Miguel Yuste, 12 - 4ª planta

28037 Madrid
(+34) 914 401 224
aenorlaboratorio@aenor.com
www.aenor.com

SUMMARY OF VALIDATION UNE-EN ISO 16140

METHOD EASY QFAST®

DETERMINATION OF SALMONELLA spp.

CUALITATIVE METHOD

Edition 4 (08-05-2017)
INDEX

1.- INTRODUCTION 3

2.- ALTERNATIVE METHOD AND REFERENCE METHOD 4

2.1.- PRINCIPLE OF THE ALTERNATIVE METHOD EASY QFAST

4
SALMONELLA
2.2.- PRINCIPLE AND PROTOCOL OF ALTERNATIVE METHOD HARD EASY
8
VETERINARY QFAST

2.3.- REFERENCE METHOD 13

3.- COMPARATIVE STUDY OF THE METHODS 13

3.1.-RELATIVE ACCURACY, RELATIVE SPECIFICITY AND RELATIVE
13
SENSITIVITY

3.2.- RELATIVE DETECTION LIMIT (LDR) 27

3.3.- INCLUSIVITY AND EXCLUSIVITY 28

4.- COLABORATORY STUDY 30

4.1.- ORGANIZATION OF THE STUDY 30

4.2.- RESULTS OF THE ANALYSIS 32

4.3.- CALCULATIONS 33

5.- INTERPRETATION 37

6.- AUDITS 40

7.- CONCLUSION 41
ANNEXES

Annexe 1 – Protocol of alternative method EASY QFast® 42

Annexe 2 – Reference method ISO 6579:2002-Microbiology of Food and animal

feeding stuffs. 44
Horizontal method for the detection of Salmonella spp.

Annexe 3 – Reference method ISO 6579/A1-Microbiology of Food and animal

feeding stuffs.
Horizontal method for the detection of Salmonella spp. Amedement
45
1: Annex D.
Detection of Salmonella spp in faeces ans enviromental samples from
primary production stage

Page 2 of 45
1.- INTRODUCTION

The results obtained that appear in the corresponding tables as well as their treatment have been
performed according to the norm UNE-EN ISO 16140.

Manufacturer: iMICROQ, Integrated Microsystems for Quality of Life, S.L.

Polígon Industrial Riu Clar

C/ Ferro 6 (nau 7)
43006 Tarragona, Spain

Certification Body: Asociación Española de Normalización y Certificación (AENOR)

C/ Génova 6
28004 Madrid, España

Expert laboratory: AENORlaboratorio

Miguel Yuste, 12, 4ª planta.
28037 Madrid

Method to validate: Method Easy QFast®, Fast method for detection of Salmonella spp., fully validated
according UNE-EN ISO 16140:2003. “Microbiology of Food and animal feeding stuffs. Protocol for validation
of alternative methods” (UNE-EN ISO 16140:2003/A1:2012).

The validation has comprised two stages:

 Comparative study of the alternative method versus the reference method performed by
AENORLaboratory
 Collaborative study of each of the two methods: Alternative method and Reference method.

Validation Reference: UNE-EN ISO 16140:2003. Microbiology of Food and animal feeding stuffs. Protocol
for validation of alternative methods.
UNE-EN ISO 16140:2003/A1:2012

Reference Methods: UNE-EN ISO 6579: 2003. Microbiology of Food and animal feeding sutffs. Horizontal
method for the detection of Salmonella spp.
UNE-EN ISO 6579 (2003). Erratum: 2007 V2. Horizontal method for the detection of Salmonella spp.
UNE-EN ISO 6579:2003/A1:2007. Microbiology of Food and animal feeding sutffs. Horizontal method for
the detection of Salmonella spp. Amendement 1: Annex D. Detection of Salmonella spp. in animal faeces
and in environmental samples from the primary production stage.

Scope of the validation:

 Animal feed samples.

 Veterinary samples (faeces, shoe covers,necks)
 Environmental samples originating from primary production (poultry industry).
 General Food: fruits and vegetables, dairy products and various products, including spices,
mayonnaise and eggs; and meat.
Page 3 of 45
2.- ALTERNATIVE METHOD AND REFERENCE METHOD

2.1.- PRINCIPLE AND PROTOCOL OF ALTERNATIVE METHOD EASY QFAST SALMONELLA EXCEPT
FOR VETERINARY SAMPLES

2.1.1.- PRINCIPLE

a) Pre-enrichment in medium 1
Add to 225 ml of pre-enrichment medium the adequate quantity of sample and incubate at
37ºC ± 1ºC for 16 ±1 hours.
b) Enrichment in medium 2:
Take 10 µl of the pre-enriched sample and incubate at 41.5 ºC ± 1ºC for 7 hours ± 1 hour.
c) Reading
Add to the eppendorf tube, 1 drop of reaction solution and 1 drop of the enrichment medium
with the sample with a steril Pasteur pipete; mix and vortex and incubate at 37 ºC ± 1ºC for 30
to 60 minutes. Add 10 µl in the sensor electromagnetic reader.

2.1.2.-PROTOCOL

a) Pre-enrichment

1- Preparation of the samples, in the laminar flow cabinet or when it is not available, a Bunsen
Burner.

Animal Feeding Samples

Weigh, with a precision of ± 1%, 25 g of sample in the flask that contains Soft medium 1. If the
Soft media does not contain supplements, add them.

Environmental Samples (supports, Box covers)

• Supports:

Submerge the 4 shoe covers in the flask that contains Hard medium 1. If the Hard medium does
not contain supplements, add them.

• Box covers:

Cut the sample to obtain 25 g of sample, with sterile material, submerge in the flask that contains
Hard medium 1. If the Hard medium does not contain supplements, add them.

Food Samples

Weigh, with a precision of ± 1%, 25 g of sample in the flask that contains Soft medium 1. If the
Soft medium does not contain supplements, add them. In the case of meat products use the Hard
medium.
Page 4 of 45
In the case of foods that contain inhibitor substances, such as spices, the preparation of the
samples is carried out according to the norm UNE-EN ISO 6887-4: 2003 “Microbiology of food for
human use and animal food. Preparation of the samples for analysis, initial suspension and decimal
dilutions for microbiological examination. Section 4: Specific rules for the preparation of products
different to milk and lactose product, meat and meat products and fish and fished products”.

2- Once the samples are prepared as indicated in point 1, proceed in the same manner for all of
them.

Agitate manually and horizontally the flask that contains the sample and the Pre-enrichment
medium.

Incubate the sample at 37ºC ± 1 ºC for 16 hours ± 1 hour.

b) Enrichment in medium 2

Wait until the enrichment medium reaches room temperature (between 18 and 25ºC) before use.

Remove the sample from the incubator and agitate the flask manually and horizontally.

Then, take previously 1 ml from the flask, with a pipette Pasteur and add to a sterile Eppendorf
tube; subsequently agitate and vortex. Take 10 µl of this tube, with fixed pipette and add to
medium 2.

Agitate with the vortex. Incubate at 41.5 ºC ± 1ºC for 7 hours ± 1 hour, in a shaking incubator
iMICROQ.

c) Reading Reaction

Wait until the solution reached room temperature (between 18ºC and 25ºC) before use.
Add to an Eppendorf tube a drop of the reaction solution and a drop of enrichment medium with
the sample using a Pipette Pasteur, agitate with the vortex and incubate at 37ºC ± 1 ºC for 30-60
minutes in the shaking incubator iMICROQ. In the case where the reaction solution is concetrated,
a drop of the reaction solution is added to the eppendorf contained in medium 2.

