hygiena KIT 2300 92 Listeria Monocytogenes Detection LyoKit User Manual

hygiena KIT 2300 92 Listeria Monocytogenes Detection LyoKit

Listeria monocytogenes Detection LyoKit

The Listeria monocytogenes Detection LyoKit is a product designed for the qualitative detection of Listeria monocytogenes. It comes with three different order numbers: KIT 2300 92, KIT 2300 93, and KIT 2300 94. The product has received approval with license number 070401 and the manual is version 4, dated February 2022.

Overview

General Information

The product is designed for the qualitative detection of Listeria monocytogenes and comes with a predetermined number of reactions, which vary based on the order number. The LyoKit tube profiles and storage and stability information are also provided i  this section.

Applicability

The Listeria monocytogenes Detection LyoKit is applicable for use in detecting Listeria monocytogenes.

Kit Contents

The kit contains a microplate with 12 x 8-tube strips that are prefilled with lyophilized ready-to-use PCR mix.

Instructions

Required Material

Additional materials needed are not provided in the kit and must be obtained separately. These materials include DNA extraction kits, PCR machines, and other laboratory equipment.

Precautions and Preparations

Before use, ensure that all required materials are available and in good condition. Follow all safety precautions when handling the product and wear appropriate personal protective equipment.

Enrichment and DNA Extraction

Use certified methods for enrichment and DNA extraction. These methods are not provided in the kit and must be obtained separately.

Procedure

The procedure is divided into three sections: workflow, program setup, and data interpretation. Follow the steps in each section carefully to ensure accurate results.

 Troubleshooting

If any issues arise during the procedure, refer to the troubleshooting section for guidance on how to resolve the issue.

Support

If additional support is needed, contact the manufacturer for assistance.

Additional Information

Testing Principle

The testing principle is not provided in the manual.

Trademarks

The manual does not mention any trademarks.

 Reference Number

The reference number is 14.

Change Index

The change index is 14.

OVERVIEW

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® R2 and a few other cyclers, please use clear low-profile tubes (DP). For a detailed overview, please have a look at our compatibility chart.

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).

Kit Contents

A schematic representation of the foodproof® Listeria monocytogenes Detection LyoKit with all its components.

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.
3| 2 x H2O PCR-grade (colorless cap)| 1 ml nuclease-free, for use as a PCR run negative control.

4

| Control Template (purple cap)| 250 µl, contains a stabilized solution of DNA for use as a PCR run positive control.

INSTRUCTIONS

Required Material
Most of the required equipment and reagents are available through HygienaTM. Please contact us for further information.

• Use a real-time PCR cycler suitable for the detection of respective probes as well as for using low or regular-profile strip tubes.
• In case the strip tubes don´t fit the instrument, the samples should be transferred to appropriate PCR vessels after resuspension of the lyophilized PCR mix.

Material

• Nuclease-free, aerosol-resistant pipette filter tips.
• 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

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 kinds 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).

• Keep the PCR mix away from light and moisture.

• For more information, please refer to the appropriate safety data sheet (SDS). The SDS is available online at www.bc-diagnostics.com.

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 kinds of foods and primary production stage (PPS) samples that are potentially contaminated with Listeria monocytogenes. The kit must not be used in diagnostic procedures.

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 (license 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 DNA extraction procedures please refer to ANNEX 1 and ANNEX 2 at the end of the manual.

Procedure

Workflow

PLACE STRIPS IN THE RACK
Take the needed number of PCR tube strips out of the aluminum bag.
Important: close the bag tightly afterward. 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

DECAP
Open strips carefully and direct before filling and discarding caps.
Important: do not leave it open longer than necessary.

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.

SEAL
Seal the tubes with the provided 8-cap strips tightly.

MIX
Resuspend the pellet after sealing by mixing thoroughly. Alternatively, resuspend the pellet by pipetting up and down multiple times in step 3.

CENTRIFUGE
Briefly spin strips, e.g., 5 sec at 500 – 1,000 x g, in a suitable centrifuge.

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

Program Setup
Program your real-time PCR instrument before setting up the PCR reactions. Select the following channels: FAM (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 a 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”.

Data Interpretation
Verify results of positive (Control Template) and negative controls (H2O), before interpreting sample results. Always compare samples to the positive and negative control. Review data from each channel and interpret results as described in the table.

Troubleshooting

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.
Problem| Possible Cause| Recommendation
---|---|---
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.

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 appl feedback.

ADDITIONAL INFORMATION

Testing Principle
The foodproof® kit provides all necessary reagents and a control template for reliable interpretations of results. To ensure the 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 to 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 PCRs. 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.

Trademarks

foodproof®, microproof®, vetproof®, ShortPrep®, RoboPrep®, and LyoKit® are trademarks of BIOTECON Diagnostics GmbH. HygienaTM is a registered trademark of Hygiena. Other brand or product names are trademarks of their respective holders.

Reference Number
The reference number and original BIOTECON Diagnostics article numbers: R 602 23 -1, R 602 23 -2, and R 602 23 -3.

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.

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
---|---|---

food proof® 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

food proof® 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

food proof® 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

food proof® 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

food proof® 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, dairy, fruit/ juices Sample size: 25 g / 225 ml broth
Reference method: ISO 11290:1996/Amd 1:2004

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
---|---|---|---
food proof® StarPrep Two Kit Extraction Procedure A STANDARD|

22 h ± 2 h

|

25 h ± 1 h

|

5 µl + 20 µl PCR- grade water

food proof® StarPrep Two Kit Extraction Procedure B RAPID

|

—

|

48 h ± 2 h

|

5 µl + 20 µl PCR- grade water

food proof® 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

• composite foods/ready-to-eat and ready-to-reheat
• meat products
• milk and dairy products
• vegetables
• seafood and fishery products
• environmental samples

Sample size

• 25 g + 150 ml Actero Listeria Enrichment Media or 225 ml Half Fraser broth
• 1 swab + 10 ml Actero Listeria Enrichment Media or 10 ml Half Fraser broth
• 1 sponge + 100 ml Actero Listeria Enrichment Media or 100 ml Half Fraser broth
• 1 wipe + 225 ml Actero Listeria Enrichment Media or 225 ml Half Fraser broth
• For sampling after the 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)Hygiena LLC

• CA 93012 Camarillo USA www.hygiena.com/technical-support-request
• Manufactured by
• BIOTECON
• Diagnostics GmbH
• Hermannswerder 17
• 14473 Potsdam
• Germany
• bcd@bc-diagnostics.com
• www.bc-diagnostics.com

References

• Home - Hygiena
• Home - Hygiena
• Support | Hygiena

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