Hygiena foodproof 2300 92 User manual

Documentation for the qualitative detection of
Listeria monocytogenes
Listeria
monocytogenes
Detection LyoKit
MANUAL
foodproof®
Order No. KIT 2300 92 / KIT 2300 93 / KIT 2300 94

Order No.
LP: KIT 2300 92
RP: KIT 2300 93
DP: KIT 2300 94
Kit for 96 reactions (lyophilized)
for a maximum of 94 samples
Store kit at 2 °C to 8 °C
For testing of food
and environmental samples
Manual:
Version 4, February 2022
Listeria monocytogenes
Detection LyoKit
foodproof®
Approvals:
LICENSE NUMBER 070401

3
TABLE OF CONTENTS
KIT 2300 92 / 93 / 94 - Listeria monocytogenes Detection LyoKit
1. OVERVIEW ..................................................................................4
1.1 General Information ................................................................................ 4
1.2 Applicability.............................................................................................. 4
1.3 Kit Contents ............................................................................................. 5
2. INSTRUCTIONS ..........................................................................6
2.1 Required Material .................................................................................... 6
2.2 Precautions and Preparations................................................................ 7
2.3 Enrichment and DNA extraction ............................................................ 8
2.3.1 Certified Methods ................................................................................................ 8
2.4 Procedure................................................................................................. 9
2.4.1 Workflow.............................................................................................................. 9
2.4.2 Program Setup................................................................................................... 10
2.4.3 Data Interpretation............................................................................................. 10
2.5 Troubleshooting..................................................................................... 11
2.6 Support................................................................................................... 12
3. ADDITIONAL INFORMATION...................................................13
3.1 Testing Principle .................................................................................... 13
3.2 Trademarks ............................................................................................ 14
3.3 Reference Number ................................................................................ 14
3.4 Change Index......................................................................................... 14
ANNEX 1 ........................................................................................15
ANNEX 2 ........................................................................................16

4
OVERVIEW
KIT 2300 92 / 93 / 94 - Listeria monocytogenes Detection LyoKit
1. OVERVIEW
1.1 General Information
Number of Reactions
The kit is designed for 96 reactions with a final reaction volume of 25 µl each. Up to 94
samples plus positive and negative control can be analyzed per run.
Storage and Stability
Store all components at 2 °C to 8 °C. They are guaranteed to be stable through the expiration
date printed on the label. Opening of the kit does not shorten the expiration date.
The PCR strips must be stored in the provided aluminum bag. Protect from light and
moisture.
LyoKit Tube Profiles
The LyoKit is available in three different tube profiles: white low profile tubes (LP), clear
regular profile tubes (RP), and clear low profile tubes (DP).
The majority of real-time PCR cyclers use low profile tubes (LP). For the Dualo 32®R2and
a few other cyclers, please use clear low profile tubes (DP). For a detailed overview, please
have a look at our compatibility chart.
1.2 Applicability
The kit described in this Instruction Manual has been developed for real-time PCR
instruments with a FAM and a VIC/Yakima Yellow or HEX detection channel. The
performance of the kit was tested with the following real-time PCR instruments:
LightCycler®480, LightCycler®96 (Roche Diagnostics), Mx3005P (Agilent Technologies),
Applied BiosystemsTM 7500 Fast (Thermo Scientific), CFX96TM (Bio-Rad) and others.
When testing the samples on a LightCycler®480 instrument, it is recommended to carry
out the DNA extraction with a wash step (foodproof®StarPrep Two Kit, KIT 2301 77,
Procedure A: STANDARD protocol).

5
OVERVIEW
KIT 2300 92 / 93 / 94 - Listeria monocytogenes Detection LyoKit
LP: KIT 2300 92
RP: KIT 2300 93
DP: KIT 2300 94
* Tube profile and instrument compatibility chart is available online: www.bc-diagnostics.com/foodproof-compatibility-chart
1.3 Kit Contents
A schematic representation of the foodproof®Listeria monocytogenes Detection LyoKit
with all its components.
Component Details
1 Microplate
12 x 8-tube strips, prefilled with lyophilized ready-to-use PCR mix.
Available are three different tube profiles: white low profile tubes (LP),
clear regular profile tubes (RP), and clear low profile tubes (DP).*
2 12 x 8-cap strips For use in real-time PCR after addition of samples.
32 x H2O PCR-grade
(colorless cap) 1 ml nuclease-free, for use as a PCR run negative control.
4Control Template
(purple cap)
250 µl, contains a stabilized solution of DNA for use as a PCR run
positive control.
1
2
3
4

6
INSTRUCTIONS
KIT 2300 92 / 93 / 94 - Listeria monocytogenes Detection LyoKit
2. INSTRUCTIONS
2.1 Required Material
Most of the required equipment and reagents are available through HygienaTM.
Please contact us for further information.
PCR strip / plate centrifuges
• Without vortex: Mini microcentrifuge for 4 x 8-strips
• With vortex: Multispin MSC-6000 for 4 x 8-strips
• With vortex: CVP-2 for 12 x 8-strips and plates
Material
Nuclease-free, aerosol-resistant pipette filter tips.
Use a real-time PCR cycler suitable for detection of respective probes
as well as for using low or regular profile strip tubes.
In case the strip tubes don´t fit for the instrument, the samples should
be transferred to appropriate PCR vessels after resuspension of the
lyophilized PCR mix.

7
INSTRUCTIONS
KIT 2300 92 / 93 / 94 - Listeria monocytogenes Detection LyoKit
2.2 Precautions and Preparations
The kit provides all reagents required for the PCR. However, in order to achieve reliable
results, the entire assay procedure must be performed under nuclease-free conditions.
Follow the instructions below to avoid nuclease, carry-over, or cross-contamination:

Keep the kit components separate from other reagents in the laboratory.

Use nuclease-free labware (e.g., pipettes, pipette tips, reaction vials).

Wear gloves when performing the assay.

To avoid cross-contamination of samples and reagents, use fresh aerosol barrier
pipette tips.
To avoid carry-over contamination, transfer the required solutions for one experiment
into a fresh tube, rather than directly pipetting from stock solutions.

Physically separate the workplaces for DNA preparation, PCR setup, and PCR to
minimize the risk of carry-over contamination. Use a PCR hood for all pipetting steps.

Sample Material: Use any sample material suitable for PCR in terms of purity,
concentration, and absence of inhibitors.

DNA Extraction: We provide sample preparation kits suitable for all kind of food
samples and primary production stage samples.

Positive Control: Always run a positive control with the samples. Use the provided
control DNA (Control Template) or a positive sample preparation control.

Negative Control: Always run a negative control with the samples. To prepare
a negative control, replace the template DNA with PCR-grade water. Include a
negative control during sample preparation to monitor reaction purity and cross-
contamination. This extraction control can be used as an additional negative control
reaction.

Confirmation
: If required, positive results may be confirmed by appropriate methods
(e.g., reference method).

Waste Disposal:
All contaminated and potentially infectious material, like enrichment
cultures or food samples, should be autoclaved before disposal and eliminated
according to local rules and regulations. For more information, e.g., proper disposal
of unused chemicals, please refer to the appropriate safety data sheet (SDS).
For more information, please refer to the appropriate safety data sheet (SDS). The SDS is
available online at www.bc-diagnostics.com.
Keep the PCR mix away from light and moisture.

8
INSTRUCTIONS
KIT 2300 92 / 93 / 94 - Listeria monocytogenes Detection LyoKit
2.3 Enrichment and DNA extraction
The foodproof®Listeria monocytogenes Detection LyoKit is intended for the rapid
detection of Listeria monocytogenes isolated by foodproof®DNA extraction methods
from enrichment cultures of all relevant kind of foods and primary production stage (PPS)
samples that are potentially contaminated with Listeria monocytogenes. The kit must not
be used in diagnostic procedures.
2.3.1 Certified Methods
The performance of the foodproof®Listeria monocytogenes Detection LyoKit in
combination with several foodproof®DNA extraction procedures, including the
foodproof®StarPrep Two Kit (procedure A and B) and the foodproof®StarPrep Two
8-strip Kit (procedure A), has been approved in an AOAC RI Performance Tested
MethodsSM program (licence number 070401) and in a NordVal International method
extension (certificate No. 025). For the AOAC-RI PTM validation study the following
food categories were tested: raw meat, processed meat, seafood, milk and dairy,
and fruit/juices. The following categories were tested for the NordVal International
validation study: composite foods/ready-to-eat and ready-to-reheat, meat products,
milk and dairy products, vegetables, seafood and fishery products, and environmental
samples. For further information about the tested matrices, enrichment protocols and
the DNA extraction procedures please refer to ANNEX 1 and ANNEX 2 at the end of
the manual.

9
INSTRUCTIONS
KIT 2300 92 / 93 / 94 - Listeria monocytogenes Detection LyoKit
2.4.1 Workflow
2.4 Procedure
This protocol describes how to perform the analysis of DNA extracts by real-time PCR.
3. ADD SAMPLES AND CONTROLS
Pipette 25 µl of samples, negative control (colorless cap) or
Control Template (purple cap) into respective wells.
If using less volume, add PCR-grade H2O to reach 25 µl.
To reduce the risk of cross-contamination, prepare only one strip at a time.
2. DECAP
Open strips carefully direct before filling and discard caps.
Important: do not leave open longer than necessary.
1. PLACE STRIPS IN RACK
Take needed number of PCR tube strips out of aluminum bag.
Important: close bag tightly afterwards. Place strips in a suitable
PCR tube rack.
If needed, gently tap the tubes to move the lyophilized
pellets to the bottom of all tubes.
4. SEAL
Seal the tubes with the provided 8-cap strips tightly.
6. CENTRIFUGE
Briefly spin strips, e.g., 5 sec at 500 - 1,000 x g, in a suitable centrifuge.
5. MIX
Resuspend pellet after sealing by mixing thoroughly.
Alternatively, resuspend pellet by
pipetting up and down multiple times in step 3.
7. START REAL-TIME PCR RUN
Cycle samples as described in the program setup (2.4.2).
Place tubes in a vertical, balanced order into the cycler,
e.g., two strips can be placed in the first and last column.
+25 µl
PREPARATION OF THE PCR MIX
Take appropriate precautions to prevent contamination, e.g. by using filter tips and wearing gloves.

10
INSTRUCTIONS
KIT 2300 92 / 93 / 94 - Listeria monocytogenes Detection LyoKit
2.4.2 Program Setup
Program your real-time PCR instrument before setting up the PCR reactions. Select the
following channels:
uFAM (L. monocytogenes), and VIC (Internal Control).
As an alternative to VIC, HEX can be used. For the PikoReal®24, Yakima Yellow has to be
selected.
For some real-time PCR instruments the probe quencher as well as the usage of a passive
reference dye has to be specified. This kit contains probes with TAMRA as quencher and no
passive reference dye.
For users of the Agilent Mx3005P instrument: Click “Instrument” and “Filter Set Gain
Settings” to open the Filter Set Gain Settings dialog box in which the gain settings may be
viewed and modified. For FAM the Filter Set Gain Setting has to be modified to “x1”.
2.4.3 Data Interpretation
Verify results of positive (Control Template) and negative controls (H2O), before interpreting
sample results. Always compare samples to positive and negative control. Review data from
each channel and interpret results as described in the table.
Pre-incubation: 1 cycle
Step 1: 37 °C for 4 min
Step 2: 95 °C for 5 min
Amplification: 50 cycles
Step 1 : 95 °C for 5 sec
Step 2*: 60 °C for 60 sec
*Fluorescence detection
1 cycle
4 min 5 min 5 sec | 60 sec*
|
50 cycles
95 °C
60 °C
37 °C
FAM VIC Result Interpretation
+ + or - Positive for L. monocytogenes
- + Negative for L. monocytogenes
- - Invalid

11
INSTRUCTIONS
KIT 2300 92 / 93 / 94 - Listeria monocytogenes Detection LyoKit
2.5 Troubleshooting
Problem Possible Cause Recommendation
Squashed or
crooked tubes,
or open /
dislodged tube
lids after run, or
the cycler does
not open or
close properly.
Wrong tube format. Choose the correct tube format for
your cycler. Tube profile and instrument
compatibility chart is available online: www.
bc-diagnostics.com/compatibility-chart
If necessary, the samples can be
transferred to appropriate PCR vessels after
resuspension of the lyophilized PCR mix.
Wrong placement of tubes. Place tubes into the cycler in a vertical
and balanced order, as described in the
instructions for the PCR instrument.
No signal
increase is
observed, even
with positive
controls.
Incorrect detection channel has
been chosen.
Set channel settings for respective dyes
accordingly.
Pipetting errors. Check for correct reaction setup and repeat
the PCR run.
Always run a positive control along with
your samples.
No data acquisition programmed. Check the cycle programs.
A sample shows
no signals,
including the
internal control.
Positive and
negative control
have proper
signals.
Inhibitory effects of the sample
material (e.g., caused by
insufficient purification).
Use the recommended DNA extraction kit.
Dilute samples or pipette a lower amount of
sample DNA (e.g., 20 µl PCR-grade water
and 5 µl sample instead of 25 µl sample).
Negative control
samples are
positive.
Carry-over contamination. Exchange all critical solutions and reagents
for DNA/RNA extraction.
Repeat the complete experiment with fresh
batches of all reagents.
Always handle samples, kit components
and consumables in accordance with
commonly accepted practices to prevent
carry-over contamination.
Add positive controls after sample and
negative control reaction vessels have been
sealed.
Fluorescence
intensity is too
low.
Inappropriate storage of kit
components.
Store lyophilized PCR mix at 2 °C to 8 °C,
protected from light and moisture.
Low initial amount of target DNA. If possible, increase the amount of sample
DNA. Depending on the chosen DNA isolation
method, inhibitory effects may occur.
Strong decrease
of fluorescence
baseline.
Resuspension of lyophilized PCR
mix not complete.
Always resuspend lyophilized PCR mix
thoroughly.
Use the recommended vortex centrifuge
with the correct settings.
Fluorescence
intensity varies
or changes
abruptly during
the run.
Insufficient centrifugation of the
PCR strips, e.g., resuspended
PCR mix is still in the upper part of
the vessel or bubbles trapped in
the mix.
Always centrifuge PCR strips.
Use the centrifuge models and settings
recommended in this manual.
Avoid the introduction of air bubbles during
pipetting.
Outer surface of the vessel or the
seal is dirty (e.g., by direct skin
contact).
Always wear gloves when handling the
vessels and seal.
Do not mark vessels on the outside of the
tubes or directly on top of the reaction mix.
Pellets are
difficult to
dissolve.
The lyophilized PCR mix started to
rehydrate.
Store the lyophilized PCR mix always in the
aluminum bag with the silica gel pads. Make
sure that the lids are tightly closed.
Remove strips from the aluminum bag only
shortly before PCR setup.
Open strip shortly before filling.
Troubleshooting continues on the next page

