BACGene Listeria monocytogenes

TEST KIT FOR QUALITATIVE REAL-TIME PCR DETECTION OF

LISTERIA MONOCYTOGENES

Cat. No. 5123222001, 5123222011 and 5123222010

For 96 reactions, 10x 96 reactions and 10x 96 HTP reactions

EGS 38/03-01/17
ALTERNATIVE ANALYTICAL
METHODS FOR AGRIBUSINESS
www.afnor-validation.com

BACGene

ID 2622 10 Mar 2022 V3.7

 BACGene Listeria monocytogenes
Cat. No. 5123222001, 5123222011 and
5123222010

Table of Contents

1 INTRODUCTION ........................................................................................................................... 3

1.1 Certifications .................................................................................................................................. 3

1.1.1 AFNOR Certification ...................................................................................................................... 3
1.1.2 AOAC Performance Testing .......................................................................................................... 4
1.2 Test Principle ................................................................................................................................. 5
1.3 Components of the Kit ................................................................................................................... 6
1.3.1 For Lysis ........................................................................................................................................ 6
1.3.2 For PCR ........................................................................................................................................ 7
1.4 Additional Equipment, Consumables and Reagents Required ..................................................... 8
1.4.1 Sampling / Enrichment .................................................................................................................. 8
1.4.2 Lysis .............................................................................................................................................. 9
1.4.3 PCR Setup .................................................................................................................................. 10
1.4.4 Optional Equipment ..................................................................................................................... 10

2 How to use this Product .............................................................................................................. 11

2.1 Safety Precautions ...................................................................................................................... 11

2.2 Working Guidelines ..................................................................................................................... 11
2.2.1 Cleaning Protocol ........................................................................................................................ 12
2.3 Waste Disposal ........................................................................................................................... 12
2.4 Enrichment .................................................................................................................................. 13
2.5 Before You Begin ........................................................................................................................ 15
2.5.1 Sample Pre-Treatment with PREraser BACGene ...................................................................... 15
2.6 Sample Lysis ............................................................................................................................... 16
2.7 PCR ............................................................................................................................................. 17
2.7.1 Special Precautions During PCR Analysis .................................................................................. 17
2.7.2 General Information..................................................................................................................... 18
2.7.3 Control Reactions ........................................................................................................................ 18
2.7.4 PCR ............................................................................................................................................. 19

3 Agilent Aria™: Instructions for use with BACGene Listeria monocytogenes kit ......................... 22

3.1 Running a PCR ........................................................................................................................... 22

3.1.1 Running PCR Using a Template ................................................................................................. 22
3.2 Data Evaluation ........................................................................................................................... 24

4 Bio-Rad CFX96 Touch™ Instructions for use with BACGene Listeria monocytogenes Kit ........ 26

4.1 Running a PCR ........................................................................................................................... 26

4.1.1 Before You Begin ........................................................................................................................ 27
4.1.2 Running a PCR Using a Plate Layout Template ......................................................................... 28

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4.2 Data Evaluation ........................................................................................................................... 31

5 Data Interpretation....................................................................................................................... 32

6 Confirmation of Presumptive Positive Results ............................................................................ 32

6.1 Confirmation of Listeria monocytogenes Positive Results .......................................................... 32

7 TROUBLESHOOTING ................................................................................................................ 34

7.1 Problems Upon Delivery / During Storage and Reception .......................................................... 34

7.2 Problems Associated with Handling of the Plates ....................................................................... 35
7.3 Incorrect Data Export and Import to BACGene Evaluation Sheet .............................................. 36
7.4 Incorrect Run File Upload Using FastFinder Software ................................................................ 37
7.5 Inconclusive Results Post Evaluation ......................................................................................... 37
7.6 Incorrect Automated Threshold Setting in Cycler Software ........................................................ 41
7.7 Cultural Confirmation................................................................................................................... 43

8 PRODUCT WARRANTIES, SATISFACTION GUARANTEE...................................................... 44

9 PRODUCT USE LIMITATIONS .................................................................................................. 44

10 IMPORTANT NOTES .................................................................................................................. 44

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 BACGene Listeria monocytogenes
Cat. No. 5123222001, 5123222011 and
5123222010

1 INTRODUCTION

The BACGene Listeria monocytogenes detection kit provides materials for rapid detection of Listeria
monocytogenes DNA from all human food products and environmental samples from food production
facilities.
The kit is intended to be used in analytical laboratories and may also be applied for other purposes in
food product research and e.g. microbial monitoring of production processes. The kit is not intended for
diagnostic use with medical specimens.
The BACGene Listeria monocytogenes kit is validated for use with the Agilent AriaMx™ and Bio-Rad
CFX96 Touch™ Deep Well PCR platforms.

1.1 Certifications

1.1.1 AFNOR Certification

The BACGene Listeria monocytogenes kit is certified by AFNOR

Certification as an alternative method for Listeria monocytogenes
detection in all human food products and environmental samples
according to EN ISO 16140-2.
The end of the validity of the NF VALIDATION certification is
indicated on the certificate
EGS 38/03-01/17. EGS 38/03-01/17
ALTERNATIVE ANALYTICAL
METHODS FOR AGRIBUSINESS
www.afnor-validation.com

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 BACGene Listeria monocytogenes
Cat. No. 5123222001, 5123222011 and
5123222010

1.1.2 AOAC Performance Testing

The BACGene Listeria monocytogenes kit is certified by AOAC-Research Institute under the
Performance Tested MethodsSM Program for detection of Listeria monocytogenes for:

 25 g / 25 mL samples of
- Raw milk
- Ice cream with chocolate inclusions (with PREraser)
- Smoked salmon (with PREraser)
- Mayonnaise-based vegetable salad
- Frozen cantaloupe balls
- Frankfurters
- Process water
 Stainless steel environmental surface
 Ceramic environmental surface
 Plastic environmental surfaces with sponge (with PREraser)

The end of the validity of the AOAC certification is indicated on the certificate 061703.


PREraser BACGene (see sections 1.3 and 2.5.1) has been performance tested for three relevant
matrices. Therefore, provided that additional matrices are verified, PREraser BACGene can be applied
to other matrices within the scope of the method.

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 BACGene Listeria monocytogenes
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1.2 Test Principle

DNA amplification and detection methods take advantage of the nucleotide sequence conservation
found in bacterial genomes that ensures the potential for high specificity and sensitivity in detection of
food-borne, pathogenic bacteria.
After enrichment, the microbial DNA is extracted by a simple thermal / enzymatic lysis step and rapidly
analysed by real-time PCR. In this way Listeria monocytogenes is detected from enrichment cultures
with extraordinary high sensitivity.
By means of specific primers (P) a nucleotide sequence of the species Listeria monocytogenes is
amplified during PCR. Primers do not react with DNA derived from closely related species from the
Bacillales order.
The amplified fragments for Listeria monocytogenes are detected with a R6G fluorescence-labelled
hybridisation probe quenched by non-fluorescent Tide Quencher™ 2 (TQ2).
An internal positive control (IPC) is included in the MasterMix. IPC DNA is amplified in parallel and
detected using a Cy5™ fluorescence-labelled hybridisation probe quenched by non-fluorescent Tide
Quencher™ 3 (TQ3). IPC detection indicates the proper functioning of the PCR.

Listeria monocytogenes Target IPC Target

Probe
Probe

P1 R6G™ TQ2 P2 P3 Cy5™ TQ3 P4

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1.3 Components of the Kit

Important Notes
 Store all reagents as indicated below, through to the expiration date printed on the label.
Never store components of the kit together with samples or post-PCR products. Shelf life is
indicated on the labels of the components.
 Do not mix components from different kit lots.