Remove the eppendorf from the skaking incubator and take 10 µl with a fixed micropippette and
add to the sensor, that has been previously cooled down, that has previously been fixed in the
electronic reader. Read by pressing the start button.

In the case that the results of the reader indicates Positive, take with inoculating loop, from the
same eppendorf tube and inoculate on a plate with two selective agar: XLD and ASAP.

Page 5 of 45
d) Expression of the results

The PRESENCE of Salmonella spp. is considered in the part of the analysis (specifying the weight in
grams, of the sample analysed or surface of the sample), when:

The electrochemical sensor indicates: Positive

Note: if the sensor indicates “retest” measure again with another sensor with the remaining sample
in the eppendorf, taking the second measurement as the definitive one.

A result with a positive value indicates that the sample is contaminated with Salmonella.

In this case, the result should be confirmed.

The ABSENCE of Salmonella spp. in the part analysed (specifying the weight in grams, of the
sample or the surface sampled), when:

The electrochemical reader indicates: Negative

A result with a Negative test indicates that the sample does not contain Salmonella at a
concentration lower than the detection limit

Confirmation of positive results

All of the positive results of the method QFast® Salmonella should be confirmed.

The confirmation should be carried out using the liquid reaction prior to reading and should be
started after the read. Isolate specific plates, as for example XLD and ASAP. Incubate the plates at
37ºC for 24 hours. Confirm the suspicious colonies via adequate biochemical and/or serological
tests.

Confirmation of positive results

All of the positive results of the method Easy QFast® Salmonella should be confirmed.

The confirmation should be carried out using the liquid reaction prior to reading and should be
started after the read. Isolate specific plates, as for example XLD and ASAP. Incubate the plates at
37ºC for 24 hours. Confirm the suspicious colonies via adequate biochemical and/or serological
tests.

Page 6 of 45
Conservation of the enrichment medium and the reagents of the Easy QFast® method

Medium 1: Pre-enrichment medium (HARD):

• Presentation in flasks with 225 ml of medium con supplements. Preservation between 2 and 8 ºC.
Expiration date 15 months from production.
• Presentation in flasks with 225 ml of medium without supplements. Preservation between 2 and 8
ºC. Expiration date 15 months from production.
• Presentation bottles of dehydrated pre-enrichment medium without supplements. Preservation
between 4 and 30 ºC. Expiration date 3 years from production.
• Medium Supplement 1 in glass packaging with a dropper. Preservation between 4 and 30 ºC.
Expiration date 15 months from production.

Medium 1: Pre-enrichment medium (SOFT):

• Presentation in flasks with 225 ml of the medium with supplements. Preservation between 2 and 8
ºC. Expiration date 15 months from production.
• Presentation in bottles of dehydrated pre-enrichment medium without supplements. Preservation
between 4 and 30 ºC. Expiration date 3 years from production.
• Supplement medium 1 in glass packaging with a dropper (only for the dehydrated format).
Conservation between 4 and 30 ºC. Expiration date 1 year after production.

Medium 2: Enrichment medium

• Presentation in eppendorf tubes with 1 ml of medium. Preservation between 2 and 8 ºC. Expiration
date 15 months from production.
• Dehydrated format in aluminium bags of 2.8 g. Preservation between 4 and 30 ºC. Expiration date
three years from production.

Reaction Solution:

• Presentation a bottle of 5 ml with a dropper. Preservation between -21 y -5ºC. Expiration date one
year from production.
• Presentation in two bottles of 3 ml with a dropper; one with the dehydrated reaction solution and
another with the diluent solution. Preservation between -21 and -5ºC. Expiration date one year
from production.

Sensor:
 Presentation in plastic packaging with silicagel. Preservation between 2 and 8 ºC. Expiration date
one year from production.

Page 7 of 45
2.2.- PRINCIPLE AND PROTOCOL OF ALTERNATIVE METHOD HARD EASY VETERINARY QFast
SALMONELLA FOR VETERINARY SAMPLES .

2.2.1.- PRINCIPLE

a) Pre-enrichment in Medium 1 and Medium 1-P

This step is necessary for proper recovery and pre-enrichment of Salmonella, while limiting the
excessive proliferation of other accompanying flora. Pre-enrichment is done in two stages.

b) Enrichment in Medium 2

This second incubation step reduces significantly the accompanying flora, maintaining or increasing
the population of Salmonella.

c) Reading

Using specific and typical enzymes of Salmonella isolated in the preceding step and an enzymatic
substrate, a measurable and quantifiable signal is obtained on the electronic reader to detect the
presence or absence of Salmonella in the sample. Positive results must be confirmed at a later
stage.

2.2.2.-PROTOCOL

a) First pre-enrichment stage in liquid medium (MEDIUM 1 HARD)

Wait for the pre-enrichment medium to reach room temperature (between 18 and 25 °C) before
use.

Add 1ml of SUPPLEMENT FOR MEDIUM 1 HARD in the container of MEDIUM 1 HARD.

Page 8 of 45
Veterinary Samples (faeces, neck, shoe covers)

• Faeces:

Weigh, with a precision of ± 1%, 25 g of sample in the flask that contains Hard medium 1
previously supplemented.

• Neck:

Weigh, with a precision of ± 1%, 25 g of sample in the flask that contains Hard medium 1
previously supplemented.

• Shoe covers:

Add the number of the shoe covers necessary in the flask that contains Hard medium 1 previously
supplemented.

Mix manually the plastic container that contains the sample and the pre-enrichment medium,
strongly and horizontally while maintaining the cap up and suitably closed.

Incubate the sample at 37ºC ± 1 ºC for 18 hours ± 1 hour.

NOTE: If required, the sample incubated in this point a) can be refrigerated (between 2 and 8ºC)
for a maximum of 48 hours before continuing with the procedure.

b) Second pre-enrichment stage in liquid medium (MEDIUM 1-P)

NOTE: The first time you use the kit or before performing the test, dispense aseptically 1 ml of
MEDIUM 1-P into the sterile microtubes supplied, and keep them refrigerated if they will not be
used immediately. Dispensed medium can be stored refrigerated (2 °C to 8 °C) and away from
light.

Wait for the microtube with MEDIUM 1-P previously dispensed to reach room temperature
(between 18 and 25 °C) before use.

Remove the container containing the sample and the pre-enrichment medium MEDIUM 1 HARD
from the incubator and mix manually and horizontally.

Take 100 μl of the previous container with a micropipette and transferred to the microtube that
contains MEDIUM 1-P. The solution is inverted and incubated at 41,5ºC ± 1ºC for 7 hours ± 1
hour, in iMICROQ incubator under agitation.

NOTE: In the event that due to the nature of the sample, for the presence of foam or particulate
matter or difficulty taking 100 µl with a micropipette directly from the container obtained in second
paragraph of the point b), take under sterile conditions 1 ml from the container obtained in this
point, place it in a sterile container and stir with a vortex. Then follow the instructions in third
paragraph of the point b).

Page 9 of 45
c) Enrichment in selective liquid medium (MEDIUM 2)

NOTE: Take into account the provisions of Annex 1 before using MEDIUM 2 provided.

Wait for the enrichment medium to reach room temperature (between 18 and 25 °C) before use.
Add under sterile conditions 1mL of MEDIUM 2 in the sterile microtube supplied. Remove the
container containing the sample and the second pre-enrichment medium (MEDIUM 1-P) from the
incubator.

