hygiena KIT 2300 99 Salmonella Detection LyoKit Instruction Manual
- June 10, 2024
- Hygiena
Table of Contents
hygiena KIT 2300 99 Salmonella Detection LyoKit
Salmonella Detection LyoKit
Product Information
The Salmonella Detection LyoKit is a product designed for the qualitative detection of Salmonella spp. It comes in four different versions, identified by Order No. KIT 2300 99 / KIT 2301 00 / KIT 2301 01 / KIT 2301 02. The product has been approved under LICENSE NUMBER 120301 and manual version 4 is valid from February 2022.
Approvals:
- LICENSE NUMBER 120301
Manual:
- Version 4, February 2022
Kit Contents:
- Microplate with 12 x 8-tube strips, prefilled with lyophilized ready-to-use PCR mix
- 12 x 8-cap strips for use in real-time PCR after addition of samples
Product Usage Instructions
Step 1: Gather the required materials for the testing process.
- Salmonella Detection LyoKit
- PCR machine
- Samples
- Pipettes and tips
- DNA extraction kit (if not using certified methods)
Step 2: Take all necessary precautions and preparations before beginning the testing process.
- Clean and disinfect all surfaces and equipment to avoid contamination.
- Wear appropriate personal protective equipment (PPE) such as gloves, lab coat, and eye protection.
- Follow all safety guidelines and regulations.
Step 3: Enrichment and DNA extraction
- If using certified methods, follow the instructions provided with the kit.
- If not using certified methods, extract DNA using a DNA extraction kit according to the manufacturer’s instructions.
Step 4: Procedure
- Set up the program on the PCR machine according to the instructions provided in the manual.
- Add the extracted DNA samples to the lyophilized PCR mix in the microplate.
- Seal the microplate with the cap strips.
- Place the microplate in the PCR machine and run the program according to the instructions provided in the manual.
Step 5: Data Interpretation
- After running the program, analyze the data generated by the PCR machine to determine if Salmonella spp. is present in the samples.
- Refer to the instructions provided in the manual for data interpretation guidelines.
Step 6: Troubleshooting and Support
- If any issues arise during the testing process, refer to the troubleshooting section in the manual for guidance.
- If further assistance is needed, contact customer support for additional help.
OVERVIEW
General Information
Number of Reactions
The kit is designed for 96 reactions or 480 reactions respectively with a
final reaction volume of 25 μl each. Up to 94 samples or 470 samples (single
sample preparation) 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 foodproof® Salmonella Detection LyoKit is intended for the rapid detection
of Salmonella spp. isolated from enrichment cultures by using the DNA
extraction methods above for all relevant kinds of foods, feeds, environmental
samples and samples from the primary production stage (PPS) that are
potentially contaminated with Salmonella. The foodproof® Salmonella Detection
LyoKit is intended for the food and feed industry and for food testing
laboratories. The limit of detection is 1 – 10 cfu / 25 g sample.
The kit described in this Instruction Manual has been developed for real-time
PCR instruments with a FAM and a VIC 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) and others.
Kit Contents
A schematic representation of the foodproof® Salmonella Detection LyoKit with
all its components.
LP: KIT 2300 99
RP: KIT 2301 01
DP: KIT 2301 02
| Component| Details
---|---|---
1| Microplate| 12 x 8-tube strips, prefilled with lyophilized ready-to-use PCR
mix.
Available are three different tube profiles: white low profile tubes (LP), clear regular profile tubes (RP), and clear low profile tubes (DP).*
2| 12 x 8-cap strips| For use in real-time PCR after addition of samples.
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)| 900 µl, contains a stabilized solution of DNA for use as a PCR run positive control.
Tube profile and instrument compatibility chart is available online: www.bc- diagnostics.com/foodproof-compatibility-chart
LP: KIT 2301 00
| Component| Details
---|---|---
1| 5 x Microplate| 12 x 8-tube strips, prefilled with lyophilized ready-to-use
PCR mix. Available as white low profile tubes (LP).*
2| 5 x Bag Cap Strips| 12 x 8-cap strips, for use in real-time PCR after
addition of samples.
3| 10 x H2O PCR-grade (colorless cap)| 1 ml nuclease-free, for use as a PCR
run negative control.
4
| 2 x Control Template (purple cap)| 500 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 detection of respective probes as well
as for using low or regular profile strip tubes.
In case the strip tubes don´t fit for the instrument, the samples should be
transferred to appropriate PCR vessels after resuspension of the lyophilized
PCR mix.
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 kind of food samples and primary production stage samples.
- Positive Control: Always run a positive control with the samples. Use the provided control DNA (Control Template) or a positive sample preparation control.
- Negative Control: Always run a negative control with the samples. To prepare a negative control, replace the template DNA with PCR-grade water. Include a negative control during sample preparation to monitor reaction purity and cross-contamination. This extraction control can be used as an additional negative control reaction.
- Confirmation: If required, positive results may be confirmed by appropriate methods (e.g., reference method).
- Waste Disposal: All contaminated and potentially infectious material, like enrichment cultures or food samples, should be autoclaved before disposal and eliminated according to local rules and regulations. For more information, e.g., proper disposal of unused chemicals, please refer to the appropriate safety data sheet (SDS).