1.3.1 For Lysis

Cat. no. 5123222001 (96 reactions)

 1x Lysis plate for sample preparation, empty, rippable (high profile)
 1x Domed caps*, for use with lysis plate, set of 12 strips
 2x Lysis Buffer L, 2.5 mL, -20 °C ± 2°C or 4°C ± 2°C
 2x Proteinase K, vials with blue cap, 500 µL, store at -20°C ± 2°C
 2x Lysozyme, vials with orange cap, 500 µL, store at -20°C ± 2°C

Cat. no. 5123222011 (10x HTP)

 10x Lysis plate for sample preparation, empty, rippable (high profile)
 10x Domed caps*, for use with lysis plate, set of 12 strips
 2x Lysis Buffer L, 25 mL, -20 °C ± 2°C or 4°C ± 2°C
 2x Proteinase K, vials with blue cap, 5 mL, store at -20°C ± 2°C
 2x Lysozyme, vials with orange cap, 5 mL, store at -20°C ± 2°C

Cat. no. 5123222010 (10x 96 reactions)

 10x Lysis plate for sample preparation, empty, rippable (high profile)
 10x Domed caps*, for use with lysis plate, set of 12 strips
 20x Lysis Buffer L, 2.5 mL, -20 °C ± 2°C or 4°C ± 2°C
 20x Proteinase K, vials with blue cap, 500 µL, store at -20°C ± 2°C
 20x Lysozyme, vials with orange cap, 500 µL, store at -20°C ± 2°C

*Note: additional domed caps for lysis can be purchased in a package of 250x 8 cap strips (cat. no.
5613900301).

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Cat. No. 5123222001, 5123222011 and
5123222010

1.3.2 For PCR

Cat. no. 5123222001 (96 reactions)

 1x BACGene Listeria monocytogenes PCR plate, with pre-dispensed MasterMix and
PCR support grid. Store light protected at -20°C ± 2°C.
 1x Optical caps, for use with PCR plate (1 bag with 120 strips)
 2x Listeria positive control plasmid DNA, vial with yellow cap, 50 µL, store
at -20°C ± 2°C.
Note: Do not freeze/thaw more than 6 times.

Cat. no. 5123222011 (10x HTP)

 10x BACGene Listeria monocytogenes PCR plate, with pre-dispensed MasterMix and
PCR support grid. Store light protected at -20°C ± 2°C.
 10x Optical caps, for use with PCR plate (1 bag with 120 strips)
 20x Listeria positive control plasmid DNA, vial with yellow cap, 50 µL, store
at -20°C ± 2°C.
Note: Do not freeze/thaw more than 6 times.

Cat. no. 5123222010 (10x 96 reactions)

 10x BACGene Listeria monocytogenes PCR plate, with pre-dispensed MasterMix and
PCR support grid. Store light protected at -20°C ± 2°C.
 10x Optical caps, for use with PCR plate (1 bag with 120 strips)
 4x Listeria positive control plasmid DNA, vial with yellow cap, 50 µL, store
at -20°C ± 2°C.
Note: Do not freeze/thaw more than 6 times.

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 BACGene Listeria monocytogenes
Cat. No. 5123222001, 5123222011 and
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1.4 Additional Equipment, Consumables and Reagents Required

1.4.1 Sampling / Enrichment

 Neutralizing buffer, e.g.

- Dey-Engley
- Letheen broth
- HiCap broth
 Swabs / sponges / wipes / cloths (for environmental samples)*
- Eurofins Technologies EZ Reach™ Sponge Sampler, Polyurethane, 10 mL HiCap
Neutralizing Broth, 24 oz bag, 100 pcs, cat. no. EUR EZ-10HC-PUR
 Stomacher® or paddle blender
 Enrichment bags with side filter
 Actero™ Listeria Enrichment Media (by FoodChek™)
 PREraser BACGene, Eurofins GeneScan Technologies, cat. no. 5123222301 or 5123222311
- For the sample pre-treatment after enrichment and prior to lysis.

*Note: Products pre-moistened with neutralising buffers containing aryl sulfonate (sodium
dodecylbenzenesulfonate) should not be used in conjunction with this kit. The use of Dey-Engley
neutralizing buffer, Letheen broth or HiCap broth is recommended.

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1.4.2 Lysis

Equipment
 2x Block heater (e.g. block heater, digital, VWR cat. no. 460-0350).
 2x Insert for block heater, 0.2 mL, 96 wells (e.g. double block, 1×96-well PCR plate,
VWR cat. no. 460-3226).
 1x 96 well cooling block (e.g. PCR-Cooler, VWR cat. no. 479-1162).
 1x Lysis support grid, blue, (e.g. cat. no. 5617702209, pack of 8 grids).
 1x Lysis rack (e.g. cat. no. 5613901401).
 1x Strip centrifuge:
Capacity of 2x 8-well strips: (e.g. Carl Roth GmbH, Rotilabo® centrifuge with butterfly
rotor cat. no. T465.1).
Capacity of 4x 8-well strips: (e.g. IKA, Mini Centrifuge Mini G cat. no. 0003958000
or VWR, MiniStar silverline cat. no. 521-2844P).
OR
 1x Plate centrifuge:
Capacity of two times 12x 8-well strips: (e.g. Benchmark Scientific, PlateFuge™
microplate microcentrifuge cat. no. C2000).
 1x Capping/Uncapping tool for 8-well strips (e.g. Thermo Fisher Scientific,
MicroAmp® Cap Installing Tool, cat. no. 4330015).
 1x Stepper pipette (with 1 mL tip), (e.g. HandyStep® S Brand®, cat. no. 705110)
 1x Single channel pipette, (e.g. Brand®, Transferpette® S 100 - 1000 µL,
cat. no. 705880).
 1x Single channel pipette, (e.g. Brand®, Transferpette® S 10 - 100 µL,
cat. no. 705874)
 1x Single channel pipette, (e.g. Brand®, Transferpette® S, 0.5 - 10 µL,
cat. no. 705870)
 1x Pipette filler, (e.g. VWR, Pipetboy acu 2 for serological pipettes, cat. no. 612-0926)

Consumables and material not contained in the kit

 Pipette tips, DNA-/Nuclease-free pipette tips
 Stepper tips, 1 mL
 Sterile container for enrichment storage
 Serological pipettes
 Gloves, powder-free

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Cat. No. 5123222001, 5123222011 and
5123222010

1.4.3 PCR Setup

Equipment
 1x PCR support grid, yellow (e.g. cat. no. 5617702208, pack of 8 grids).
Note: 1x PCR support grid is provided with each kit.
 1x PCR rack (e.g. cat. no. 5613901301).
 1x Capping/Uncapping tool for 8-well strips (e.g. Thermo Fisher Scientific,
MicroAmp® Cap Installing Tool, cat. no. 4330015).
 1x Single channel pipette, (e.g. Brand®, Transferpette® S, 0.5 - 10 µL,
cat. no. 705870).
AND/OR
 1x Multichannel pipette, (e.g. Brand®, Transferpette® S-8 Kanal / 0.5 – 10 µL,
cat. no. 705900)
 1x Disposable transfer rack (e.g. an empty tip box) as mount for the yellow support grid
and the PCR plate
 1x Real-time PCR Thermocycler:
- Agilent AriaMx™ (cat no.: G8830A) (up to Software v.1.5), with FAM™ and Cy5™
filter set.
- Bio-Rad CFX96 Touch™ (CFX Manager™ Software v. 3.1 / CFX MaestroTM Software
1.1).
- Bio-Rad CFX96 Touch™ Deep Well (CFX Manager™ Software v. 3.1 / CFX
MaestroTM Software 1.1).
 Windows 7/10 PC with Microsoft Excel 2010 32 bit (Excel Version 14.7140.xxxx or later).
 BACGene Evaluation Sheet version 2
OR
FastFinder automated PCR analysis software (UgenTec NV, Hasselt, Belgium) with
BACGene Listeria monocytogenes plugins with version 1

Consumables and material not contained in the kit

 BACGene PCR Negative Control, Eurofins GeneScan Technologies, cat. no. 5617702500
(optional)
 Roti® Nucleic Acid-free (C. Roth GmbH, cat. no. HP69) or 1% HCl (for DNA
decontamination)
 Pipette tips, DNA-/Nuclease-free pipette tips
 Nuclease free water, molecular biology grade
 Gloves, powder-free

1.4.4 Optional Equipment

 MALDI biotyper of Bruker

(software version: Flex Control 3.4, Flex Analysis 3.4 MBT Compass explorer 4.1)

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2 HOW TO USE THIS PRODUCT

2.1 Safety Precautions

 All samples should be handled with caution as they are potentially infectious.
 In order to safeguard the health of laboratory personnel, it is essential that the whole of this
method be carried out only by skilled personnel using good laboratory practices.
 A BSL-2 laminar flow biosafety cabinet is recommended for activities with potential for
producing aerosols of pathogens.
 Relevant national Health and Safety Regulations relating to this organism must be adhered to.
 Care must be taken in the disposal of all infectious materials.
 Do not eat, drink or apply cosmetics in the work area in which the test is performed.
 Do not pipette by mouth.
 Avoid contact of kit components with injured skin.
 Listeria monocytogenes should not be handled by pregnant women, children, the elderly and
immuno-compromised individuals due to the high infection risk and fatal health consequences
for this group, in particular for the unborn child in case of pregnant women.
 The BACGene Listeria monocytogenes kit contains enzymes (proteinase K and lysozyme)
which may cause allergic reactions.
 Lysis buffer L can cause serious eye irritation.
 Always wear appropriate Personal Protective Equipment (PPE).
 The remaining components of the BACGene Listeria monocytogenes kit are not classified
according to Regulation (EC) No. 1907/2006 (REACH). For more information, please refer to
the kit’s safety data sheet (SDS).

2.2 Working Guidelines

 Comply with Good Laboratory Practice (refer to EN ISO 7218 standard).

 Refer to EN ISO 22174:2005 for the general requirements for the in vitro amplification of
nucleic acid sequences.
 Refer to associated guidelines for lab equipment maintenance e.g. ISO/DIS20836 for thermal
performance testing of thermal cyclers.
 Perform cleaning protocol (outlined in next section).
 Clearly separate the area for taking samples from the enrichment, from the area where lysis
and particularly PCR setup are performed.
 Use different equipment (e.g. pipettes) for sampling the enrichment, performing lysis and PCR
 Use DNA, nuclease-free and sterile lab ware.
 Wear gloves.
 Change gloves after removing samples from the enrichment, before starting the lysis protocol.

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2.2.1 Cleaning Protocol

For use of this kit, two different types of contamination can impair results: Bacterial contamination is
prevented by regular disinfection, DNA contamination is prevented by regular decontamination.
Before commencing work and after completion of work, ensure workspaces to be clean:

Workspace Cleaning protocol

Enrichment preparation area Disinfect surfaces with 80% ethanol.
Lysis area Disinfect surfaces with 80% ethanol. Decontaminate surface with
Roti® Nucleic Acid-free or 1% HCl.
Pipettes used during lysis should also be decontaminated with
Roti® Nucleic Acid-free.
PCR area Decontaminate surfaces with
Roti® Nucleic Acid-free or 1% HCl.

2.3 Waste Disposal

Dispose of any waste which is potentially contaminated with pathogenic bacteria according to your
internal and local regulations.

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2.4 Enrichment

Enrichment samples shall be prepared in pre-warmed (37 ± 1 °C) Actero™ Listeria Enrichment Media.
Please ensure that the prepared medium is stored at 2° – 8 °C under light protection.
For preparation of initial suspensions follow instructions of EN ISO 11290 and EN ISO 6887standards.
For sampling of environmental samples follow instructions EN ISO 18593 standard.
It is strongly recommended to prepare samples in stomacher bags containing a side filter. It is also
strongly recommended to prepare enrichment controls (positive and negative) in parallel with the
samples. Growth in the negative enrichment control indicates contamination of the medium used and
therefore enrichment should be repeated.
After the enrichment protocol, samples can be stored at 2-8 °C for up to 72 h before testing.

Certified matrices by AOAC (License No: 061703)

Enrichment Volume for Analysis
Matrix
Medium Temp Time Enrichment Lysate

Raw milk,
Smoked salmon (incl.
PREraser),
Mayonnaise-based
vegetable salad,
Frozen cantaloupe 1:10
balls, Actero™ Listeria
Frankfurters, Enrichment Media
Ice-cream with (Pre-warmed to 37 °C
chocolate inclusions ± 1 °C)
(incl. PREraser)
25 g

Process water
25 mL
37 ± 1 °C 18 - 24 h 30 µL 5 µL
10 mL
Stainless steel
environmental surface Actero™ Listeria
1” x 1” area Enrichment Media
Tool: swab (Pre-warmed to 37 °C
± 1 °C)
Ceramic environmental
surface
4” x 4” area 100 mL
Tool: sponge Actero™ Listeria
Plastic environmental Enrichment Media
surface (incl. (Pre-warmed to 37 °C
PREraser) ± 1 °C)
4” x 4” area
Tool: sponge

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Certified categories by AFNOR (EGS 38/03-01/17)

Enrichment Volume for Analysis
Sample Category
Medium Temp Time Enrichment Lysate

All human food 1:10

products
Actero™ Listeria
25 g Enrichment Media
Dusts, process water (Pre-warmed to 37°C ±
25 g / mL 1°C)

10 mL
Swab Actero™ Listeria
Enrichment Media
1
(Pre-warmed to 37°C ±
1°C) 37 ± 1°C 18 - 24 h 30 µL 5 µL
225 mL
Wipe, cloth Actero™ Listeria
Enrichment Media
1
(Pre-warmed to 37°C ±
1°C)
100 mL
Sponge Actero™ Listeria
Enrichment Media
1
(Pre-warmed to 37°C ±
1°C)

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2.5 Before You Begin

1. Place cooling block for lysis at 4°C ± 2°C overnight

2. Turn on heating block to 37°C ± 2°C and 95°C ± 2°C
3. Thaw proteinase K and lysozyme just before adding it to Lysis buffer L
4. Prepare final lysis buffer
Cat no. 5123222001:
Add 500 µL proteinase K (vial with blue cap) and 500 µL lysozyme (vial with orange cap) to
2.5 mL Lysis buffer L (transparent container).

Cat no. 5123222010 (10x):

Add 5 mL proteinase K (small transparent container) and 5 mL lysozyme to 25 mL Lysis buffer L.
(transparent container).
5. Mix gently by inverting 5 times.
6. Dispense the final lysis buffer by placing the lysis plate into the lysis rack and aliquoting 70 µL of
the final lysis buffer into each well.
7. Seal wells with domed caps.
8. Mark date of preparation and name on the label provided with kit and place onto the storage rack
for lysis buffer plate/strips.
9. The final lysis buffer can be stored for 2 weeks at 4°C ± 2°C.

Note: The volume of Lysis buffer L of the kits with cat nos. 5123222001 and 5123222011 (3.5 mL) is
sufficient for 48 reactions. Lysis buffer L volume of the kit with cat no. 5123222010 (35 mL) is sufficient
for 480 reactions or five 96 well plates.

2.5.1 Sample Pre-Treatment with PREraser BACGene

An optional elimination of free DNA from enrichments prior to the lysis step can be incorporated into the
workflow and has been AOAC-RI certified with the use of the kit.
PREraser BACGene can be applied for each sample in case of sample types with expected high levels
of free DNA (e.g. environmental samples) or after a first PCR positive prior to a second BACGene PCR
to exclude the presence of free DNA.
Please refer to the PREraser BACGene user’s manual to include PREraser BACGene to the workflow.
PREraser BACGene is not part of the AFNOR certified workflow for Listeria monocytogenes.
See section 1.4.1 for ordering information.