Take 10 μl of the previous container with a micropipette and transferred to MEDIUM 2. The solution
is inverted and incubated at 41,5ºC ± 1ºC for 18 hours ± 1 hour, in iMICROQ incubator under
agitation.

d) Reaction and reading

NOTE: The first time you use the kit, reconstitute the reaction solution according to the provisions
of Annex 1 and the product label.

Wait for the reaction solution (REACTION SOLUTION EASY) and the SENSOR to reach room
temperature (between 18 and 25 °C) before use. Add in the microtube containing the sample with
medium 2 after the incubation, one droplet of REACTION SOLUTION EASY, vortex and incubate at
37ºC ± 1 ºC for 30 minutes in iMICROQ incubator under agitation.

Afterwards, the tube is removed from the incubator and, immediately, homogenised and 10 μl of
the solution is taken with a micropipette and is introduced in a SENSOR that has been placed in the
electronic reader. The reading is carried out by pressing Start key in the electronic reader.

e) Expression of the results

The PRESENCE of Salmonella spp. is considered in the part of the analysis (specifying the weight in
grams, of the sample analysed or surface of the sample), when:

The electrochemical sensor indicates: Positive

Note: if the sensor indicates “retest” measure again with another sensor with the remaining sample
in the eppendorf, taking the second measurement as the definitive one.

A result with a positive value indicates that the sample is contaminated with Salmonella.

In this case, the result should be confirmed.

The ABSENCE of Salmonella spp. in the part analysed (specifying the weight in grams, of the
sample or the surface sampled), when:

The electrochemical reader indicates: Negative

Page 10 of 45
 A result with a Negative test indicates that the sample does not contain Salmonella at a
concentration lower than the detection limit

Confirmation of positive results

All of the positive results of the method QFast® Salmonella should be confirmed.

The confirmation should be carried out using the liquid reaction prior to reading and should be
started after the read. Isolate specific plates, as for example XLD and ASAP. Incubate the plates at
37ºC for 24 hours. Confirm the suspicious colonies via adequate biochemical and/or serological
tests.

Confirmation of positive results

All of the positive results of the method Easy QFast® Salmonella should be confirmed.

The confirmation should be carried out using the liquid reaction prior to reading and should be
started after the read. Isolate specific plates, as for example XLD and ASAP. Incubate the plates at
37ºC for 24 hours. Confirm the suspicious colonies via adequate biochemical and/or serological
tests.

Conservation of the enrichment medium and the reagents of the Hard Easy Veterinary QFast®
Salmonella method

Medium 1: Pre-enrichment medium (HARD):

• Presentation in flasks with 225 ml of medium con supplements. Preservation between 2 and 8 ºC.
Expiration date 15 months from production.
• Presentation in flasks with 225 ml of medium without supplements. Preservation between 2 and 8
ºC. Expiration date 15 months from production.
• Presentation bottles of dehydrated pre-enrichment media without supplements. Preservation
between 4 and 30 ºC. Expiration date 3 years from production.
• Medium Supplement 1 in glass packaging with a dropper. Preservation between 4 and 30 ºC.
Expiration date 15 months from production.

Medium 1-P: Second pre-enrichment:

• Liquid medium in plastic bottle (50ml). Preservation between 2 and 8 ºC. Expiration date 15
months from fabrication.

Medium 2: Enrichment medium

• Presentation in eppendorf tubes with 1 ml of medium. Preservation between 2 and 8 ºC. Expiration
date 15 months from production.
• Presentation in plastic bottle of 50 ml. Preservation between 2 and 8 ºC. Expiration date 15 months
from production.

Page 11 of 45
Reaction Solution:

• Presentation a bottle of 5 ml with a dropper. Preservation between -21 y -5ºC. Expiration date one
year from production.
• Presentation in two bottles of 3 ml with a dropper; one with the dehydrated reaction solution and
another with the diluent solution. Preservation between -21 and -5ºC. Expiration date one year
from production.
Sensor:
 Presentation in plastic packaging with silicagel. Preservation between 2 and 8 ºC. Expiration date
one year from production.

The following table presents a summary of the description of the method together with the application field
and the corresponding reference method.

Table 1: Summary of the description of the method with the application field

Description Application Field Reference method

Método alternativo EASY
Veterinary samples from the poultry
QFast® HARD para la Norm UNE-EN ISO
detección rápida de industry 6579:2003/A1:2007
Salmonella spp
Environmental samples from primary Norm UNE-EN ISO 6579:2003
production (poultry)

Meats
Alternative Method HARD
EASY Veterinary QFast® Veterinary samples Norm UNE-EN ISO
Salmonella SOFT for fast 6579:2003/A1:2007
detection of Salmonella
spp
Alternative Method EASY
Animal Feeding Samples
QFast® Salmonella SOFT Norm UNE-EN ISO 6579:2003
for fast detection of
Food samples except meat
Salmonella spp

Page 12 of 45
2.3.- REFERENCE METHODS

The reference methods are the following:

 UNE-EN ISO 6579 (2003): Microbiology of Food and animal feeding stuffs. Horizontal method for
the detection of Salmonella spp.
 UNE-EN ISO 6579 (2003). Erratum: 2007 V2. Horizontal method for the detection of Salmonella
spp.
 UNE-EN ISO 6579/A1 (2007): Microbiology of Food and animal feeding stuffs. Horizontal method
for the detection of Salmonella spp. Amendement 1: Annex D. Detection of Salmonella spp. in
animal faeces and in environmental samples from the primary production stage.

3.- COMPARATIVE STUDY OF THE METHODS

The following study has been performed:

 Relative Efficiency (AC), Relative Specificity (SP) and Relative Sensitivity (SE).
 Relative detection level
 Inclusivity and exclusivity

3.1.- RELATIVE ACCURATY, RELATIVE SPECIFICITY AND RELATIVE SENSITIVITY

3.1.1 Matrix for performing the validation

In this study they analysed 480 samples, both with alternative EASY Salmonella Qfast as well as with the
reference method UNE-EN ISO 6579 for Food and Animal Food and UNE-EN ISO 6579/A1 for the
environmental samples. In the table 2, the distribution of the samples by category is established.

Table 2- Distribution of samples by category in the validation

Positive Negative
Category Total
samples (1) Samples

Environmental (support dust and box covers) 40 30 70

Animal Feeding (corn, rye and soya) 69 60 129

Food (Dairy products, fruits and vegetables,
163 118 281
and others, and meat)
Vaterinary samples (faeces, necks, cover
76 36 112
shoes)
TOTAL 348 244 592
(1)Samples positive by one method or another

Page 13 of 45
3.1.2 Natural samples with Salmonella spp.

They have analysed 31 naturally contaminated samples, which amounts to 8,9 % of the total number of
with Salmonella spp. analysed.

When it has not been possible to obtain a sufficient number of natural samples with Salmonella spp., the
natural samples are inoculated, as described in the UNE-EN ISO 16140.

According to that indicated in the UNE-EN ISO 16140, prior to the inoculation of the natural samples, the
strain is put under stress.

3.1.3 Preparation of the sample for analysis

The UNE-EN ISO 16140 has established with regard to the reference and the alternative method that the
validation should be carried out, whenever possible, with the same sample. In this case, it is not possible
and they have proceeded according to the described in the second case of the point 5.1.1.2.3 of the UNE-
EN ISO 16140.

3.1.4 Results of the tests

In the following tables the paired results of the reference and alternative method are shown; In addition,
they have also calculated the parameters relative sensitivity (SE), relative specificity (SP) and relative
accuracy (AC).

The following shows the definitions of each of the abbreviations:

A+ = Total number of Positive results by the alternative method

A - = Total number of Negative results by alternative method
R+ = Total number of Positive results by the reference method
R - = Total number of Negative results by the reference method

PA = Concordance of Positive results

NA = Concordance of Negative results
PD = Positive Deviation
ND = Negative Deviation

In the following tables the results by category and the global results are shown.

Page 14 of 45
 Table 3- Global Results

Method ISO 6579 and ISO 6579/A1

All categories
Reference method (R+) Reference method (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA=300 PD=22
EASY
Qfast®
Negative Alternative
Negative deviation (A-/R+) Negative concordance (R-/A-)
method (A-)
ND=28 NA=242

CATEGORY 1: ANIMAL FEEDING SAMPLES

Table 4- Results of the category 1: samples of Food animal

Method UNE-EN ISO 6579

Category 1: Animal Feeding
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA= 59 PD= 8
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND= 4 NA= 58

Table 5- Results in corn. Soft medium

Method UNE-EN ISO 6579

Corn
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA= 10 PD= 2
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND= 2 NA= 10

Table 6- Results in rye. Soft medium

Method UNE-EN ISO 6579

Rye
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) (PA=10 PD=3
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND=1 NA= 10

Page 15 of 45
 Table 7- Results in soya. Soft medium

Method UNE-EN ISO 6579

Soya
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA=9 PD=1
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND=1 NA= 10

Table 8- Results in corn. Hard medium

Method UNE-EN ISO 6579

Corn
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA=10 PD=2
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND=0 NA= 8

Table 9- Results in rye. Hard medium

Method UNE-EN ISO 6579

Rye
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA=10 PD=0
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND=0 NA= 10

Table 10- Results in soya. Hard medium

Method UNE-EN ISO 6579

Soya
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA=10 PD=0
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND=0 NA= 10

Page 16 of 45
CATEGORY 2: ENVIRONMENTAL SAMPLES

Table 11- Results of the category 2: Environmental samples

Method UNE-EN ISO 6579/A1

Category 2: Environmental
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA= 38 PD= 0
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND= 2 NA= 30

Table 12- Results in support dust

Method UNE-EN ISO 6579

Support dust
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA=23 PD=0
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND=2 NA= 15

Table 13- Results in box covers

Method UNE-EN ISO 6579

Box covers
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA=15 PD=0
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND=0 NA= 15

Page 17 of 45
CATEGORY 3: GENERAL FOOD

Table 14- Results of the category 3: Food samples

Method UNE-EN ISO 6579

Category 3: Food Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA= 135 PD= 8
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND= 20 NA= 118

Table 15- Results dairy products

Method UNE-EN ISO 6579

Dairy products
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA=37 PD=0
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND=0 NA= 30

Table 16- Results vegetables products

Method UNE-EN ISO 6579

Vegetables products
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA=37 PD=0
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND=7 NA= 30

Table 17- Results different products

Different products Method UNE-EN ISO 6579

(egg, mayonnaise, spices) Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA=32 PD=0
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND=5 NA= 30

Page 18 of 45
 Table 18- Results Meat

Method UNE-EN ISO 6579

Meat
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA=29 PD=8
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND=8 NA= 28

CATEGORY 4: VETERINARY SAMPLES

Table 19- Results of the category 4: Veterinary samples

Method UNE-EN ISO 6579

Category 4: Veterinary
samples Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

EASY method (A+) PA= 68 PD= 6
Qfast®
Negative
Veterinary
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND= 2 NA= 36

Table 20- Results faeces

Method UNE-EN ISO 6579

Faeces
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA=19 PD=0
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND=1 NA= 12

Table 21- Results cover shoes

Method UNE-EN ISO 6579

Cover shoes
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA=29 PD=6
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND=1 NA= 12

Page 19 of 45
 Table 22- Results Necks

Method UNE-EN ISO 6579

Necks
Reference method (R+) Alternative (R-)

Positive Alternative Positive concordance (A+/R+) Positive deviation (R-/A+)

method (A+) PA=20 PD=0
EASY
Qfast® Negative
Alaternative method Negative deviation (A-/R+) Negative concordance (R-/A-)
(A-) ND=0 NA= 12

3.1.5 Calculation and interpretation of the results and Confidence intervals.

Calculation of the relative accuracy (AC), relative specificity (SP) and relative sensitivity (SE).

The results of the validation are shown in table 23.

Table 23- Results of relative accuracy (AC), relative specificity (SP) and relative sensitivity (SE)

Accuracy Specificity Sensitivity

NA ND PA PD Total (AC) (SP) (SE)
Animal Food samples 58 4 59 8 129 90,70% 87,88% 93,65%
Food samples 118 20 135 8 281 90,04% 93,65% 87,10%
Environmental
30 2 38 0 70 100,00% 100,00% 95,00%
samples
Veterinary samples 36 2 68 6 112 92,86% 85,71% 97,14%
Total 206 26 232 16 480 91,55% 91,67% 91,46%

Page 20 of 45
The table 24 shows the obtained results of AC, SP y SE (%) for all the categories with their lower
confidence limit (LCL) for the extension results.

Table 24- Results of the AC, SP y SE wit their lower confidence limit

Reference methods UNE-EN ISO 6579 or UNE-EN ISO 6579/A1 // Method Easy Qfast®

Categories AC (%) LCL LCS SP (%) LCL LCS SE (%) LCL LCS

Category 1. Animal Feeding samples 90,70 85,40 - 87,88 80,02 95,74 93,65 88,82 -
Category 2. Environmental samples 100,00 - - 100,00 - - 95,00 92,50 -
Category 3. Foods samples 90,04 84,03 - 93,65 88,82 - 87,10 75,25 98,95
Category 4. Veterinary samples 92,86 88,43 - 85,71 81,40 90,02 97,14 94,70 -
All categories 91,55 87,10 - 91,67 87,34 - 91,46 89,92 -

Statistical Test according to annex F of the norm UNE-EN ISO 16140

The test used for the study of discordant results discordant is:

After counting the total number of discordant results of Y in the following manner:

Y= PD + ND

Check if the two methods can be different by the balance of the sensitivity versus the specificity:

- Case 1: For Y < 6, less than 6 discordant results there is no test available.
- Case 2: For 6 ≤ Y ≥ 22, between 6 and 22 discordant results, the m is calculated as the smaller of
the two values PD and ND and the binomial law is applied according to the following table 25:

If m ≥ M for a given Y, the two METHODS are different α < 0.05 (bilateral).

Disagreements
Y= PD + ND 6a8 9 a 11 12 a 14 15 a 16 17 a 19 20 a 22
M= máx (m)
for α <0,05 0 1 2 3 4 5

Page 21 of 45
- Case 3: For Y > 22, more than 22 discordant results, the MCNemar test is used with the chi-square
distribution for 1 degree of freedom:

X2 = d2/Y, with d= |PD - ND| and Y= PD + ND

The two methods are different if α <0.05 (bilateral) if X2 > 3,841

In the validation, Y value is 50, so third case must be applied (McNemar Test).

The obtained results of the test are:

d = |PD - ND| = |22 - 28| = 6; d2 = 6 x 6=36

Y= PD + ND = 22+28 = 50

X2 = d2/Y = 36/50 = 0,72  X2 < 3,841  The two methods are not different.