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® Salmonella Detection LyoKit is intended for the rapid detection of Salmonella spp. DNA isolated from enrichment cultures prepared by valid methods and inoculated with all relevant kinds of foods and primary production stage (PPS) samples.
- Certified Methods
The foodproof® Salmonella Detection LyoKit is AOAC RI Performance Tested MethodsSM program validated (licence number 120301) for a variety of foods including: custard, ground beef, chocolate ice cream, mayonnaise, pet food, primary production stage samples (boot socks with environmental material).
Furthermore, the foodproof® Salmonella Detection LyoKit in combination with the foodproof® Magnetic Preparation Kit I (S 400 11) for automated sample preparation and the foodproof® StarPrep One Kit (S 400 07) for manual sample preparation is validated according to ISO 16140-2 by MicroVal studies (2011LR40 and 2011LR42). The validation was done with the following food categories: fish and seafood products, chocolate and bakery products, egg products, feed samples, meat and meat products, milk and dairy products and primary production stage samples (PPS). The extension of the Salmonella detection method for the foodproof® Salmonella Detection LyoKit was done with the following PCR instruments: CFX96TM from Bio-Rad (software version CFX Manager 2.0) and LightCycler® 480 II (software version 1.5.1) from Roche.
In the table below an overview is shown how the samples were processed within
the validation studies.
Protocols MicroVal projects 2011LR40 / 2011LR42 – foodproof® Salmonella
Detection LyoKit
**Category***
|
Enrichment
| Second enrichment| DNA
extraction
|
Confirmation
---|---|---|---|---
meat and meat products/
milk and dairy products/egg products/feed samples/ fish and seafood products
|
25 g + 225 ml BPW
for 20 h ± 2 h at 37 °C ± 1 °C
| none| food proof® StarPrep One|
according EN ISO 6579-
1:2017
cocoa and cocoa-contai- ning products| 25 g + 225 ml skimmed milk +
Brilliant Green
for 20 h ± 2 h at 37 °C ± 1 °C
| none| food proof® StarPrep One| according EN ISO 6579-
1:2017
meat and meat products/
milk and dairy products/egg products/feed samples/ fish and seafood products
| 25 g + 225 ml BPW
for 19 h – 20 h at 37 °C ± 1 °C
| none| food proof® Magnetic Preparation Kit I| according EN ISO 6579-
1:2017
PPS| 25 g + 225 ml BPW (preheated) for 16 h – 17 h at 37 °C ± 1 °C| 1 ml + 9 ml RVS broth 16 h – 24 h
at 41.5 °C
| food proof® StarPrep One on 500 µl enrichment culture|
according EN ISO 6579-
1:2017
*Preparation of test samples according to the appropriate part of ISO 6887.
Procedure
This protocol describes how to perform the analysis of DNA extracts by real- time PCR.
-
Workflow
-
PLACE STRIPS IN RACK
Take needed number of PCR tube strips out of aluminum bag. Important: close bag tightly afterwards. Place strips in a suitable PCR tube rack.
If needed, gently tap the tubes to move the lyophilized pellets to the bottom of all tubes. -
DECAP
Open strips carefully direct before fi lling and discard caps. Important: do not leave 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 pellet after sealing by mixing thoroughly.
Alternatively, resuspend pellet by pipetting up and down multiple times in step 3. -
CENTRIFUGE
Briefl y 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 fi rst and last column. -
Program Setup
Program your real-time PCR instrument before setting up the PCR reactions. Select the following channels:
FAM (Salmonella), and VIC (Internal Control).
As an alternative to VIC, HEX can be used. For the PikoReal® 24, Yakima Yellow has to be selected.
Pre-incubation: 1 cycle
Step 1: 37 °C for 4 min
Step 2: 95 °C for 5 min
Amplifi cation: 50 cycles
Step 1 : 95 °C for 5 sec
Step 2 : 60 °C for 60 sec
Fluorescence detection
For some real-time PCR instruments the probe quencher as well as the usage
of a passive reference dye has to be specified. This kit contains probes with
TAMRA as quencher and no passive reference dye.
For users of the Agilent Mx3005P instrument: Click “Instrument” and “Filter
Set Gain Settings” to open the Filter Set Gain Settings dialog box in which
the gain settings may be viewed and modified. For FAM the Filter Set Gain
Setting has to be modified to “x1”.
Data Interpretation
Verify results of positive (Control Template) and negative controls (H2O),
before interpreting sample results. Always compare samples to positive and
negative control. Review data from each channel and interpret results as
described in the table.
| FAM | VIC | Result Interpretation |
|---|---|---|
| + | + or – | Positive for Salmonella |
| – | + | Negative for Salmonella |
| – | – | Invalid |
Troubleshooting
| Problem | Possible Cause | Recommendation |
|---|
Squashed or crooked tubes, or open / dislodged tube lids after run, or the cycler does not open or close properly.| Wrong tube format.| Choose the correct tube format for your cycler. Tube profile and instrument
compatibility chart is available online: www. bc- diagnostics.com/compatibility-chart
If necessary, the samples can be transferred to appropriate PCR vessels after resuspension of the lyophilized PCR mix.