12
INSTRUCTIONS
KIT 2300 92 / 93 / 94 - Listeria monocytogenes Detection LyoKit
2.6 Support
If you have questions or experience any problems with our products, please contact us:
www.hygiena.com/technical-support-request
Our aim is to provide you with a solution as quickly and effectively as possible. We would
also like you to contact us if you have any suggestions for improving the product or in case
you would like to use our product for a different application. We highly value your feedback.
Problem Possible Cause Recommendation
Squashed or
crooked tubes,
or open /
dislodged tube
lids after run, or
the cycler does
not open or
close properly.
Wrong tube format. Choose the correct tube format for
your cycler. Tube profile and instrument
compatibility chart is available online: www.
bc-diagnostics.com/compatibility-chart
If necessary, the samples can be
transferred to appropriate PCR vessels after
resuspension of the lyophilized PCR mix.
Wrong placement of tubes. Place tubes into the cycler in a vertical
and balanced order, as described in the
instructions for the PCR instrument.
No signal
increase is
observed, even
with positive
controls.
Incorrect detection channel has
been chosen.
Set channel settings for respective dyes
accordingly.
Pipetting errors. Check for correct reaction setup and repeat
the PCR run.
Always run a positive control along with
your samples.
No data acquisition programmed. Check the cycle programs.
A sample shows
no signals,
including the
internal control.
Positive and
negative control
have proper
signals.
Inhibitory effects of the sample
material (e.g., caused by
insufficient purification).
Use the recommended DNA extraction kit.
Dilute samples or pipette a lower amount of
sample DNA (e.g., 20 µl PCR-grade water
and 5 µl sample instead of 25 µl sample).
Negative control
samples are
positive.
Carry-over contamination. Exchange all critical solutions and reagents
for DNA/RNA extraction.
Repeat the complete experiment with fresh
batches of all reagents.
Always handle samples, kit components
and consumables in accordance with
commonly accepted practices to prevent
carry-over contamination.
Add positive controls after sample and
negative control reaction vessels have been
sealed.
Fluorescence
intensity is too
low.
Inappropriate storage of kit
components.
Store lyophilized PCR mix at 2 °C to 8 °C,
protected from light and moisture.
Low initial amount of target DNA. If possible, increase the amount of sample
DNA. Depending on the chosen DNA isolation
method, inhibitory effects may occur.
Strong decrease
of fluorescence
baseline.
Resuspension of lyophilized PCR
mix not complete.
Always resuspend lyophilized PCR mix
thoroughly.
Use the recommended vortex centrifuge
with the correct settings.
Fluorescence
intensity varies
or changes
abruptly during
the run.
Insufficient centrifugation of the
PCR strips, e.g., resuspended
PCR mix is still in the upper part of
the vessel or bubbles trapped in
the mix.
Always centrifuge PCR strips.
Use the centrifuge models and settings
recommended in this manual.
Avoid the introduction of air bubbles during
pipetting.
Outer surface of the vessel or the
seal is dirty (e.g., by direct skin
contact).
Always wear gloves when handling the
vessels and seal.
Do not mark vessels on the outside of the
tubes or directly on top of the reaction mix.
Pellets are
difficult to
dissolve.
The lyophilized PCR mix started to
rehydrate.
Store the lyophilized PCR mix always in the
aluminum bag with the silica gel pads. Make
sure that the lids are tightly closed.
Remove strips from the aluminum bag only
shortly before PCR setup.
Open strip shortly before filling.
Problem Possible Cause Recommendation
Squashed or
crooked tubes,
or open /
dislodged tube
lids after run, or
the cycler does
not open or
close properly.
Wrong tube format. Choose the correct tube format for
your cycler. Tube profile and instrument
compatibility chart is available online: www.
bc-diagnostics.com/compatibility-chart
If necessary, the samples can be
transferred to appropriate PCR vessels after
resuspension of the lyophilized PCR mix.
Wrong placement of tubes. Place tubes into the cycler in a vertical
and balanced order, as described in the
instructions for the PCR instrument.
No signal
increase is
observed, even
with positive
controls.
Incorrect detection channel has
been chosen.
Set channel settings for respective dyes
accordingly.
Pipetting errors. Check for correct reaction setup and repeat
the PCR run.
Always run a positive control along with
your samples.
No data acquisition programmed. Check the cycle programs.
A sample shows
no signals,
including the
internal control.
Positive and
negative control
have proper
signals.
Inhibitory effects of the sample
material (e.g., caused by
insufficient purification).
Use the recommended DNA extraction kit.
Dilute samples or pipette a lower amount of
sample DNA (e.g., 20 µl PCR-grade water
and 5 µl sample instead of 25 µl sample).
Negative control
samples are
positive.
Carry-over contamination. Exchange all critical solutions and reagents
for DNA/RNA extraction.
Repeat the complete experiment with fresh
batches of all reagents.
Always handle samples, kit components
and consumables in accordance with
commonly accepted practices to prevent
carry-over contamination.
Add positive controls after sample and
negative control reaction vessels have been
sealed.
Fluorescence
intensity is too
low.
Inappropriate storage of kit
components.
Store lyophilized PCR mix at 2 °C to 8 °C,
protected from light and moisture.
Low initial amount of target DNA. If possible, increase the amount of sample
DNA. Depending on the chosen DNA isolation
method, inhibitory effects may occur.
Strong decrease
of fluorescence
baseline.
Resuspension of lyophilized PCR
mix not complete.
Always resuspend lyophilized PCR mix
thoroughly.
Use the recommended vortex centrifuge
with the correct settings.
Fluorescence
intensity varies
or changes
abruptly during
the run.
Insufficient centrifugation of the
PCR strips, e.g., resuspended
PCR mix is still in the upper part of
the vessel or bubbles trapped in
the mix.
Always centrifuge PCR strips.
Use the centrifuge models and settings
recommended in this manual.
Avoid the introduction of air bubbles during
pipetting.
Outer surface of the vessel or the
seal is dirty (e.g., by direct skin
contact).
Always wear gloves when handling the
vessels and seal.
Do not mark vessels on the outside of the
tubes or directly on top of the reaction mix.
Pellets are
difficult to
dissolve.
The lyophilized PCR mix started to
rehydrate.
Store the lyophilized PCR mix always in the
aluminum bag with the silica gel pads. Make
sure that the lids are tightly closed.
Remove strips from the aluminum bag only
shortly before PCR setup.
Open strip shortly before filling.

13
ADDITIONAL INFORMATION
KIT 2300 92 / 93 / 94 - Listeria monocytogenes Detection LyoKit
3.1 Testing Principle
3. ADDITIONAL INFORMATION
The foodproof®kit provides all necessary reagents and a control template for reliable
interpretations of results. To ensure maximum reliability of the kit and to prevent
misinterpretation of negative results due to inhibition of the amplification, an Internal
Control (IC) is included. A hydrolysis probe was designed to bind specifically the IC,
allowing detection in the respective channel, whereas the target DNA is detected in another
channel. In case of a negative result due to inhibition of the amplification by the sample
DNA of interest, the amplification of the IC is suppressed as well, whereas a negative result
for the sample DNA of interest and amplification of the IC clearly indicates the absence of
parameter in the sample. The real-time PCR kit minimizes contamination risk and contains
all reagents (except for template DNA) needed for the detection of target DNA. Primers
and probes provide specific detection of target DNA in food and environmental samples,
including primary production stage samples. The described performance of the kit is
guaranteed for use only on the real-time PCR instruments listed above.
Step-by-Step Procedure
1.
Using the kit‘s sequence-specific primers in a polymerase chain reaction (PCR), the
PCR instrument and the supplied reagents amplify fragments of specific sequences for
target DNA.
2.
The PCR instrument detects these amplified fragments in real time through
fluorescence generated by cleavage of the hybridized probe due to the 5´-nuclease
activity of the Taq DNA polymerase. The probe is labeled at the 5´-end with a reporter
fluorophore and at the 3´-end with a quencher.
3.
During the annealing/elongation phase of each PCR cycle, the probe hybridizes to
an internal sequence of the amplicon and is cleaved by the 5’ nuclease activity of the
Taq DNA polymerase. This cleavage of the probe separates the reporter dye from the
quencher dye, increasing the reporter dye signal.
4.
The PCR instrument measures the emitted fluorescence of the reporter dye.
Prevention of Carry-Over Contamination
The heat-labile Uracil-DNA N-Glycosylase (UNG) is suitable for preventing carry-over
contamination between PCR’s. This technique relies on the incorporation of deoxyuridine
triphosphate (dUTP) during all amplification reactions, and the pretreatment of all successive
PCR mixtures with the heat-labile UNG. The UNG cleaves DNA at any site where a
deoxyuridine residue has been incorporated. The resulting abasic sites are hydrolyzed
due to the high temperatures during the initial denaturation step, and can no longer serve
as PCR templates. The heat-labile UNG is inactivated during the initial denaturation step.
Native DNA (e.g., the isolated target genomic DNA) does not contain uracil and is therefore
not degraded by this procedure. Since dTTP is replaced with dUTP and UNG is included in
this kit, decontamination can be achieved with the provided reagents.

14
ADDITIONAL INFORMATION
KIT 2300 92 / 93 / 94 - Listeria monocytogenes Detection LyoKit
3.3 Reference Number
The reference number and original BIOTECON Diagnostics article numbers:
R 602 23 -1, R 602 23 -2, and R 602 23 -3.
3.4 Change Index
Version 4, February 2022:
Rebranding, new document layout and content, new order number.
Introduction of NordVal International logo.
Introduction of ANNEX 2: NordVal #025 Extension Table and additional information in the
Applicability Statement.
R 602 23 20 (5)
3.2 Trademarks
foodproof®, microproof®,vetproof®, ShortPrep®, RoboPrep®, and LyoKit®are trademarks
of BIOTECON Diagnostics GmbH.
Hygiena
TM is a registered trademark of Hygiena.
Other brand or product names are trademarks of their respective holders.

