hygiena KIT230099 Salmonella Detection LyoKit Instruction Manual
- June 17, 2024
- Hygiena
Table of Contents
hygiena KIT230099 Salmonella Detection LyoKit
Product Information
Specifications
- Product Name: Salmonella Detection LyoKit
- Product Number: KIT230099 / KIT230100 / KIT230101 / KIT230102
- Approvals: LICENSE NUMBER 120301
- Revision: A, September 2023
Overview
General Information
The Salmonella Detection LyoKit is a qualitative detection kit used to
detect Salmonella spp. It provides reliable results for the presence of
Salmonella in various samples.
Applicability
The Salmonella Detection LyoKit is applicable for use in laboratories and
food testing facilities. It can be used for the detection of Salmonella in
food samples.
Kit Contents
The Salmonella Detection LyoKit includes the following components:
- Microplate: A 12 x 8-tube strips microplate, prefilled with lyophilized ready-to-use PCR mix. Three different tube profiles are available: white low profile tubes (LP), clear regular profile tubes (RP), and clear low profile tubes (DP).
Product Usage Instructions
Required Material
Before starting the detection process, ensure you have the following
materials:
- Salmonella Detection LyoKit
- Sample collection containers
- Lysis buffer
- DNA extraction kit
- Pipettes and tips
- Thermal cycler
- DNA detection system
Precautions and Preparations
Before proceeding with the detection process, take the following
precautions and preparations:
- Wear appropriate personal protective equipment, such as gloves and lab coat.
- Ensure the work area is clean and free from contamination.
- Read the entire instruction manual before starting the procedure.
Enrichment and DNA Extraction
The enrichment and DNA extraction process is crucial for accurate
detection. Follow the certified methods provided with the Salmonella Detection
LyoKit. The certified methods ensure reliable results.
Certified Methods
The Salmonella Detection LyoKit provides certified methods for enrichment
and DNA extraction. Follow these methods for accurate detection:
- Collect the sample in a suitable container.
- Add lysis buffer to the sample container.
- Perform DNA extraction using a DNA extraction kit according to the manufacturer’s instructions.
Procedure
-
Workflow
Follow the workflow below to perform the Salmonella detection using the LyoKit: -
Prepare the PCR reaction mix by rehydrating the lyophilized PCR mix in the microplate tubes.
-
Add the extracted DNA to the PCR reaction mix in the microplate tubes.
-
Place the microplate tubes in a thermal cycler and run the PCR program.
-
Analyze the PCR results using a DNA detection system.
-
Program Setup
Set up the thermal cycler with the appropriate PCR program for Salmonella detection. Refer to the thermal cycler manual for instructions on program setup. -
Data Interpretation
Interpret the PCR results obtained using a DNA detection system. Follow the guidelines provided with the DNA detection system for accurate interpretation of results.
Troubleshooting
If you encounter any issues during the Salmonella detection process, refer
to the troubleshooting section of the instruction manual. It provides
solutions to common problems and guidance for resolving issues.
Support
If you need further assistance or have any questions regarding the
Salmonella Detection LyoKit, contact our support team. They will provide you
with the necessary support and guidance.
Additional Information
Testing Principle
The Salmonella Detection LyoKit is based on a specific testing principle
that ensures accurate and reliable detection of Salmonella spp. in various
samples. Refer to the instruction manual for detailed information on the
testing principle.
Trademarks
Certain trademarks mentioned in the instruction manual are the property of
their respective owners.
Reference Number
The reference number for the Salmonella Detection LyoKit is provided for
identification and documentation purposes.
Change Index
The change index section of the instruction manual lists any revisions or
updates made to the product instructions. Refer to this section for
information on changes made to the instructions.
FAQ
-
What is the purpose of the Salmonella Detection LyoKit?
The Salmonella Detection LyoKit is used for qualitative detection of Salmonella spp. in various samples. -
What materials are required for the detection process?
The detection process requires the Salmonella Detection LyoKit, sample collection containers, lysis buffer, DNA extraction kit, pipettes and tips, thermal cycler, and DNA detection system. -
How can I troubleshoot issues during the detection process?
The instruction manual provides a troubleshooting section that offers solutions to common problems encountered during the Salmonella detection process. -
How can I contact support for further assistance?
If you need support or have any questions, please contact our support team for assistance.
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 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, 0.1 mL (LP), clear regular profile tubes, 0.2 mL (RP) and clear low profile tubes, 0.1 mL (DP).
- The majority of real-time PCR cyclers use low profile tubes, 0.1 mL (LP). For the Dualo 32® R2 and a few other cyclers, please use clear low profile tubes, 0.1 mL (DP). For a detailed overview, please reference 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), and Applied BiosystemsTM 7500 Fast (Thermo Scientific).
- The kit must not be used in diagnostic procedures.
Kit Contents
A schematic representation of the foodproof Salmonella Detection LyoKit with
all its components.
LP: KIT230099 RP: KIT230101 DP: KIT230102
-
Microplate 12 x 8-tube strips, prefilled with lyophilized ready-to-use PCR mix.
Three different tube profiles are available: white low profile tubes (LP), clear regular profile tubes (RP) and clear low profile tubes (DP).* -
12 x 8-cap strips For use in real-time PCR after addition of samples.
-
2 x H2O PCR-grade (colorless cap) 1 mL nuclease-free, for use as a PCR run negative control.
-
Control Template (purple cap) 900 μL, contains a stabilized solution of DNA for use as a PCR run positive control.
LP: KIT230100
-
5 x Microplate 12 x 8-tube strips, prefilled with lyophilized ready-to-use PCR mix.
Available as white low profile tubes (LP).* -
5 x Bag Cap Strips 12 x 8-cap strips, for use in real-time PCR after addition of samples.
-
10 x H2O PCR-grade (colorless cap) 1 mL nuclease-free, for use as a PCR run negative control.
-
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 Hygiena®.
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 or 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
-
DNA extraction kits
- foodproof StarPrep One (KIT230175 – 100 reactions; KIT230176 – 500 reactions) or
- foodproof StarPrep Three (KIT 230187) or
- foodproof Magnetic Preparation Kit I (KIT 230180)
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 nucleases,
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 and other 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.
- Confi rmation: If required, positive results may be confi rmed 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.
Enrichment and DNA extraction
The foodproof Salmonella Detection LyoKit is intended for the rapid detection
of Salmonella spp. DNA isolated from enrichment cultures. For DNA extraction,
please use the kits mentioned in 2.1 Required Material.
Certified Methods
- The foodproof Salmonella Detection LyoKit is AOAC RI Performance Tested MethodsSM program validated (License no. 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 (KIT230180) for automated sample preparation and the foodproof StarPrep One Kit (KIT230175) 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
*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 filling 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
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 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.
- 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 a non-fluorescent (“dark”) 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 control (H2O),
before interpreting sample results. Always compare samples to positive and
negative controls. Review data from each channel and interpret results as
described in the table.
Troubleshooting
Support
- If ave questions or experience any problems with our products, please contact us: www.hygiena.com/support
- 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 suggestio n case you would like to use our product for a different application. We highly value your 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 amplifi cation, an Internal Control (IC) is included. A hydrolysis probe
was designed to bind specifi cally 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 amplifi cation by the
sample DNA of interest, the amplifi cation of the IC is suppressed as well,
whereas a negative result for the sample DNA of interest and amplifi cation 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 specifi c 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 in 1.2
Applicability. For other instruments, please contact us.
Step-by-Step Procedure
- Using the kit‘s sequence-specifi c primers in a polymerase chain reaction (PCR), the PCR instrument and the supplied reagents amplify fragments of specifi c sequences for target DNA.
- The PCR instrument detects these amplifi ed 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.
- 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 PCRs. This technique relies on the
incorporation of deoxyuridine triphosphate (dUTP) during all amplifi cation
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®, StarPrep®, RoboPrep® and LyoKit® are trademarks of Hygiena Diagnostics GmbH.
- Hygiena® is a registered trademark of Hygiena.
- Other brand or product names are trademarks of their respective holders.
Reference Number
The reference number and original Hygiena Diagnostics GmbH article numbers:
R 602 27-1 (KIT23099), R 602 27-2 (KIT230101), R 602 27-3 (KIT230102), and R
602 27-1 L (KIT230100).
ABOUT COMPAN6Y
- Hygiena Diagnostics GmbH
- Hermannswerder 17
- 14473 Potsdam
- Germany
- www.hygiena.com
- diagnostics.support@hygiena.com
References
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