Wrong placement of tubes.| Place tubes into the cycler in a vertical and
balanced order, as described in the instructions for the PCR instrument.
No signal increase is observed, even with positive controls.| Incorrect
detection channel has been chosen.| Set channel settings for respective dyes
accordingly.
Pipetting errors.| Check for correct reaction setup and repeat the PCR run.
Always run a positive control along with your samples.
No data acquisition programmed.| Check the cycle programs.
A sample shows no signals, including the internal control. Positive and
negative control have proper signals.| Inhibitory effects of the sample
material (e.g., caused by insufficient purification).| Use the recommended DNA
extraction kit.
Dilute samples or pipette a lower amount of sample DNA (e.g., 20 µl PCR-grade water and 5 µl sample instead of 25 µl sample).
Negative control samples are positive.| Carry-over contamination.| Exchange all critical solutions and reagents for DNA/RNA extraction.
Repeat the complete experiment with fresh batches of all reagents.
Always handle samples, kit components and consumables in accordance with commonly accepted practices to prevent carry-over contamination.
Add positive controls after sample and negative control reaction vessels have been sealed.
Fluorescence intensity is too low.| Inappropriate storage of kit components.|
Store lyophilized PCR mix at 2 °C to 8 °C, protected from light and moisture.
Low initial amount of target DNA.| If possible, increase the amount of sample
DNA. Depending on the chosen DNA isolation method, inhibitory effects may
occur.
Strong decrease of fluorescence baseline.| Resuspension of lyophilized PCR mix
not complete.| Always resuspend lyophilized PCR mix thoroughly.
Use the recommended vortex centrifuge with the correct settings.
---|---|---
Fluorescence intensity varies or changes abruptly during the run.|
Insufficient centrifugation of the PCR strips, e.g., resuspended PCR mix is
still in the upper part of the vessel or bubbles trapped in the mix.| Always
centrifuge PCR strips.
Use the centrifuge models and settings recommended in this manual.
Avoid the introduction of air bubbles during pipetting.
Outer surface of the vessel or the seal is dirty (e.g., by direct skin contact).| Always wear gloves when handling the vessels and seal.
Do not mark vessels on the outside of the tubes or directly on top of the reaction mix.
Pellets are difficult to dissolve.| The lyophilized PCR mix started to rehydrate.| Store the lyophilized PCR mix always in the aluminum bag with the silica gel pads. Make sure that the lids are tightly closed.
Remove strips from the aluminum bag only shortly before PCR setup.
Open strip shortly before filling.
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 maximum reliability of the kit and to prevent misinterpretation of negative results due to inhibition of the amplification, an Internal Control (IC) is included. A hydrolysis probe was designed to bind specifically the IC, allowing detection in the respective channel, whereas the target DNA is detected in another channel. In case of a negative result due to inhibition of the amplification by the sample DNA of interest, the amplification of the IC is suppressed as well, whereas a negative result for the sample DNA of interest and amplification of the IC clearly indicates the absence of parameter in the sample. The real-time PCR kit minimizes contamination risk and contains all reagents (except for template DNA) needed for the detection of target DNA. Primers and probes provide specific detection of target DNA in food and environmental samples, including primary production stage samples. The described performance of the kit is guaranteed for use only on the real-time PCR instruments listed above.
Step-by-Step Procedure
-
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.
-
The PCR instrument detects these amplified fragments in realtime 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. -
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.
-
The PCR instrument measures the emitted fluorescence of the reporter dye.
Prevention of Carry-Over Contamination
The heat-labile Uracil-DNA N-Glycosylase (UNG) is suitable for preventing
carry-over contamination between PCR’s. This technique relies on the
incorporation of deoxyuridine triphosphate (dUTP) during all amplification
reactions, and the pretreatment of all successive PCR mixtures with the heat-
labile UNG. The UNG cleaves DNA at any site where a deoxyuridine residue has
been incorporated. The resulting abasic sites are hydrolyzed due to the high
temperatures during the initial denaturation step, and can no longer serve as
PCR templates. The heat-labile UNG is inactivated during the initial
denaturation step. Native DNA (e.g., the isolated target genomic DNA) does not
contain uracil and is therefore not degraded by this procedure. Since dTTP is
replaced with dUTP and UNG is included in this kit, decontamination can be
achieved with the provided reagents.
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 27 -1, R 602 27 -2, R 602 27 -3, and R 602 27 L.
3.4 Change Index
Version 4, February 2022:
Rebranding, new document layout and content, new order number.
Addition of the MicroVal method.
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
foodproof®
Salmonella
Detection LyoKit
Order No.
LP: KIT 2300 99
RP: KIT 2301 01
DP: KIT 2301 02
Kit for 96 reactions (lyophilized) for a maximum of 94 samples
LP: KIT 2301 00
Kit for 480 reactions (lyophilized) for a maximum of 470 samples
Store kit at 2 °C to 8 °C
For testing of food and environmental samples
Approvals: