hygiena KIT230117 Vibrio Detection LyoKit Instruction Manual

: June 22, 2024
: Hygiena

Table of Contents

hygiena KIT230117 Vibrio Detection LyoKit
Product Information
Product Usage Instructions
OVERVIEW
INSTRUCTIONS
Troubleshooting
ADDITIONAL INFORMATION
Read User Manual Online (PDF format)
Download This Manual (PDF format)

hygiena KIT230117 Vibrio Detection LyoKit

Specifications

Product : Vibrio Detection LyoKit
Product No.: KIT230117 / KIT230118
Approval : LICENSE NUMBER 061901
Revision : A, April 2024

Product Information

Overview
The Vibrio Detection LyoKit is designed for the qualitative detection of Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae, and their associated toxin genes tdh, trh1, trh2, and ctx.

Applicability
The kit is not intended for diagnostic procedures.

Kit Contents
The LyoKit includes components such as microplate, cap strips, PCR-grade water, and control template. It is available in two tube profiles: white low profile tubes (LP) and clear regular profile tubes (RP).

Product Usage Instructions

Required Material
Use a real-time PCR cycler suitable for detection of respective probes and compatible with low or regular profile strip tubes. If strip tubes do not fit, transfer samples to appropriate PCR vessels after resuspending the lyophilized PCR mix.

Precautions and Preparations
Follow standard laboratory safety protocols. Ensure all materials are at room temperature before starting the procedure.

Enrichment and DNA Extraction
Follow certified methods for enrichment and DNA extraction as specified in the instructions provided.

Procedure

Workflow: Follow the step-by-step instructions provided in the manual for sample preparation, PCR setup, and data interpretation.
Program Setup: Configure the PCR cycler with appropriate settings for the detection of Vibrio species and toxin genes.
Data Interpretation: Analyze the results based on the guidelines provided to determine the presence of target genes.

Troubleshooting
If encountering issues during the procedure, refer to the troubleshooting section in the manual or contact customer support for assistance.

Support
For any queries or assistance, reach out to customer support using the contact information provided in the manual.

FAQ

Q: Can this kit be used for diagnostic purposes?
- A: No, the kit is not intended for diagnostic procedures.

foodproof®

Vibrio Detection LyoKit

PRODUCT INSTRUCTIONS
Documentation for the qualitative detection of
Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae and their associated toxin genes tdh, trh1, trh2 and ctx

Product No. KIT230117 / KIT230118

foodproof®
Vibrio
Detection LyoKit
Product No.
LP : KIT230117
RP : KIT230118
Kit for 96 reactions (lyophilized) for a maximum of 94 samples
Store kit at 2 to 8 °C

For testing of food and environmental samples

Approval

PRODUCT INSTRUCTIONS
Revision A, April 2024

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 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 two different tube profiles: white low profile tubes (LP) and clear regular profile tubes (RP).

The majority of real-time PCR cyclers use white low profile tubes (LP). However, the Dualo 32® R2 and a few other cyclers require clear low profile tubes (DP) or clear regular profile tubes (RP). For a detailed overview, please have a look at our compatibility chart.

Applicability
The foodproof® Vibrio Detection LyoKit is intended for the qualitative detection of the three major human pathogenic Vibrio species, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae, isolated from enrichment cultures prepared by valid methods with all relevant kinds of samples that are potentially contaminated with these microorganisms, e.g., seafood. In addition, presence of the pathogenicity associated toxin genes thermostable direct hemolysin (tdh), tdh-related hemolysin (trh1 and trh2) and cholera toxin (ctx) in isolates may be qualitatively assessed by melting curve analysis. To be used in the analytical laboratory for food, bioburden and environmental testing applications. The kit must not be used in diagnostic procedures. The kit described in this instruction manual has been developed for real-time PCR instruments with FAM, HEX, ROX and Cy5 detection channels, and capable of performing a melting curve analysis.