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2.6 Sample Lysis

1. Label lysis buffer plate/strips to ensure correct orientation when pipetting samples.
2. Using a serological pipette, remove an aliquot of enrichment culture and dispense into a sterile
container. This step is highly recommended to avoid cross-contamination, as opposed to inserting
a micropipette directly into the enrichment bag.
3. Use an Un-/Capping Tool (with blue lysis label, dedicated to lysis area) to open the first lysis strip.
Note: Open each strip individually, add samples and close. Only then proceed to the next strip. The
correct method to use this tool is to place the “teeth” of the tool under the connection between the
caps and lever the caps open.
4. Transfer 30 µL of enrichment into appropriate wells.
Note: Do not add enrichment samples to wells A1 and B1 to ensure the same layout in the following
PCR (A1: positive control, B1: negative control).
5. Seal the strip using the Un-/Capping Tool.
6. Proceed to the next lysis strip until all samples have been included.
7. Place lysis plate/strips with blue frame onto the 37°C (± 2°C) heating block for 20 min.
8. Transfer lysis plate/strips with blue frame onto the 95°C (± 2°C) heating block for 10 min.
9. Finally transfer lysis plate/strips with blue frame onto the blue 96 well cooling block (stored at 4°C ±
2°C) for 5 min.
10. Ensure that no condensation remains in the lids by transferring the lysis plate/strips with the blue
frame to a 96-well plate centrifuge or a centrifuge with adapter for 0.2 mL in 8-strip format. Spin
down samples for 30 sec within the range of 400 x g to 2000 x g. If condensate remains in the lids,
please repeat this step.
11. If particles are visible in the lysis vessel, centrifuge the plate/strip following the procedure in the step
above.
12. Transfer lysis plate and blue frame into the working rack with transparent inlay.

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2.7 PCR

2.7.1 Special Precautions During PCR Analysis

PCR is an exponential reaction. The detection of single DNA targets is possible. The extreme sensitivity
requires special precautions for handling and equipment. After a successful amplification, several billion
amplicons are present in the reaction tube. Each of them might lead to a false positive result when
contaminating sample material, i.e. by spreading in aerosols.

The most important rules to avoid PCR contamination:

 Separate the different procedures spatially.
Ideally use separate rooms for sample preparation and PCR setup, or at least dedicated
different areas, equipment and consumables for each procedure.
 Use DNA/Nuclease-free filter tips.
 Wear disposable powder-free gloves.
 Store BACGene Listeria monocytogenes kit components for PCR and lysis only in dedicated
areas, and apart from enrichment preparation areas and sample storage.
 Always use PCR controls and preferably also enrichment controls to monitor for
contamination.
 Consider the PCR/post-PCR area/room where the PCR cycler is located as potentially
contaminated with DNA-, decontaminate regularly and make sure to avoid carry over to other
areas by using separate equipment, lab coats, shoes.
 Dispose of used PCR plates/strips very carefully, make sure that caps do not open, move
waste only in tightly closed bags.
 Use separate equipment and materials for cleaning of different areas, in particular for the PCR
cycler room/area and for PCR setup. Instruct cleaning personnel accordingly.
 Control all areas for DNA/amplicon contamination on a regular basis (swabs/PCR analysis).

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2.7.2 General Information

PCR is performed in a volume of 25 µL in 0.1 mL reaction tubes/plates according to the real-time PCR
cycler instructions.
Test samples:
20 µL MasterMix + 5 µL lysate per reaction

Positive control (C+):

20 µL MasterMix + 5 µL Listeria monocytogenes positive control plasmid DNA

Negative control (C-):

20 µL MasterMix + 5 µL heat-inactivated lysis buffer (prepared alongside samples in well B1,
37°C for 20 min and 95°C for 10 min)
Note: BACGene PCR Negative Control (Eurofins GeneScan Technologies, cat. no. 5617702500) can
be used as C- as an alternative to heat-inactivated lysis buffer.

2.7.3 Control Reactions

Important
For every PCR, it is necessary to prepare a positive and a negative control reaction. These are required
for the evaluation of the PCR results! The layout should start with the positive control in A1, the negative
control in B1, samples in the following wells.

Required:
 1 Positive control (C+):
5 µL Listeria monocytogenes positive control plasmid DNA
 1 Negative control (C-):
5 µL heat-inactivated lysis buffer (or BACGene PCR Negative Control) added to MasterMix

Recommended:
 1 Negative enrichment control (E-):
Listeria monocytogenes free enrichment medium control
 1 Positive enrichment control (E+):
Lysed enrichment medium inoculated with Listeria monocytogenes

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2.7.4 PCR

Plate Setup
The following plate document shows the recommended distribution of reactions:

1 2 3 4 5 6 7 8 9 10 11 12
A C+ 7 15 23 31 39 47 55 63 71 79 87
B C- 8 16 24 32 40 48 56 64 72 80 88
C 1 9 17 25 33 41 49 57 65 73 81 89
D 2 10 18 26 34 42 50 58 66 74 82 90
E 3 11 19 27 35 43 51 59 67 75 83 91
F 4 12 20 28 36 44 52 60 68 76 84 P-*
G 5 13 21 29 37 45 53 61 69 77 85 E-
H 6 14 22 30 38 46 54 62 70 78 86 E+

Plate layout for 91/92 samples;

C+ = positive control
C- = negative control
E- = negative enrichment control
E+ = positive enrichment control
P- = negative control for PREraser treatment

*The use of a PREraser negative control (C- or E- with PREraser) is recommended when applying a PREraser
treatment to the samples.

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PCR Setup
Before you begin
 Make sure you have switched on the computer, the PCR instrument and ensure the sample
layout for the PCR plate is suitably documented and programmed.
 Only remove the number of PCR strips, containing pre-dispensed MasterMix, necessary for
testing the amount of samples to be analysed and store the remainder at -20 °C ± 2 °C.
Important: In case of cutting an 8-strip into smaller parts it is crucial to let the plastic warm up
at room temperature for around 1 minute prior to cutting. Do not place the strip into a cooling
block.
Note: Frequent freezing and thawing might cause inactivation of the reagents. Do not
freeze/thaw more than 3 times.
 Spin down the strips with MasterMix for 30 sec between 400  2000 × g.
 Visually check that all MasterMix is in the bottom of the wells.
1. Place PCR strips, together with the yellow PCR support grid, into an appropriate support rack. Avoid
exposing the PCR plate to light for long periods of time.
2. The lysis and PCR plate should be arranged in the same orientation in order to facilitate handling
with the multichannel pipette and in order to avoid cross contamination of wells (place the lysis plate
i.e. behind the PCR plate).
3. Open lysis plate as described in section 2.6.
4. Open the corresponding PCR strip by fixing the upper and lower end with one hand and removing
the foil starting with the loose end from top to bottom in 180 degree angle (see Figure 1A).
Note: Open each strip individually, add samples and close. Only then proceed to the next strip.
5. Transfer 5 µL of each lysate by using an 8-well multichannel pipette to the corresponding PCR strip.

Note: Make sure that the lysate is taken from the upper half of the lysis volume, avoid touching a
potential pellet of debris. Ensure the lysate is pipetted directly on top of the MasterMix.

6. Add 5 µL positive control DNA (vial with yellow cap) to well A1.

Note: Do not freeze/thaw positive control more than 6 times.

7. Close the PCR strip with an optical cap strip with clean, gloved hands (see Figure 1C).
8. Re-close the lysis strip with fresh domed caps (not supplied with the kit).
9. Repeat steps 3 - 8 with the remaining strips.
10. Transfer the PCR plate to the real-time instrument and start the run (refer to section 3 for Agilent
AriaMx™ + section 4 for Bio-Rad CFX96 Touch™).
11. Store the lysis plate at -20 ± 2 °C in case a re-run of the PCR is required.

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Figure 1 A. Removing foil from PCR strips. B. Incorrect way to close the lids in one direction. C. Correct
way to close lids by softly fixing the ends of the cap strip to the reaction tubes, then applying even
pressure to tightly close the PCR strips.

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3 AGILENT ARIA™: INSTRUCTIONS FOR USE WITH BACGENE LISTERIA

MONOCYTOGENES KIT

The BACGene Listeria monocytogenes kit is validated for the use on the Agilent AriaMx™ with software
versions up to 1.5.
The PCR platform should be prepared prior to commencing the practical work. For a more detailed
description of the instrument programming please refer to the user manual of the Agilent AriaMx™.
BACGene offers two methods for evaluating the final results. Users can choose between the automated
evaluation software FastFinder or the BACGene Evaluation Sheet. Evaluation specific comments are
marked in the protocol below in coloured boxes:
Evaluation sheet only

FastFinder only

3.1 Running a PCR

The following instructions outline how to set up a PCR run on the Agilent AriaMx™ using an external
computer.
Note: If you are using the cycler via the touch screen without an external computer please contact us at
kits@eurofins.com for more information.
Note: The Aria software is able to interface with a Laboratory Information Management System “LIMS”.
Please contact kits@eurofins.com for more information.

3.1.1 Running PCR Using a Template

Note: The necessary run template (.amxt) file is available via kits@eurofins.com. Alternatively, a run
template can be generated from LIMS, please contact us for more information.
1. Turn on Agilent AriaMx™ cycler and the remote computer.
2. Open the Agilent AriaMx™ software and continue via the software:
3. Go to “File”, then “Open…”
4. Browse to find the required run template file.
Evaluation sheet only

For evaluation with BACGene Evaluation Sheet: “BACGene_EV_Listeria_monocytogenes.amxt”

FastFinder only

For evaluation with FastFinder: “BACGene_FF_Listeria_monocytogenes.amxt”

5. Then click “Open”.
6. In the opened experiment tab sample names should be added to the field “Well Name”. Alternatively,
sample names can be added later during the evaluation of the run.
7. Please check that the following parameters are included in the run template:
 “Plate Setup” on the right side of the interface under the header “Wells”:
- ”Add Dyes”:

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 HEX™ and Cy5™ are selected for all relevant 96 wells

 “Target Name”- in order to check target names (table below) click on the arrow next to “Target”
on the right side of the screen. If more than one system is analysed on one plate, please use
the following target name for respective wells of the BACGene Listeria monocytogenes kit and
verify the target names for the other assays with the respective assay manual:

BACGene Listeria Detection Evaluation sheet only FastFinder only

monocytogenes Channel Target Name Target Name
Listeria L.mono.
HEX™ 2018a
monocytogenes
IPC Cy5™ 2018i IPC

Note: The target names have to be correct to ensure the correct evaluation using the BACGene
Evaluation Sheets or FastFinder software. The correct target names have already been assigned to the
wells in run templates provided. For more information please contact us via kits@eurofins.com.
 Check sample type definitions
Evaluation sheet only

Sample type definitions for evaluation with BACGene Evaluation Sheet:

- Well type of the positive control should be defined as “Calibrator” (e.g. well A1)
- Well type of the negative control should be defined as “Buffer” (e.g. well B1)
- Well type of all other wells should be defined as “Unknown”
FastFinder only

Sample type definitions for evaluation with FastFinder software:

- Well name of positive control should start with “C+” (e.g. well A1)
- Well name of negative control should start with “C-“ (e.g. well B1)
Important: For mixed plates please confirm the parameters using the manuals for each of the assays
included.
8. Change to the “Thermal Profile” view on the left side of the screen (under “Setup”).
9. Confirm cycler protocol

1 HOLD 42 CYCLES
enzyme activation denaturation annealing & extension
15 min at 95°C 15 sec at 95°C 60 sec at 60°C
no data collection no data collection data collection

Important: The run should always be initiated using a template containing the required information!
10. Select “Run” in the “Thermal Profile” tab to open the “Instrument Explorer”.
11. Select the correct AriaMX cycler and click on “Send Config”
12. If required, enter your login details for the selected instrument.
13. Save the experiment under the desired name and in the desired location.

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FastFinder only

Note: For the use with FastFinder save run files in the folder defined as “To be processed” in the
software (see separate short guide “FastFinder for BACGene kits”).
14. The run information has now been sent to the selected instrument.
15. The following icon will appear on the bottom right of the screen of the cycler:

16. Click the icon and press “Open Primed Experiment” on the pop-up.
Note: In case “Sync Plate” message appears, please confirm and continue.
17. The experiment will open on the screen of the cycler.
18. Under “Thermal Profile” click “Run Experiment”.

3.2 Data Evaluation

After run completion, data should be evaluated using either the BACGene Evaluation Sheet or the
FastFinder software. The data export for the evaluation using the BACGene Evaluation Sheet is outlined
below.
The evaluation using FastFinder is done using the run files directly without requiring an additional data
export, for more details refer to separate short guide “FastFinder for BACGene kits”.
Note: Please do not use the “Disc Symbol” option on the touch screen for saving runs on a USB stick.
Only use the “Copy & paste” option as described in the instrument documentation.
Evaluation sheet only

Export of Data for the Evaluation Using the BACGene Evaluation Sheet
12. Open Agilent AriaMx™ software (up to v 1.5) on your PC and open the run file by going to “File”,
“Open…” and browsing to the correct location.
Note: If you are using the cycler via the touch screen without an external computer please contact us
at kits@eurofins.com for more information.
13. Ensure all relevant wells are selected in the “Plate Setup” screen.
14. As a final check before export go to “Graphical Displays” under the “Analysis” header and ensure
that the “Background Based Threshold” is set to Cycle Range “5 thru 9” and “Sigma Multiplier 10”.
This option can be found by clicking the arrow next to “Amplification Plots” on the right side of the
software interface.

15. To prepare a default configuration for result export, follow these instructions. This step only needs
to be carried out the first time you use the software. If the default configurations have already been
selected continue to step 5.
 Go to “Export Data” tab.

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 To set up a default export content:

- From the “Definition” drop down menu select “Add new”.
- An “Add New Definitions” window opens up, uncheck “Use Current Settings”
- Name this “BACGene Export”.
- In “File Type” select “Text”.
- In items list, select “Amplification plots” and “Tabular Results”.
- Edit “Tabular Results” by clicking on the blue pen and click “Select All” in the “Column
Options” pop-up.
- Press disk icon beside the “Definition” drop down menu to save this export
configuration.

16. To export results, follow these instructions

 Go to “Export Data” under the header “Results”.
 Select “BACGene Export” from the Definition drop-down menu.
 Select “Export Data” and save to chosen location.
17. Proceed to “BACGene Evaluation Sheet” for data interpretation.
FastFinder only

For evaluation using FastFinder refer to the separate short guide “FastFinder for BACGene kits”.

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4 BIO-RAD CFX96 TOUCH™ INSTRUCTIONS FOR USE WITH BACGENE

LISTERIA MONOCYTOGENES KIT

The BACGene Listeria monocytogenes kit is validated for use with the Bio-Rad CFX96 Touch™ and
Bio-Rad CFX96 Touch™ Deep Well with CFX Manager™ software version 3.1 / CFX Maestro™
software version 1.1.
The PCR platform should be prepared prior to commencing the practical work. For a more detailed
description of the instrument programming please refer to the user manual of the Bio-Rad CFX96
Touch™ or CFX96 Touch™ Deep Well.
BACGene offers two methods for evaluating the final results. Users can choose between the automated
evaluation software FastFinder or the BACGene Evaluation Sheet. Evaluation specific comments are
marked in the protocol below in coloured boxes:
Evaluation sheet only
FastFinder only

4.1 Running a PCR

The following instructions outline how to set up a PCR run on the Bio-Rad CFX96 Touch™ and Bio-Rad
CFX96 Touch™ Deep Well with an external computer.
Note: If you are using the cycler via the touch screen without an external computer please contact us
at kits@eurofins.com for more information.

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4.1.1 Before You Begin

Note: The required protocol and plate layout templates are available via kits@eurofins.com.
Furthermore, the CFX Manager™/ CFX Maestro™ 1.1 software can be configured for use with a
Laboratory Information Management System “LIMS”. Please contact kits@eurofins.com for more
information.

Note: This procedure only needs to be carried out the first time you are running the assay.

1. Turn on Bio-Rad CFX96 Touch™ (Deep Well).

2. Open the Bio-Rad CFX Manager™ 3.1 / CFX Maestro™ 1.1.
3. Go to “File”.
4. Select “Open” and “Protocol”.
5. Browse to select the desired protocol file “BACGene_protocol.prcl” or
“BACGene_DW_protocol.prcl” (depending on the instrument used).
6. Click “Open”.
7. In the newly opened window, displaying the protocol selected in step 5, select “File” and then “Save
As”.
8. All protocols have to be saved in:
C:\Users\Public\Documents\Bio-Rad\CFX\Users\admin\ExpressLoad
Enter “BACGene_protocol” or “BACGene_DW_protocol” as the protocol name in the CFX software,
click on “Save” and close the window.
9. Repeat steps 3 – 8 for the respective plate layout template. Depending on the evaluation method
(evaluation sheet or FastFinder software) make sure to import and save the correct plate layout
template:
Evaluation sheet only

For evaluation with BACGene Evaluation Sheet:

“BACGene_EV_Listeria_monocytogenes_Plate.pltd”
FastFinder only

For evaluation with FastFinder: “BACGene_FF_Listeria_monocytogenes_Plate.pltd”

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4.1.2 Running a PCR Using a Plate Layout Template

Make sure that the correct protocol and plate layout templates have been imported into the Express
Load folder according to section 4.1.1. Alternatively, a LIMS template import can be generated directly
from LIMS, for more information please contact us at kits@eurofins.com.
1. Open the CFX Manager™ 3.1 or CFX Maestro™ 1.1 software.
2. In the “Startup wizard” select the correct instrument in the “Select instrument” drop-down menu.
3. Under “Select run type” click “User defined”
4. In the “Run setup” window select the BACGene protocol template from the “Express load” drop-
down menu.
Important: The run should always be initiated using the correct protocol and layout templates.
5. Prior to starting the run check that the following parameters are correct:
 In the “Protocol” tab check:
- Sample volume: 25 µL
- Thermal cycler times and temperatures
- Thermal Profile:

1 HOLD 41 REPETITIONS
enzyme activation denaturation annealing & extension
15 min at 95°C 15 sec at 95°C 60 sec at 60°C
no data collection no data collection data collection

6. Click on “Next” and open the plate layout template from the “Express load” drop-down menu:
Evaluation sheet only

For evaluation with BACGene Evaluation Sheet:

“BACGene_EV_Listeria_monocytogenes_Plate.pltd”
FastFinder only

For evaluation with FastFinder: “BACGene_FF_Listeria_monocytogenes_Plate.pltd”

7. Go to “Edit selected...” to open the “Plate editor”.
8. Click on “Spreadsheet View/Importer” to add sample names.

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9. Sample names can be added either manually, via copy and paste or importing a “*.csv” file (click on
“Import”).

10. After adding sample names, close the spreadsheet view by clicking “OK”.
11. In the “Plate editor” check:
 Plate Type: BR White
 Scan Mode: All Channels
 Fluorophores: VIC™ and Cy5™ are selected for all relevant 96 wells
 Target Names: To check, click “Edit Selected” and the target names are shown on the right
side of the screen, compare with target names in table below. If more than one system is
analysed on one plate, please use the following target names for respective wells of the
BACGene Listeria monocytogenes kit and verify the target names for the other assays with
the respective assay manual:

BACGene Listeria Detection Evaluation sheet only FastFinder only

monocytogenes Channel Target Name Target Name
Listeria L.mono.
VIC™ 2018a
monocytogenes
IPC Cy5™ 2018i IPC

Note: The target names have to be correct to ensure the correct evaluation using the BACGene
Evaluation Sheet or FastFinder software. The correct target names are assigned to the wells in
templates provided by us. For more information please contact us via kits@eurofins.com
 Check sample type definitions:
Evaluation sheet only

Sample type definitions for evaluation with BACGene Evaluation Sheet:

- Positive control should be defined as “Positive control” (e.g. well A1)
- Negative control should be defined as “Negative control” (e.g. well B1)
- All other wells should be defined as “Unknown”
FastFinder only

Sample type definitions for evaluation with FastFinder software:

- Name of positive control should start with “C+” (e.g. well A1)
- Name of negative control should start with “C-“ (e.g. well B1)
Important: For mixed plates please confirm the parameters using the manuals for each of the assays
included.

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12. Click “OK” to close the “Plate Editor” and select “Next” in the “Run setup” tab to get to the “Start
Run” tab.
13. Select the cycler from the list.
14. Click “Open Lid”, load the plate into the cycler and then press “Close Lid”
15. Click “Start Run”.
16. In the directory, choose the location to save the run file and then click “Save”

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4.2 Data Evaluation

After run completion, data should be evaluated using the BACGene Evaluation Sheet or FastFinder
software. The data exports from the Bio-Rad CFX96 Touch™ or Bio-Rad CFX96 Touch™ Deep Well
for the BACGene Evaluation Sheet are described below.
The evaluation using FastFinder is described in the separate short guide “FastFinder for BACGene kit”.
Evaluation sheet only

Export of Data for the Evaluation Using the BACGene Evaluation Sheet
1. Open Bio-Rad CFX Manager™ software version 3.1 / CFX Maestro™ software 1.1 and select the
saved run. This can be done using one of the following options:
a. Double click on the saved run file. This will also automatically open the Bio-Rad CFX Manager™
3.1 / CFX Maestro™ 1.1 software.
a. Files can be dragged and dropped into the open Bio-Rad CFX Manager™ 3.1 / CFX Maestro™
1.1 software.
b. In the open software, go to “File”, then click “Open” and select the run file via the directory.
Note: If you are using the cycler via the touch screen without an external computer please contact us
at kits@eurofins.com for more information.
2. Once the run file is opened, go to “Settings” tab and ensure the following options are selected:
 Cq Determination Mode = Single Threshold.
 Baseline Setting = Baseline Subtracted Curve Fit.
 Ensure that “Apply Fluorescence Drift Correction” is selected under “Baseline Settings”.
3. To export results, follow these instructions:
 Go to “Export” tab.
 Select “All Data Sheets” and export as text files (*.txt). Choose a location for the files.
 Got to “Export” and select “Custom Export” choose text format (*.txt) with the following columns
and save in chosen location:
- Well
- Fluorophore
- Target Name
- Content
- Sample Name
- Cq
- End RFU
Important: The order of the exported columns is mandatory.
Note: Save all export files in the same folder.
4. Click “Export”
5. In the “Export Complete” pop-up, click “Ok”
6. Proceed to BACGene Evaluation Sheet for data interpretation.

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FastFinder only

For evaluation using FastFinder refer to the separate short guide “FastFinder for BACGene kits”.

5 DATA INTERPRETATION

Data is interpreted using the FastFinder evaluation software or ‘BACGene Evaluation Sheet’ provided.
For FastFinder evaluation please refer to the “FastFinder for BACGene kits” manual.
For an evaluation using the evaluation sheet upload data as described in ‘BACGene Evaluation Sheet’
file.
Interpretation of final sample results is summarized in the following table:

Listeria monocytogenes Internal Positive Control Final results

Reaction positive valid positive for Listeria monocytogenes
Reaction positive invalid positive for Listeria monocytogenes
Reaction negative valid negative for Listeria monocytogenes
Reaction negative invalid Questionable*

*Refer to troubleshooting section (see section 7)

6 CONFIRMATION OF PRESUMPTIVE POSITIVE RESULTS

In the context of NF VALIDATION and AOAC-PTM certification, all samples identified as positive by the
BACGene Listeria monocytogenes kit must be confirmed by (one of) the following test(s):

6.1 Confirmation of Listeria monocytogenes Positive Results

Option 1:
Using the conventional tests described in the methods standardized by CEN or ISO from colonies
(including the purification step). The confirmation step must start from the enrichment broth or from
typical colonies isolated on selective media.

Option 2:
Direct streaking of 10 µL from enrichment in Actero™ Listeria Enrichment Media (by FoodChek™) onto
O&A agar.
Incubation of the O&A plates at 37°C ±1°C for 24 ± 3h and if necessary (negative) further 24h ± 3h.
Check for the presence of characteristic colonies which are presumed to be Listeria monocytogenes.
The presence of characteristic colonies is sufficient to confirm the presence of Listeria monocytogenes.

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Option 3 (validated in the scope of NF VALIDATION, only):

Using the MALDI biotyper of Bruker (software version: Flex Control 3.4, Flex Analysis 3.4 MBT Compass
explorer 4.1) for confirmation on isolated characteristic colonies which are presumed to be Listeria
monocytogenes on O&A. If characteristic colonies are not well isolated, confirmation may also be
performed after purification on TSYEA plates. This option is dedicated to the confirmation of Listeria
monocytogenes.

Option 4 (in the scope of NF VALIDATION, only):

Using any other method certified according to NF VALIDATION or AOAC, respectively, the principle of
which must be different from the BACGene Listeria monocytogenes kit. The protocol of detection of the
second validated method used for the confirmation shall be followed entirely. All steps which are before
the step from which the confirmation is started shall be common to both methods. The BACGene Listeria
monocytogenes kit and the second validated method must have common first steps, for instance the
same enrichment broth.
Independent of the confirmation method used, in the event of discordant results (presumptive positive
with the alternative method, non-confirmed by one of the means described above, in particular by the
latex test) the laboratory must follow the necessary steps to ensure the validity of the result obtained.

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7 TROUBLESHOOTING

BACGene offers two methods for evaluating the final results. Users can choose between the automated
evaluation software FastFinder or the BACGene Evaluation Sheet. Evaluation specific troubleshooting
comments are marked below in colored boxes:
Evaluation sheet only

FastFinder only

7.1 Problems Upon Delivery / During Storage and Reception

Observation:
MasterMix plate is not frozen upon arrival and without appropriate cooling (dry ice)

Possible Cause Solution

Plates defrosted during Contact Eurofins GeneScan Technologies Customer Service
shipping. (kits@eurofins.com or +49–(0)761–5038–200) or your local
distributor.

Observation
MasterMix was thawed.

Possible Cause Solution

Freezer problems Install an external alarm system on your freezers in order to get
notice if there are problems.

GeneScan Technologies takes no liability for products stored

incorrectly. If you nevertheless would like to test the performance of
the product, contact Eurofins GeneScan Technologies Customer
Service (kits@eurofins.com or +49–(0)761–5038–200) for
guidelines.
MasterMix storage was GeneScan Technologies takes no liability for products stored
forgotten upon reception incorrectly. If you nevertheless would like to test the performance of
and/or not stored at -20°C. the product, contact Eurofins GeneScan Technologies Customer
Service (kits@eurofins.com or +49–(0)761–5038–200) for
guidelines.

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7.2 Problems Associated with Handling of the Plates

Observation
Frozen MasterMix film covering the well

Possible Cause Solution

Plates were not thawed Use fresh strips, thaw and spin down before removing the seal.
and spun down before
removing the seal.

Observation
Liquid MasterMix film covering the well

Possible Cause Solution

Plates were not spun down Use fresh strips and spin down before opening the seal.
after thawing and prior to
removing the seal.

Observation
MasterMix is spilling during removing the seal

Possible Cause Solution

MasterMix plate is not fixed Make sure to fix the strip with one hand and remove seal strictly in
appropriately within the 180-degree angle (not pulling upwards, but towards yourself).
PCR rack. Review instructions in section 2.7.4.

Observation
Optical cap strips do not align with wells and plastic is damaged

Possible Cause Solution

The strip was closed in one To facilitate closing the cap strips on PCR strips first softly fix the
direction. ends of the cap strip to the reaction tubes, than apply even pressure
to tightly close the PCR strips.

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7.3 Incorrect Data Export and Import to BACGene Evaluation Sheet

Evaluation sheet only

Observation
Data import into the BACGene Evaluation Sheet was not successful/possible.

Possible Cause Solution

The content has not been Ensure the content is enabled by following instructions in links
enabled and Macros are provided in the “BACGene Evaluation Sheet” or on the official
not running properly. Microsoft Office website (http://office.microsoft.com/). Then repeat
the evaluation.
Data files are not Agilent AriaMx™:
recognized for the import. Ensure that “Text” is selected as file type in the “Export Data”
section.

Bio-Rad CFX96 Touch™ (Deep Well):

Ensure that “Text” (as Export Format) and “Tab” (as Column
Separator) are selected in the “Custom Export” section.
Relevant data is missing in Ensure that all export files are saved in the same folder and the file
the export files. names are not changed during/after export.
When prompted, manually chose the respective files or repeat the
export.

Agilent AriaMx™:
Ensure that all relevant fluorophores/targets are selected in the
“Graphical Displays” section for all relevant wells.
Ensure that all columns are selected for export in “Tabular results”
file (see section 3.2).

Bio-Rad CFX96 Touch™ (Deep Well):

Ensure that all relevant fluorophores/targets are selected for all
relevant wells in the “Plate Editor” section.
Ensure that the correct columns were selected during the “Custom
Export” from the CFX software (see section 4.2).

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7.4 Incorrect Run File Upload Using FastFinder Software

FastFinder only

Observation
The samples are not automatically labelled (sample definition and/or assay type) on the plate
layout in the FastFinder analysis

Possible Cause Solution

Run file templates are not Use the templates provided to correctly label your samples and
used controls automatically.
or If you are not using the templates check if the sample type
nametags are not correctly definitions and target names (nametags) are correctly assigned for
applied the use with FastFinder (section 3.1.1 and 4.1.2).

Sample type definitions and target assays can also be assigned

manually in FastFinder by dragging and dropping sample definitions
to the respective wells or selecting the wells to be edited and
choosing the correct sample definitions.

7.5 Inconclusive Results Post Evaluation

Observation
No positive/negative controls set. Evaluation is not possible.

Possible Cause Solution

C+ or C- have not been Evaluation sheet only
assigned.
Go to the “Results tab view” worksheet and define the correct task
(C+ and/or C-) via the “Task” drop-down menu (Column G of the
Evaluation Sheet) for the corresponding wells.

Alternatively: Define the respective Tasks in the original run file and
repeat the export and evaluation.
FastFinder only
Restart analysis and identify/select correct location of C+ and C-.

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Observation
The negative control (C-) is positive for at least one target assay.

Possible Cause Solution

Cross contamination Follow decontamination procedure.
occurred.
Repeat lysis of enrichment and repeat PCR using fresh aliquots of
all reagents. If C- continues to show a positive signal, contact
Eurofins GeneScan Technologies (kits@eurofins.com or +49–
(0)761–5038–200).
C- has not been assigned Make sure that the correct well for C- is selected.
correctly.

Observation
C- not in range (in the IPC system) - evaluation not possible.
Evaluation of the IPC is dependent on the C- control reaction. If this reaction is not in range, the IPC
cannot be evaluated in the sample reactions.

Possible Cause Solution

MasterMix plate was The MasterMix plate should not be frozen and thawed more than 3
freeze-thawed more than 3 times as it may cause inactivation of the reagents.
times
95°C lysis step was not Ensure the heating block reaches the correct temperature
performed correctly. (95 °C ± 5 °C) by measuring with a thermometer. Ensure this step is
performed for the correct duration (10 min).
The MasterMix was not in Always visually check the level of the MasterMix before use. If the
the bottom of the PCR MasterMix is not at the bottom of the tubes, spin down before use.
tube, but in the lid.
C- has not been assigned Make sure that the correct well for C- is selected.
correctly.

Automated threshold Evaluation sheet only

calculation failed.
Refer to section 7.6.

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Observation
C+ is negative/too late in the required target system - evaluation is not possible.
Note: The warning message „Late Cq value of C+“ is an indication reduced performance of the C+.
However, evaluation of the results is still valid. Also review troubleshooting recommendations below.

Possible Cause Solution

Pipetting error - no positive Repeat PCR ensuring 5 µL of the positive control material is
control or too little positive pipetted into appropriate well (A1).
control material was added.

The positive control was If incorrect storage is suspected, use a fresh tube of positive control
not stored correctly. material. When starting a new kit, always use the positive control
material from that kit.
The positive control Use a fresh tube of positive control material. Do not freeze/thaw
material has undergone too more than 6 times. If C+ continues to show a negative signal,
many freeze-thaw cycles. contact Eurofins GeneScan Technologies.
The frozen MasterMix was Always visually check the level of the MasterMix before use. If the
not in the bottom of the MasterMix is not at the bottom of the tubes, allow to thaw, spin
PCR tube, but in the lid. down and re-freeze before use.
C+ has not been assigned Make sure that the correct well for C+ is selected.
correctly.

Automated threshold Evaluation sheet only

calculation failed. Refer to section 7.6.

Evaluation sheet only

Observation
Cq value of the C+ is too early (in the required target system) – evaluation is not possible.

Possible Cause Solution

C+ has not been assigned Make sure that the correct well for C+ is selected.
correctly.
Automated threshold Refer to section 7.6.
calculation failed.

Observation
Linear, straight lines instead of amplification curves or amplification curves that lay completely
below the threshold

Possible Cause Solution

Fluorescence drift was not Bio-Rad CFX96 Touch™ (Deep Well):
automatically removed Ensure the “Apply Fluorescent Drift Correction” is selected and
from analyses repeat the export of results. Then continue with the evaluation.
Automated threshold Refer to section 7.6.
calculation failed.

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Observation
A “Q” or invalid result is observed for sample

Possible Cause Solution

Missing PCR tubes. The analysed well might actually be empty. Check layout of tubes
in PCR platform to ensure it matches the analysed layout.
The frozen MasterMix was Always visually check the level of the MasterMix before use. If the
not in the bottom of the PCR MasterMix is not at the bottom of the tubes, allow to thaw, spin
tube, but in the lid. down and re-freeze before use.
95 °C lysis step was not Confirm that the heating block reaches the correct temperature
performed correctly. (95 °C ± 5 °C) by measuring with a thermometer. Repeat lysis of
enrichment and repeat the PCR.
Sample lysate was not taken Repeat lysis of enrichment and repeat PCR. Ensure to take
from upper half of tube when sample from upper half of tube.
transferring to PCR.
Inhibition of PCR or Note: Leaving the enrichment bag or aliquot in sterile container to
inconclusive results (refer to rest for 5 – 10 minutes after agitation and before lysis to allow
“Data interpretation” table, sedimentation of crude suspended particles might reduce the
section 5). inhibitory effect of some matrices.

If PCR remains inhibited, repeat lysis of enrichment and repeat

PCR.

If PCR remains inhibited, dilute the lysate sample with DNA-

/Nuclease free water (one part sample plus four parts water and
one part sample plus nine parts water).

If inhibition still persists, review instructions of the respective target

specific reference method and the ISO 6887 series for preparation
of initial suspensions.
Check if the matrix type has been validated for use with the
BACGene kit. The method should only be applied to validated
matrices.

Automated threshold Evaluation sheet only

calculation failed.
Refer to section 7.6.

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Evaluation sheet only

7.6 Incorrect Automated Threshold Setting in Cycler Software

Important: Before manually adjusting any settings, please make sure that all steps described in the
data export section 3.2 (for AriaMx™) and 4.2 (for Bio-Rad CFX96 Touch™ Standard and Deep Well)
were followed.
The interpretation of real-time PCR runs depends on automated algorithms that analyse and interpret
the measured fluorescence data.
These algorithms are thoroughly validated by the manufacturers of the PCR cyclers and the respective
evaluation software and provide reliable results for the vast majority of analyses.
In rare cases, when the automated threshold calculation of the cycler software fails, manual adjustments
are reasonable and justified in order to get a correct interpretation of the measured data (e.g. automatic
threshold settings might calculate a threshold which is not set in the exponential phase of the
amplification curves).

Examples of threshold settings for Agilent AriaMx™ and Bio-Rad CFX™:

In both software platforms the threshold can be adjusted via drag & drop if the automatic threshold
calculation fails.
In case of failure of the automatic threshold settings, it is possible to review the curves and set the
threshold within the exponential phase (phase 2; Figure 2 and Figure 3) of the amplification curve (signal
intensity doubling in each cycle) before it gets into a phase with steady linear increase of the signal
intensity (phase 3; Figure 2 and Figure 3).
Note: The adjustment is described for the linear view. For AriaMx™ make sure Graph Type “Linear” is
selected. For Bio-Rad CFX™ ensure that the box “Log Scale” is unchecked.
Agilent AriaMx™ software:

Figure 2. Exemplary amplification plot of a real -time PCR in linear scale (Graph Type “Linear”). 1,
background; 2, exponential amplification phase; 3, linear amplification phase; 4, threshold line.


not part of the AOAC validation study.

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Bio-Rad CFX™ software:

Figure 3: Exemplary amplification plot of a real-time PCR in linear scale (Box “Log Scale” is unchecked).
1, background; 2, exponential amplification phase; 3, linear amplification phase; 4, threshold line.

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7.7 Cultural Confirmation

Observation
The positive result in the Listeria monocytogenes PCR assay(s) cannot be confirmed by culture
(no isolated typical colonies).

Possible Cause Solution

Pre-enrichment bag was Always mix the pre-enrichment thoroughly (as target organisms can
not mixed thoroughly settle) before taking out the portion to streak onto selective agar
before taking the portion to plates.
streak onto selective agar
plates.
The volume streaked onto Always make sure the loop for streaking is completely covered with
the selective agar plates 10 µL of the culture, if this does not work, use 50µL of the culture for
was not sufficient. plating.

Quality of selective agar Make sure agar plates are quality tested (e.g. according to ISO
plates is not sufficient. 11133).

The detection level of the Subculture 100 µL of the pre-enrichment in 9.9 mL Full Fraser and
confirmation method is not incubate up to 48h at 37°C ± 1°C or/and spread 100 µL pre-
reached (e.g. for slow- enrichment onto O&A plates (for samples with low levels of bacterial
growing Listeria background).
monocytogenes serovars).
Target organism cannot be Make sure the selective agar plates are prepared and stored
detected on plates as they according to the manufacturer’s instructions and quality-controlled
are overgrown with (e.g. according to ISO 11133).
background flora.
Contamination with Make sure all recommendations regarding DNA contamination
genomic (free DNA from prevention are followed and take measures to identify the source of
dead bacteria) in the a possible contamination (i.e. environmental swabbing for DNA
environment causes contamination and/or block contamination test for Agilent AriaMx).
positive PCR results.
Free DNA from dead target General:
organism cell in the sample Use of PREraser BACGene is recommended for samples in which a
produces a positive PCR high level of free DNA from dead cells is expected. PREraser is
result. applied before lysis of the living cells in order to eliminate signals
from free DNA. Refer to the PREraser BACGene manual and
relevant certifications for details about the scope and applicability of
the method for the matrix in question.

Environmental samples:
Environmental samples should be taken in a way that reduces
sampling of dead bacteria, e.g. by rinsing of surfaces after
decontamination to remove dead bacteria.
Make sure that swabs are enriched according to certified protocol.

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8 PRODUCT WARRANTIES, SATISFACTION GUARANTEE

Eurofins GeneScan Technologies GmbH (“GeneScan”) warrants the products manufactured by it will
be free of defects in materials and workmanship when used in accordance with the applicable
instructions before the expiration date marked on the product packaging and when stored under the
storage conditions recommended in the instructions and/or on the package. GeneScan makes no other
warranty, expressed or implied. There is no warranty of merchantability or fitness for a particular
purpose.
GeneScan’s sole obligation with the respect to the foregoing warranties shall be, at its option, to either
replace or to refund the purchase price of the product(s) or part thereof that proves defective in materials
or workmanship within the warranty period, provided the customer notifies GeneScan promptly of any
such defect. GeneScan shall not be liable for any direct, indirect or consequential damages resulting
from economic loss or property damages sustained by buyer or any customer from the use of the
product(s). A copy of Eurofins GeneScan Technologies GmbH terms and conditions can be obtained
on request, and is also provided in our price lists.

9 PRODUCT USE LIMITATIONS

This kit is developed, designed, and sold for research purposes only. It is not to be used for diagnostic
purposes or analysis of food and feed unless expressly cleared for that purpose by the competent
regulatory authorities in the country of use. All due care and attention should be exercised in the handling
of the materials described in this text.

10 IMPORTANT NOTES

Registered names, trademarks, etc. used in this document, even when not specifically marked as such,
are not to be considered unprotected by law.
Eurofins GeneScan Technologies GmbH is only responsible for the content of this manual in its original
language (English) and not liable for any translations.

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 TECHNICAL SUPPORT SERVICE
For technical assistance and more information please reach out to your local sales
support as stated on the Eurofins Technologies website or reach out to your local
distributor.
Eurofins Technologies
Fóti út 56.
1047 Budapest
Hungary
https://www.eurofins-technologies.com/contacts
© Eurofins GeneScan Technologies GmbH, all rights reserved.

ID 2622 10 Mar 2022 V3.7