Page 22 of 45
3.1.6 Study of discordant results

Negative deviations (ND)

In relation to the negative deviation, there were 28. The information about these samples is shown in table
26.
Table 26- Discordant Results obtained: negative deviations
ISO 6579/A1
Nº Easy Qfast Reading
Matriz XLD ASAP or ISO 6579
Sample Result sensor
result
1297 Shoulder of lamb Absence/ 25 g 466 nA Positive Positive Presence/ 25 g
Absence/ 25 g Positive Positive
1314 Chopsticks 447 nA Presence/ 25 g
Absence/ 25 g Positive Positive
1315 Chopsticks skirt 378 nA Presence/ 25 g

1316 Shoulder of lamb Absence/ 25 g 837 nA Positive Positive Presence/ 25 g

1228 Pork chop Absence/ 25 g 487 nA Positive Positive Presence/ 25 g
1273 Pork chop Absence/ 25 g 650 nA Positive Positive Presence/ 25 g

1274 Pork chop Absence/ 25 g 485 nA Positive Positive Presence/ 25 g

Absence/ 25 g Positive Positive
1308 743 nA Presence/ 25 g
Pork Stew
496 Granulated garlic Absence/ 25 g 620 nA Negative Negative Presence/ 25 g
Absence/ 25 g
497 Granulated garlic 648 nA Negative Negative Presence/ 25 g
Absence/ 25 g
498 Orégano 378 nA Negative Negative Presence/ 25 g

Absence/ 25 g
503 Tabasco 446 nA Negative Negative Presence/ 25 g
Absence/ 25 g
504 Tabasco 354 nA Negative Negative Presence/ 25 g

449 Gourmet salad Absence/ 25 g 571 nA Positive Positive Presence/ 25 g

452 Mezclum salad Absence/ 25 g 727 nA Positive Positive Presence/ 25 g

Absence/ 25 g
741 444 nA Positive Positive Presence/ 25 g
Red Grape
Absence/ 25 g
742 282 nA Positive Positive Presence/ 25 g
Red Grape
Absence/ 25 g
745 617 nA Positive Positive Presence/ 25 g
Pear
458 Apple juice Absence/ 25 g 739 nA Positive Negative Presence/ 25 g

510 Apple juice 639 nA Positive Negative Presence/ 25 g

Absence/ 25 g
Support sponge Ausencia/
560 372 nA Positive Positive Presence/ support
powder soporte
Support sponge Ausencia/
562 221 nA Positive Positive Presence/ support
powder soporte
354 Soya Ausencia/ 25 g 383 nA Positive Positive Presence/ 25 g

369 Barley Ausencia/ 25 g 485 nA Positive Positive Presence/ 25 g

344 Corn Absence/ 25 g 377 nA Positive Positive Presence/ 25 g

345 Corn 553 nA Positive Positive Presence/ 25 g

Absence/ 25 g
Positive (1
1506 Faeces Absence/ 25 g 167 nA Negative Presence/ 25 g
colony)
Absence/ 4 Error/546 Presence/ 4 shoe
1562 Shoe covers Negative Negative
shoe covers nA covers
nA: nano amperios
Page 23 of 45
Comments:

The eight meat samples indicated with negative deviations, were packaged in a modified atmosphere that
minimises the growth of microbes. In this type of samples, with a low microbial load, for which the hard
medium is used such as the pre-enrichment medium, the Salmonella spp. can remain metabolically
inactive due to the potent effect of the medium. This means that the enzyme reaction does not occur at
the time of reading in the sensor, giving a negative read but subsequently producing the growth of
Salmonella spp. in the XLD and ASAP plates.

In the two samples of the tabasco species (503 y 504), the two samples of powdered garlic (496 y 497)
and oregano (498) there was no growth of characteristic colonies of Salmonella spp., in any of the two
confirmation plates; both samples were inoculated using the strain (CV14), this strain is used in the
inclusiveness study, which was satisfactory.

The spices Chile Tabasco, the garlic and the oregano, had natural substances with anti-microbial effects.
The norm UNE-EN ISO 6887-4: 2003 “Microbiology of the food for human consumption and animal food.
Preparation of the samples for analysis, initial suspension and decimal dilutions to examine microbiology”
establish that the preparation of the samples of species for their subsequent microbiological analysis by the
ISO methods, require the addition of potassium sulphite to the peptone water diluent (APT) with the aim of
reducing the antimicrobial activity of the inhibitor substances that contain these types of matrices. This
addition is carried out in the spice samples for their subsequent analysis by the reference method but not
for the samples analysed by the alternative method. This explains that these samples by the alternative
method have obtained false negatives. As a consequence of these results the protocol of the method
should include that for the case of this type of samples their preparation should be performed according to
the indications in the norm UNE-EN ISO 6887-4.

In the two environmental samples (560, 562) there was a low growth of characteristic colonies of
Salmonella spp. in the two plates; after the corresponding biochemical and serological conformations that
confirmed that they were Salmonella spp. The samples were inoculated with 2.7 cfu/ powder based
sponge, the characteristics of this matrix do not ensure the homogeneity of such low contamination levels.
Thus, we conclude that the quantity of microorganisms at the time of the read was not sufficient for the
reader to detect them and if incubated for a further 24 hours at 37ºC ± 1ª C, the microorganism would
have been visible on the XLD and ASAP plates.

In the case of the Animal Food samples (344, 345, 354, 369), the read was performed after incubating the
sample for 30 minutes with the reaction solution. After obtaining these negative deviations, the samples
were incubated for a further 30 minutes and they were read again, the sensor indicating the presence; on
this basis, it is recommended that the samples should be incubated for the maximum possible time
established in this phase of the method.

Page 24 of 45
In the case of the two samples of red grapes, the reader gave a negative response, however, there was
growth of characteristic colonies both on the XLD plates as well as the ASAP plates. The red grapes have
polyphenols, substances whose antimicrobial effect is described in the literature. The growth of Salmonella
spp. is affected by this antimicrobial effect, and is less than that expected after the development of the
procedure of the alternative test and does not reach the threshold level required so that the sensor gives a
positive response, but subsequently there is growth of Salmonella spp. on the XLD and ASAP plates.

In the samples of IV gamma (salads packaged in modified atmospheric packaging) the presence of
Salmonella spp. was natural; the quantity of Salmonella spp. at the time of the read was probably lower
than the threshold required for the reader to give a positive response, but subsequently there was growth
of Salmonella spp. on the XLD and ASAP plates.

In the present study, 12 apple juice samples were analysed that were inoculated with Salmonella spp. on
three different days, of which, two of them had not given the expected results. The apple juice has an acid
pH (around of 3.5), the conditions are not adequate for the microbial growth; an insufficient spike in the
two samples combined with the low pH could result in a negative result as there is not sufficient
Salmonella spp. at the time of the reading.

Finally, a false negative has been obtained in a pear sample, with the growth of characteristic colonies on
both plates. The spike of these samples was lower than hoped from the control inoculation that has given
rise to this deviation; there was growth on the XLD and ASAP plates, which has also happened in the
relative detection limit study in the expected levels between 0.5 and 2 cfu/25 g.

In faeces sample, there is a low growth of Salmonella spp. it is detected, due to just a characteristic colony
has been obtained on the XLD plate and No characteristic colony growth in the ASAP chromogenic, both
facts indicate the low concentration of inoculated Salmonella in the sample.
The discordance between both methods should be due to the fact that the analysis has been performed in
two different sample portions. The same thing has occurred with the cover shoes sample in which one, no
characteristic colony has growth in any of the two plates.

Page 25 of 45
Positive Deviations (PD)

With regard to the positive deviations, there were 22; the information about these samples are shown in
table 27.