The performance of the kit was tested with the following real-time PCR instruments: LightCycler® 480 (I and II), LightCycler® 96 (Roche Diagnostics), Mx3005P®, AriaMx (Agilent Technologies), Applied BiosystemsTM 7500 Fast, QuantStudio™ 5 (Thermo Scientific) and CFX96TM (Bio-Rad).

The kit must not be used in diagnostic procedures.

Kit Contents
A schematic representation of the foodproof Vibrio Detection LyoKit with all its components.

LP : KIT230117
RP: KIT230118

| Component| Details
---|---|---
1| Microplate| 12 x 8-tube strips, prefilled with lyophilized ready-to-use PCR mix.

Different tube profiles are available: white low profile tubes (LP) and clear regular profile tubes (RP).*

Tube profile and instrument compatibility chart is available online

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 in 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 and reagents

foodproof StarPrep Three (KIT 230187) or
foodproof StarPrep Three 8-Strip Kit (KIT 230188)
Reagent D (Kit 230001 or KIT230003)

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.
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.hygiena.com/sds.

Enrichment and DNA extraction
The enrichment can be done for 16 to 18 hours in media according to e.g., ISO/TS 21872-1:2017 (Alkaline Saline Peptone Water, ASPW) or FDA BAM Chapter 9 (Alkaline Peptone Water, APW).

Certified Methods
The foodproof Vibrio Detection LyoKit was validated according the AOAC RI Performance Tested MethodsSM program (License No. 061901) for raw shrimp, raw oysters, cooked octopus, raw scallops and raw fish. 25 g samples were enriched in APW for 16 to 18 hours at 36 ± 1 °C. For raw shrimp, enrichment for 6 to 8 hours can also be used. DNA extraction was performed using foodproof StarPrep® Three Kit (single tubes and 8-strip protocols) and Reagent D, according to the respective product instructions.

For the AOAC RI validation, the performance of the kit was tested with the following real-time PCR instruments: LightCycler® 480, LightCycler® 96 (Roche Diagnostics), AriaMx (Agilent Technologies), Applied Biosystems™ 7500 Fast (Thermo Scientific) and CFX96™ (Bio-Rad).

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 (V. parahaemolyticus, tdh), HEX (V. vulnificus, trh1 and trh2), ROX (V. cholerae, ctx), and Cy5 (Internal Control).

Fluorescence detection Fluorescence detection during 37 – 65 °C ramp with 2 – 4 measurements/°C

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

Melting Curve: 1 cycle
Step 1 : 95 °C for 50 sec
Step 2 : 37 °C for 50 sec
**Step 3** : ramp up to 65 °C

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.

A Color Compensation is necessary for users of the LightCycler 480 System : Color Compensation Set 3 (KIT230005) or 5 (KIT 230011) will work for LightCycler 480 I. For the LightCycler 480 II, only Color Compensation Set 5 will work properly.

For the Mx3005P instrument : Choose Experiment Type ‘SYBR® Green (with Dissociation Curve)’ and add HEX, ROX, and Cy5 channels for data collection in the setup section. Click ‘Instrument’ and ‘Filter Set Gain Settings’ to open the Filter Set Gain Settings dialog box. For FAM and HEX the Filter Set Gain Setting must be modified to ‘x4’. For ROX and Cy5 the Filter Set Gain Setting must be modified to ‘x1’. In addition, the amplification step 1 must be set to 10 seconds (Step 1: 95 °C for 10 sec) instead of only 5 seconds.

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 tables.

Amplification curves

FAM	HEX	ROX	Cy5	Result Interpretation
+	+	+	+ or –	Positive for V. parahaemolyticus , V. vulnificus and

V. cholerae

–| +| +| + or –| Positive for V. vulnificus and V. cholerae
+| –| +| + or –| Positive for V. parahaemolyticus and V. cholerae
+| +| –| + or –| Positive for V. parahaemolyticus and

V. vulnificus

–| +| –| + or –| Positive for V. vulnificus
+| –| –| + or –| Positive for V. parahaemolyticus
–| –| +| + or –| Positive for V. cholerae
–| –| –| +| Negative for V. parahaemolyticus , V. vulnificus and

V. cholerae

–| –| –| –| Invalid

The Control Template contains all target sequences and usually generates higher fluorescent values than positive samples. This can affect positive/negative calls in automatic analysis of amplification curves by the respective instrument software. Always check results visually for plausibility.