15
ANNEX
KIT 2300 92 / 93 / 94 - Listeria monocytogenes Detection LyoKit
ANNEX 1
AOAC-RI #070401 Extension Table for the foodproof®
Listeria monocytogenes Detection LyoKit
The following table shows the recommended enrichment time for different food matrices
with enrichment in Half Fraser broth in combination with different foodproof®DNA extraction
procedures that have been validated for the AOAC-RI PTM extension of the foodproof®
Listeria monocytogenes Detection LyoKit.
For further information regarding the DNA extraction procedures below, please refer to the
appropriate BIOTECON Diagnostics package inserts on: www.bc-diagnostics.com.
DNA Extraction Enrichment time in
Half Fraser broth at 30°C
Enrichment time per
matrix
foodproof®ShortPrep II Kit 24 h – 48 h
minced meat 48 h
melon 24 h
raw fish 48 h
sausage 48 h
Harzer cheese 48 h
foodproof®StarPrep Two
Kit Extraction Procedure A 24 h
minced meat 24 h
melon 24 h
raw fish 24 h
sausage 24 h
Harzer cheese 24 h
foodproof®StarPrep Two
Kit Extraction Procedure B 24 h – 48 h
minced meat 48 h
melon 24 h
raw fish 48 h
sausage 48 h
Harzer cheese 48 h
foodproof®StarPrep Two
8-strip Kit 48 h
minced meat 48 h
melon 48 h
raw fish 48 h
sausage 48 h
Harzer cheese 48 h
foodproof®Magnetic Pre-
paration Kit II 24 h – 48 h
minced meat 48h
melon 24 h
raw fish 24 h
sausage 48 h
Harzer cheese 48 h
Tested food categories: raw meats, processed meats, seafood, milk and dairy, fruit/ juices
Sample size: 25 g / 225 ml broth
Reference method: ISO 11290:1996/Amd 1:2004

16
ANNEX
KIT 2300 92 / 93 / 94 - Listeria monocytogenes Detection LyoKit
ANNEX 2
NordVal #025 Extension Table for the foodproof®
Listeria monocytogenes Detection LyoKit
The following table shows the recommended enrichment time for the tested food categories
and environmental samples in Actero Listeria Enrichment Media (ALEM) and Half Fraser
broth in combination with different foodproof®DNA extraction procedures that have
been validated for the NordVal International method extension of the foodproof®Listeria
monocytogenes Detection LyoKit (certificate No. 025).
For further information regarding the DNA extraction procedures below, please refer to the
appropriate BIOTECON Diagnostics package inserts on: www.bc-diagnostics.com.
DNA Extraction Enrichment time in
ALEM at 36°C
Enrichment time in
Half Fraser broth
at 30°C
DNA extract for
PCR
foodproof®
StarPrep Two Kit
Extraction
Procedure A
STANDARD
22 h ± 2 h 25 h ± 1 h 5 µl + 20 µl PCR-
grade water
foodproof®
StarPrep Two Kit
Extraction
Procedure B RAPID
--- 48 h ± 2 h 5 µl + 20 µl PCR-
grade water
foodproof®
StarPrep Two
8-strip Kit
Extraction
Procedure A
STANDARD
22 h ± 2 h 25 h ± 1 h 5 µl + 20 µl PCR-
grade water
Tested categories:
xcomposite foods/ready-to-eat and ready-to-reheat
xmeat products
xmilk and dairy products
xvegetables
xseafood and fishery products
xenvironmental samples

17
KIT 2300 92 / 93 / 94 - Listeria monocytogenes Detection LyoKit
Sample size:
x25 g + 150 ml Actero Listeria Enrichment Media or 225 ml Half Fraser broth
x1 swab + 10 ml Actero Listeria Enrichment Media or 10 ml Half Fraser broth
x1 sponge + 100 ml Actero Listeria Enrichment Media or 100 ml Half Fraser broth
x1 wipe + 225 ml Actero Listeria Enrichment Media or 225 ml Half Fraser broth
xFor sampling after cleaning process, premoisten:
- 1 swab + 1 ml broth universal neutralizing (+ 9 ml ALEM or Half Fraser)
- 1 sponge + 10 ml broth universal neutralizing (+ 90 ml ALEM or Half Fraser)
- 1 wipe + BPW + 10 % neutralizing agent (+ 225 ml ALEM or Half Fraser)
Reference method: ISO 11290-1/A1 (May 2017)

Manufactured by
BIOTECON
Diagnostics GmbH
Hermannswerder 17
14473 Potsdam
Germany
[email protected]
www.bc-diagnostics.com
Hygiena LLC
CA 93012 Camarillo
USA
www.hygiena.com/technical-support-request