Table 27- Discordant results obtained: positive deviations

Nº
Matriz Easy Qfast result Reading sensor XLD ASAP ISO 6579
sample
Result
1027 Minced pork Presence/25g 1020Rt/1209 nA Negative Negative Negative/25g

1285 Beef tenderloin Presence/25g 1491 nA Negative Negative Negative/25g

1209 Bovine fillet Presence/25g 1,68 uA Negative Negative Negative/25g

1212 Lamb fillet Presence/25g 1,16 uA Negative Negative Negative/25g

1213 Pork fillet Presence/25g 1446 nA Negative Negative Negative/25g

940rt/1025rt nA Negative
1023 Minced beef Presence/25g Negative Negative/25g

1026 Minced beef Presence/25g 1310 nA Negative Negative Negative/25g

1028 Minced beef Presence/25g 1437 nA Negative Negative Negative/25g

529 Feed on soya Presence/25g 1,91 uA Negative Negative Negative/25g

373 Feed on corn Presence/25g 1368 nA Negative Negative Negative/25g

375 Feed on corn Presence/25g 4,88 uA Negative Negative Negative/25g

523 Feed on barley Presence/25g 2,30 uA Negative Negative Negative/25g

524 Feed on barley Presence/25g 4,56 uA Negative Negative Negative/25g

531 Feed on barley Presence/25g 5,25 uA Negative Negative Negative/25g

1413 Corn Presence/ 25 g 1341 nA Negative Negative Negative/25g

1416 Corn Presence/ 25 g 956rt/872 rt Negative Negative Negative/25g

Presence/4 shoe Negative Negative/4
1685 Shoe covers 1,86 uA Positive
covers shoe covers
Presence/4 shoe Negative/4
1686 Shoe covers 2,66 uA Positive Negative
covers shoe covers
Presence/4 shoe Negative/4
1687 Shoe covers 2,54 uA Positive Positive
covers shoe covers
Presence/4 shoe Negative/4
1688 Shoe covers 3,25 uA Positive Positive
covers shoe covers
Presence/4 shoe Negative/4
1692 Shoe covers 3,56 u A Positive Positive
covers shoe covers
Presence/4 shoe Negative/4
1693 Shoe covers 3,10 uA Positive Positive
covers shoe covers
uA : microamperios
nA: nanoamperios

Comments:

With regard to the positive deviations obtained in the samples, there was no growth of characteristic
colonies on the XLD or ASAP plates; however, the identification of the colonies present on both plates was
made via the biochemical gallery API 20E.

Page 26 of 45
A series of samples were identified Enterobacter cloacae and/or Hafnia alvei. In the exclusivity study, the
strain of the collection Enterobacter cloacae (CECT 194) and Hafnia alvei (CECT 157) were analysed, which
were absent both on the soft and hard medium. In this case the strains were identified in both medium
with wild strains present naturally in the samples analysed; it could be that in this case, the wild strains
had behaved differently to the strains in the collection. In other samples, the identification gave rise to
microorganisms such as Cronobacter spp., Enterobacter aurigenes, Proteus mirabilis, Citrobacter coseri o
farmeri, Klebsiella pneumoniae.

In all samples where a positive deviation was obtained, after the compulsory confirmation the conclusion
was made that the results was presumptive positive and negative confirmed.

3.2.- RELATIVE DETECTION LIMIT (LDR)

The relative detection level is the smallest number of culturable microorganisms that can be detected in
the sample in 50% of occasions by the alternative and reference methods. The artificial contaminations are
performed in accordance with the requisites of the norm UNE-EN ISO 16140, previously described.

The determination of the relative detection limit was performed for six different matrices, each
encompassed a category. For each one of the categories they used a different target microorganism.

They have used 4 levels, as a minimum, of target microorganisms per matrix, including the negative
control. The analysis was performed six times by both methods, alternative and reference.

These matrices as well as the Salmonella spp. strains were:

• Dried powder: Salmonella london

• Dairy products (crude milk): Salmonella dublin
• Fresh Eggs: Salmonella enteritidis
• Vegetables (fruit): Salmonella Virchow
• Corn: Salmonella derby
• Meat: Salmonella arizonae
 Faeces: Salmonella typhimurium

The limit of detection has benn calculated as the Spearman–Kärber1 Test.

1 “Hitchins A. Proposed Use of a 50 % Limit of Detection value in Definig Uncertainty Limits in the
Validation of presence-Absence Microbial Detection Methods. Draft 10th December, 2003”.

Page 27 of 45
 Table 28- Relative Detection Level Study: Results obtained

Strain/matriz Relative detection level (cfu/25g o support)

Alternative mehotd EASY Reference method UNE-EN ISO

Salmonella Qfast® 6579 or UNE-EN ISO 6579/A1

DRIED Relative detection 0,39 0,61

SUPPORT level interval
0,16-0,93 0,27-1,38
FAECES Relative detection
level interval 5,13 3,13
3,85-6,80 1,60-6,05
FRUIT Relative detection
level interval 0,8 0,22
0,48-1,32 0,15-0,35
EGGS Relative detection 0,75 0,3
level interval
0,52-1,10 0,20-0,50
CRUDE MILK Relative detection 0,48 0,35
level interval
0,28-0,8 0,28-0,45
MEAT Relative detection 0,58 0,4
level interval
0,38-0,90 0,22-0,72
CORN Relative detection 0,4 0,4
Soft medium 1 level interval
0,25-0,62 0,25-0,62
CORN Relative detection 0,45 0,25
Hard medium level interval
1 0,25-0,8 0,15-0,40

The alternative method and the reference method have relatively similar detection levels.

In the relative detection levels of the alternative method there are between 0.16 and 6.80 cfu/25g o
support and in the case of the reference method between 0.15 and 6.05 cfu/25g o support.

3.3.- INCLUSIVITY AND EXCLUSIVITY

The inclusivity is the capacity of the alternative method to detect the analyte target between wide groups
of strains. The objective of the study is to verify that all the strains are detected by the alternative method
as there are two culture media, one for simple matrices (Soft) and other for complex matrices (Hard), the
study is performed with both medium.

The exclusivity is the absence of interference in the alternative method of an adequate group of non-target
strains. The objective of the study is to verify that the non-target strains are not detected by the
alternative method and this verifying the selectivity of the alternative method. As with the inclusiveness
Page 28 of 45
the study is performed with both broths.

3.3.1 Test protocol

The strains selected for this study are in cryoballs, which are allowed to grow in BHI broth at 37ºC for 24h.
Once this time has passed, the corresponding decimal dilutions are performed in maximum recovery
diluent (MRD), and 0.3 ml of the adequate dilution is added to 225 ml of the corresponding medium. To
control the inoculum added to the sample, 1ml of the corresponding dilution is spread on the 5 PCA plates
and they are incubated for 72h at 30ºC.

3.3.2 Results and conclusion

The results of the inclusiveness and exclusivity, both in the Hard as well as the Soft medium, are shown in
Annex 5 for the Exclusivity and Annex 6 for the Inclusivity.

The results obtained in both the Hard as well as the Soft medium were correct, both for the inclusivity as
well as the exclusivity.

Page 29 of 45
4.- COLABORATIVE STUDY

4.1.- ORGANIZATION OF THE STUDY

The collaborative exercise study was organised in November and December 2014. The number of
participating laboratories was 11.

The chronogram of the collaborative is detailed below:

• 18-21 November 2014: Reception of the samples of powdered milk in the laboratory

• Week of the 24th of November 2014: start of test lot A (Samples M1 to M12)

• Week of the 1st December 2014: start of test lot B (Samples M13 to M24)

There were no incidents in the in the planned chronogram.

4.1.1.-Nature of the preparations

A sample was selected that originated from a single lot, consistent in skimmed powdered milk.