Melting curves
Samples that show a positive amplification signal in the FAM or ROX detection channel can be further differentiated using a melting curve analysis. A prerequisite for the unambiguous detection and discrimination of the Vibrio toxin types in this multi-color experiment is a suitable calibration of the PCR instrument for channels FAM, HEX and ROX. Please refer to the Operator’s Manual of your real-time PCR cycler for further information.

The following table lists the detectable Vibrio toxin types in the respective channels and their expected melting peak temperatures (± 2 °C dependent on the real-time PCR instrument):

Vibrio

toxin

| FAM channel – melting peak temperature| HEX channel – melting peak temperature| ROX channel – melting peak temperature
---|---|---|---
tdh| 49 ± 2 °C| none| none
trh1| none| 53.5 ± 2 °C| none
trh2| none| 47 ± 2 °C| none
ctx| none| none| 49 ± 2 °C
Control Template| 49 ± 2 °C| 53.5 ± 2 °C| 49 ± 2 °C

The melting peak temperature ranges given in the above table mainly reflect the variability between instruments and their respective analysis software. The Control Template contains a mixture of all target sequences except trh2. Functionality of trh2 detection is provided by the trh1 melting curve. Mind that melt curves in channel HEX are associated with Vibrio parahaemolyticus (detected in channel FAM). tdh, trh1 and trh2 are associated with pathogenic Vibrio parahaemolyticus. ctx is associated with pathogenic Vibrio cholerae. V. vulnificus does not contain any of the toxin genes detected with this kit.

Melting peaks can occur at a temperature greater than 60 °C. These are not attributed to one of the target toxin types and can safely be ignored.

The peak height of positive samples may vary according to the initial cell concentration. Melting peaks of positive samples may be absent, if target is detected with an amplification cycle later as 30. If the melting curve is absent at an amplification cycle later as 30, please subcultivate the sample 1:10 in APW at 37 °C for 12 to 18 hours, and repeat DNA extraction and PCR. Note that the presence or absence of specific melting peaks should be checked manually for all positive samples as the peak finding algorithms of the respective PCR instrument software may not detect all relevant maxima of the melting curve. A guarantee for the identification via melting curves cannot be given.

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.
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 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.

Troubleshooting continues on the next page

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 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 suggestion 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 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 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 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 in 1.2 Applicability. For other instruments, please contact us.

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 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 a basic 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.

License

License Notice
The purchase price of this product includes limited, nontransferable rights under US Patent No. 7,687,247 owned by Life Technologies Corporation to use only this amount of the product to practice the claims in said patent solely for activities of the purchaser for bioburden testing, environmental testing, food testing, or testing for genetically modified organisms (GMO) in accordance with the instructions for use accompanying this product. No other rights are conveyed, including no right to use this product for in vitro diagnostic, therapeutic, or prophylactic purposes. Further information on purchasing licenses under the above patent may be obtained by contacting the Licensing Department, Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008. Email: outlicensing@lifetech.com.

Reference Number
The reference number and original Hygiena Diagnostics GmbH article numbers
R 602 44 -1, and R 602 44 -2.

Change Index

Version 1, July 2015:
First version of the package insert.
Version 2, March 2017:
License Notice changed.
Introduction of vortex centrifuges into the PCR Setup Procedure.
Version 3, February 2022:
Rebranding, new document layout and content, new order number.
Revision A, April 2024:
New branding.
R 602 44 20 -> INS-KIT230117-18-REVA

Hygiena®
Camarillo, CA 93012 USA diagnostics.support@hygiena.com

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