
(+/+) (-/+)
Positive (A+)
199 0
Negative Deviation Negative Agreement
Alternative Method
(+/-) (-/-)
Negative (A-)
5 116

Using the values in Table 21, the sensitivity of the reference and alternative methods
were determined, along with relative trueness and false positive rate. Calculations for
each statistic is provided below. Additionally, the requirement of fractional positive
results was obtained for both the alternative and reference method. A summary of
results in presented in Table 22.

(𝑃𝐴+𝑃𝐷)
SEalt = (𝑃𝐴+𝑃𝐷+𝑁𝐷) 𝑥 100% =

Figure 1: Calculation for the Sensitivity of the Alternative Method

(𝑃𝐴+𝑁𝐷)
SEref = 𝑥 100% =
(𝑃𝐴+𝑃𝐷+𝑁𝐷)

Figure 2: Calculation for the Sensitivity of the Reference Method

 (𝑃𝐴+𝑁𝐴)
RT = 𝑥 100% =
𝑁

Figure 3: Calculation for the Relative Trueness

Figure 4: Calculation for the False Positive Rate of the Alternative Method

Table 16: Summary of Statistical Analysis of the ILS Study

Sensitivity for the Alternative Method 97.5 %

Sensitivity for the Reference Method 100.0%
Relative Trueness 98.4%
False Positive Ratio for the Alternative Method 0.9%

5.8 Interpretation of data

For a paired study, the difference between (ND – PD) and the sum of (ND + PD) at L1
shall not be higher than the AL. The values found for (ND – PD) and (ND + PD) shall
not be higher than the acceptability limits (AL).

The acceptability limits for 13 collaborators is 4 for (ND – PD) and 5 for (ND + PD). The
results obtained from the ILS pass the criteria set forth by 16140-2.

5.9 Evaluation of the RLOD between Laboratories

The RLOD for the ILS was calculated using the available Excel spread sheet
(http://standards.iso.org/iso/16140) as listed in ISO 1614-2:2016. These results are
provided for informational purposes only. See Table 17 for results of the RLOD.

Table 17: RLOD of the ILS

RLOD RLODL RLODU b-ln(RLOD) sd(b) z-Test statistic p-value

1.113 0.821 1.510 0.107 0.152 0.703 0.482
Table 18: ILS Results by Method

LOD50% = 50% limit of detection in LOD95% = 95% limit of detection in

Log SD of log CFU per sample size CFU per sample size
Method
Method Method Method Lower Upper Lower Upper
Effect Detection Detection
Effect Effect Conf. Conf. Conf. Conf.
Limt Limit
Limit Limit Limit Limit
Reference 0.780 -0.249 0.122 0.89 0.70 1.13 3.84 3.01 4.90
Alternative 0.704 -0.351 0.122 0.98 0.77 1.26 4.26 3.33 5.44
The methods are not significantly different at the 5% significance level (change in deviance of the model with
method effects to the null model Dmethod = 0.39 with 1 degree of freedom, p-value 0.53).
Conclusion
The relative limit of detection (RLOD) of the alternative method, as compared to the reference method, is 1.11
with a 90% confidence interval of 0.83 - 1.47.

Table 19: ILS Results by Laboratories

Laboratory Number Lab Effect Log Lab Effect SD of log Lab Effect
1 ∞ ∞
2 1.040 0.039 0.318
3 ∞ ∞
4 0.693 -0.367 0.312
5 ∞ ∞
6 ∞ ∞
7 0.214 -1.541 0.361
8 0.582 -0.542 0.319
9 ∞ ∞
10 ∞ ∞
11 0.693 -0.367 0.312
12 0.291 -1.234 0.366
13 1.040 0.039 0.318
Combined Results 0.740 -0.301 0.086
The probabilities of detection (POD) of the laboratories are significantly
different at the 5% significance level (change in deviance of the model
Conclusion
with laboratory effects to the null model Dlab = 45.6 with 12 degrees of
freedom, p-value 0).

5.10 Conclusion of the Interlaboratory study

The data obtained during the ILS meets the criteria set forth in ISO 16140-2:2016
and indicates that the alternative method, the Assurance GDS for Listeria
monocytogenes Tq is considered equivalent to the ISO 11290-1:2017 reference
method.
ANNEX A: Flow diagram of the reference method

ISO 11290-1:2017

Food Types:
25 g + 225 mL Half Fraser Broth
Environmental samples:
1 swab + 10 mL Half Fraser Broth
or 1 sponge + 90 mL Half Fraser Broth
or 25 g or 25 mL + 225 mL Half Fraser Broth

Incubation 25 h ± 1 h
at 30°C

Streaking onto ALOA (OAA) + secondary agar (MOX) plates
 +
 0.1 mL + 10 mL Fraser Broth
 
 Incubation 24 h ± 2 h at 37°C
 
 Streaking FB onto ALOA (OAA)+ secondary agar
 (MOX) plates
 
FOR BOTH SETS OF ISOLATION AGAR PLATES:
Incubation 48 h ± 2 h
at 37°C
(read plates at both 24 and 48 hours)

Confirmatory test on one typical colony,
and four other colonies
(if the first one is negative
(typical colony: blue to blue-green – ALOA
gray, flat, dimpled - PALCAM)

Streaking onto TSA-YE

Incubation 18 h – 24 h
at 37°C

Gram Stain, Beta-Hemolysis, L-Rhamnose, D-Xylose
ANNEX B: Flow diagram of the alternative method

Assurance GDS Listeria monocytogenes

Food Types:
25 g + 225 mL Half Fraser Broth
Environmental samples:
1 swab + 10 mL Half Fraser Broth
or 1 sponge + 90 mL Half Fraser Broth
or 10 g or 10 mL + 90 mL Half Fraser Broth

Incubation 24 h ± 2 h at 30°C

Concentrate with Pick Pen device. Transfer 0.5 mL Half Fraser Broth

Concentrate with Pick Pen device, lyse and analyze via PCR.

Or
Store IMS Half Fraser Broth at 5 °C ± 3 °C up to 72 h.
Concentrate with PickPen device, lyse and analyze via PCR.

CONFIRM PER ISO 11290-1 (ANNEX A)

Streaking onto ALOA (OAA)+ secondary agar (PALCAM) plates

Incubation 48 h ± 2 h
at 37°C
(read plates at both 24 and 48 hours)

Confirmatory test on one typical colony, and four other colonies
(if the first one is negative)
(typical colony: blue to blue-green – ALOA, gray, flat, dimpled - MOX)

Streaking onto TSA-YE

Incubation 18 h – 24 h
at 37°C

Gram Stain, Beta-Hemolysis, L-Rhamnose, D-Xylose
ANNEX C: Kit insert