With regard to this sample, the absence of Salmonella spp., was verified via the application of the
Reference method (UNE-EN ISO 6579). In these tests, the presence of Salmonella spp. was not found in
any of the samples analysed.

Before being sent, a sufficient quantity of skimmed powdered milk packages were prepared to allow the
addition prior to sending a quantity inoculated with Salmonella spp. prepared with powdered milk,
obtaining a final weight of 25 grams.

Page 30 of 45
4.1.2.- Inoculation rate

The samples were inoculated in three different levels:

Table 29- Levels of inoculation of Salmonella spp.

Level Salmonella spp. media count in 25 g

L0 0

L1 35 ufc

L2 76 ufc

The inoculum were prepared with the addition of Salmonella entérica from the CECT 4594.

The stability tests carried out for the period of the test confirmed the survival of the microorganism in all
the samples analysed that had been inoculated.

4.1.3.- Problems with the temperature and the transport at the time of reception

The samples were coded as M1 to M24, with each laboratory receiving two packages of each code, which
were used randomly for the test with the Reference method or the alternative method.

The samples and inoculum for sending to the laboratories were stored in the freezer (-30ºC) until
dispatched and the stability of the inoculum was confirmed when these were kept in the fridge for up to 2
weeks.

The samples were sent on the 17th of November, in an isothermal packet with dry ice and all the
laboratories received them on the 18th of November. All the preparations arrived on time.

The names of the samples and the contamination level are in the table 30.

Contamination level Name of sample

L0 M1, M2, M7, M8, M13, M14, M19, M20

L1 M3, M4, M5, M6, M9, M10, M11, M12

M15, M16, M17, M18, M21, M22, M23,
L2
M24

Page 31 of 45
4.2.- RESULTS OF THE ANALYSIS

The summary of the results obtained by the laboratories are shown in table 31.

Table 31- Coincidence of the results for each of the methods applied and the inoculation level

Alternative method Reference method

Laboratory
Nº positive samples Nº positive samples
Level 0 Level 1 Level 2 Level 0 Level 1 Level 2
0/8 8/8 7/8 1/8 8/8 8/8
1
1/8 8/8 7/8 0/8 8/8 8/8
2

3 0/8 7/8 8/8 0/8 8/8 8/8

0/8 8/8 8/8 4/8 8/8 8/8

4
0/8 8/8 8/8 0/8 8/8 8/8
5

6 0/8 8/8 8/8 0/8 8/8 8/8

7 0/8 7/8 7/8 0/8 8/8 8/8

0/8 8/8 8/8 0/8 8/8 8/8

8
0/8 8/8 8/8 0/8 8/8 8/8
9
0/8 8/8 8/8 0/8 8/8 8/8
10

11 0/8 8/8 8/8 0/8 8/8 8/8

FP TP TP FP TP TP

1 86 85 5 88 88

N 88 88 88 88 88 88

FP: Number of false positives

TP: Number of true positives
N: Number of analyses carried out in each level

Page 32 of 45
4.3.- CALCULATIONS

4.3.1.- CALCULATION OF THE PERCENTAGES OF SPECIFICITY (SP), SENSITIVITY (SE) AND

RELATIVE EFFICIENCY (AC)

Calculations of Specificity (SP) and Sensitivity (SE)

The values of specificity and sensitivity in both methods are shown in table 32, including the value of the
inferior limit (LCL).

For the calculation of specificity and sensitivity, the following formula is used.

For level L0:

For levels L1 and L2:

Being:

FP: False positives

TP: True positives
N: Number of samples

Table 32: Specificity (SP) and Sensitivity (SE) of the alternative and reference method

Alternative Reference Method

Linf Linf
Easy QFast®Method UNE-EN ISO 6579
Specificity (SP)

SP (level L0) 98,86% 96,60% 94,32% 89,38%

Sensibility (SE)

SE (Level L1) 97,73% 94,55% 100,00% 100,00%

SE (Level L2) 96,59% 92,72% 100,00% 100,00%

SE (Level L1+L2) 97,16% 94,65% 100,00% 100,00%

Page 33 of 45
 Calculation of Relative Accuracy (AC)

The comparison of the pairs of results of the different levels of contamination are summarised in table 33.
Table 33: Results obtained by both methods

Reference Method
Alternative
Level UNE-EN ISO 6579
Easy QFAST® Salmonella Method
Positive samples Negative samples Total

Positive samples PA= 0 PD= 1 1

Level L0 Negative samples ND= 5 NA= 82 87

Total 5 83 88

Positive samples PA= 86 PD=0 86

Level 1 Negative samples ND= 2 NA= 0 2

Total 88 0 88

Positive samples PA= 85 PD= 0 85

Level 2 Negative samples ND= 3 NA= 0 3

Total 88 0 88

Positive samples PA= 171 PD= 0 171

Level 1+2 Negative samples ND= 5 NA= 0 5

Total 176 0 176

Note-PA: positive concordance; NA: negative concordance; ND: negative deviations; PD positive deviations.

The values of relative accuracy (AC) for the different levels of contamination are shown in table 34, together
with the corresponding lower confidence interval (LCL).

The relative efficiency is calculated according to the following formula:

Being:

PA: Number of positive concordance

NA: Number of negative concordance
N: Total number of samples analysed

Page 34 of 45
Table 34: Values of the Relative accuracy (AC) and confidence intervals

Level Parameter Result

AC 93,18%
Level 0
LCL 87,81%

AC 97,73%
Level 1
LCL 95,55%

AC 96,59%
Level 2
LCL 92,72%

AC 97,16%
Level 1+2
LCL 94,65%

Page 35 of 45
4.3.2.- STUDY OF THE DISCORDANT RESULTS

Statistical test according to Annex F of the standard UNE-EN ISO 16140

The results of the statistical test obtained are shown in table 35.

Reference Method
Alternative Easy Qfast® Method
Y= ND+PD Y=6

m lower value of PD and ND=1

Level 0 Conclusion M=0 for Y form 6 to 8
Both methods are considered as
equivalent (m>M)
Y= ND+PD Y=2 (Y<6)

Conclusion No statistical test is available.

Level 1
Both methods are considered as
equivalent

Y= ND+PD Y=3 (Y<6)

No statistical test is available.
Level 2 Conclusion Both methods are considered as
equivalent.

Y= ND+PD Y=5 (Y<6)

No statistical test is available.

Conclusion
Level 1 + Level 2 Both methods are considered as
equivalent

The method Easy Qfast® Salmonella and the Reference method UNE-EN ISO 6579 are
considered as equivalent.

Page 36 of 45
5- INTERPRETATION OF RESULTS FROM THE COLLABORATIVE SUTUDY AND COMPARISON OF
THE METHODS

5.1. COMPARISON OF RELATIVE ACCURACY (AC), RELATIVE SPECIFICITY (SP) AND RELATIVE
SENSITIVITY (SE) VALUES

In the Table 36 show the results obtained during the collaborative study carried out in powdered milk and
global validation.

Table 36: Comparison of collaborative study carried out in powdered milk and comparison of methods

Collaborative Study Comparasion of methods

Relative Accuracy(AC) 93,18% 91,25%
Relative Sensitivity (SE) 97,16% 89,92%
Relative Specificity (SP) 98,86% 92,79%

The data collected in this table shows that the values of AC, SE and SP are higher in the collaborative study
than in the comparative study; This is because the validation data correspond to a very broad scope
(environmental samples from primary production, animal feed and food samples in general).

5.2.- ACCORDANCE, CONCORDANCE AND CONCORDANCE ODDS RATIO (COR)

The Accordance is the percentage chance of finding the same result from two identical test portions (both
positive and negative), in the same laboratory under repeatibility conditions.

In order to find this information regarding the data obtained in a collaborative study, the probability that
two samples give the same result is calculated based on the labs participants. Once probabilities are
obtained this are averaged.

The Concordance is the percentage chance of finding the same result for two identical samples analysed
in two different laboratories.

For the calculation, they take the replication of each laboratory, matching the identical results with the
others obtained in the laboratory. The concordance is the average of all data matched from the same
results with all the possible pairs of data.

The Concordance odds ratio: Although in a qualitative method, it is difficult to calculate the grade of
collaborative variation, as the conformity and the concordance are strongly influenced by the efficiency

Page 37 of 45
grade, it is possible to calculate what is called the relative concordance opportunity (COR), calculated
according to the following equation:

If the concordance is smaller than the accordance, it indicates that two identical samples are more likely to
give the same result if they are analysed by the same laboratory than if they are analysed by different
ones, suggesting that there can be variability in performance between laboratories.

5.2.1- ACCORDANCE, CONCORDANCE AND COR

Table 37: Results of accordance, concordance and COR in the collaborative of the powdered milk

Easy QFast® Salmonella UNE-EN ISO 6579

Level Accordance Concordance COR Accordance Concordance COR

Level 0 98,01% 90,40% 5,24 93,47% 100,00% 1,79

Level 1 96,02% 95,51% 1,13 100,00% 100,00% 1,00

Level 2 94,03% 94,52% 0,91 100,00% 100,00% 1,00

Conclusions

The conclusions of the collaborative study in powdered milk are the following:

 With regard to the results sent by the participants:

For the normalised method reference, only two laboratories have found Salmonella spp.
corresponding to a level 0, non-inoculated level. The laboratory number 1 has found a positive
sample of the 8 non-inoculated and laboratory number 4, declares 4 positives in level 0. However,
none of these two laboratories have found the presence of Salmonella spp. in level 0 with the
alternative method Easy QFast®. Only laboratory number 2 has found a positive in the level 0
with the alternative method Easy QFast®.

In the case of the level 1 and level 2 samples of the total participants, the presence was
found with the reference method. The detection of the alternative method is, thus satisfactory,
although with some comments: laboratory number 3 did not detect a positive sample in level 1
with the alternative method Easy QFast®. The participants’ number 1 and number 2 reported a
negative result in level 2 with the alternative method Easy QFast®. The laboratory number 7
reported a negative in the level 1 and another in level 2.

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 The specificity of the alternative method is greater than 98% and the value obtained by the
reference method.

 The Sensitivity, thus, is above 95%, in all the levels studied, and very close to the values obtained
in the reference method.

 The accordance and concordance of the alternative method Easy QFast®, is above 90%. However,
we found that the differences found between the results of the identical samples distributed are
attributable to the inter-laboratory variability, more than the behaviour of the method as shown by
the COR factor. The elevated value obtained for this statistic in the alternative method Easy
Qfast® of 5.24 responds to the excellent conformity of the method, compared with the accordance.

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6. AUDITS

Introduction

AENOR performed an annual audit that verifies the maintenance that complies with the requisites
established in the “Regulation of the certification of validated alternative methods for the quality control
and food safety (RP B 59)”.

On one hand, the quality management system is audited in the manufacturing facilities with the objective
of verifying the maintenance conditions established in the rule (RP B59).

On the other hand, annual contrast tests have been performed, in a way that during the 5 years, the
certified method is verified in all the matrices at least once. Prior to the follow-up audit, AENOR prepares a
sample that is divided into aliquots. The auditor gives one of the aliquots to the manufacturer, starting the
analysis of the test in the presence of the auditor. Once the analysis is finished, the results obtained are
sent to AENOR laboratory. In parallel, AENOR laboratory performs the tests on the second aliquot. The
results are compared and report is prepared.

Results of the audit

Year 2014

 Audit: audit was performed successfully.

 Procedure test 2014: mixing matrix with Salmonella spp. added. Result correct.

Year 2015

 Audit: audit was performed successfully.

 Procedure test 2015: yoghurt matrix with Salmonella spp. added. Result correct.

Year 2016

 Audit: audit was performed successfully.

 Procedure test 2016: fresh meat with Salmonella spp. added. Result correct. Easy Qfast Salmonella
(Hard medium).
 Procedure test 2016: powder garlic without Salmonella spp. added. Result correct. Easy Qfast
Salmonella (Soft medium).

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7.- CONCLUSION

Conclusions of the Comparative Study are:

The method EasyQFast® Salmonella has a satisfactory relative accuracy, relative specificity and relative
sensitivity. With regard to the results obtained, we conclude that the method EasyQFast® Salmonella is
equivalent to the reference method. The same conclusion has been obtained for Veterinary EasyQFast®
Salmonella.
The results of the tests in necks and faeces have been refrigerated at 4-8ºC during 24h and 48h after
incubated the medium 1, the results obtained are the same that without refrigeration.

The levels of detection of the alternative method EasyQFast® and the reference method are similar.

The exclusivity and inclusiveness are totally satisfactory for both medium used and the alternative method.

The conclusions of the collaborative study are:

The results obtained in the collaborative study of efficacy, specificity and relative sensitivity are somewhat
higher than those obtained in the Comparative Study, since the data of the validation correspond to a very
broad scope.

Regarding the results obtained in the present validation, we establish that both methods are
equivalent.

The conclusions of the audit performed are:

The results of the audit performed as well as the performance tests have been satisfactory in the years
2014, 2015 and 2016.

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Annex 1–Protocol of the alternative method Easy QFast®

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Page 43 of 45
 Annex 2 – Reference Method
UNE-EN ISO 6579:2003 Microbiology of the food for human consumption and Animal Food.
Horizontal method for the detection of Salmonella spp.

xg or ml of product

Dilution to 1/10 in APT

Incubation at 37°C ± 1°C for 18 h ± 2 h

Add 0.1ml of mother solution incubated at 1 ml mother solution incubated at

10 ml RVS broth 10 ml MKTTn broth
Incubation at 41.5°C ± 1°C, 24 h ± 3 h Incubation at 37°C ±1°C, 24 h ±3 h

XLD and ASAP Agar (as second chromogenic media)

Incubation 37°C ± 1°C a 24 h ± 3 h

Isolate 1 characteristic colony

(For each agar media) and 4 colonies more in case the first one is negative

Nutrient Agar
Incubation 24 h ± 3 h a 37°C ±1°C

Biochemical Confirmation Serological Confirmation

serológica

Interpretation of the results

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 Annex 3 - Reference Method
Reference Method ISO 6579/A1- Microbiology of food and animal feeding stuffs.
Horizontal method for the detection of Salmonella spp. Amendement 1: Annex D.
Detection of Salmonella spp. in animal faeces and in environmental samples from
the primary production stage.

Pre-enrichment: APT
Dilution 1/10

Incubation at 37°C ± 1°C for18 h± 2 h

Deposit separately
3 drops on the MSRV agar
(0.1 ml in total)

Incubation at 41.5°C ± 1°C for 24 h ± 3 h

Read plates:
Positive Test: migration area white-greyish from the drop inoculated.
Negative Test: re-incubate the other plates 24 h ± 3 h

Pick with a loop of 1μl in the area outside of the migration area
Isolate on XLD agar

Incubation at 37°C ± 1°C, 24 h ± 3 h

Read
Select a characteristic colony for isolation

Isolate on nutrient agar

Incubate at 37°C ± 1°C
24 h ± 3 h

Biochemical confirmation and serological confirmation

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