Thermo Fisher Scientific RapidFinder Equine ID Kit Mode d'emploi

Marque
Thermo Fisher Scientific
Modèle
RapidFinder Equine ID Kit
Taper
Mode d'emploi
For testing of Food and Environmental samples only.
RapidFinder™ Equine ID Kit
USER GUIDE
Real-time PCR detection of equine DNA in food and feed
samples
for use with:
Applied Biosystems™ QuantStudio™ 5 Real‑Time PCR Instrument
Applied Biosystems™ 7500 Fast Real‑Time PCR Instrument
Catalog NumberA15570
Publication Number MAN0013472
Revision C.0
Health in Code S.L., Calle de la Travesia s/n, 15E Base 5, Valencia 46024, Spain
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
Revision history:none (English)
Revision Date Description
C.0 28 November
2023 •The manufacturer address was updated.
•The trademark statement was updated.
•The storage temperature for the General Master Mix was updated.
•The Specificity and Sensitivity table was added to the Appendix.
•References to RapidFinder™ Analysis Software and RapidFinder™ Express Software were removed.
B.0 8 July 2021 •ISO certification was added.
•QuantStudio™ 5 Real‑Time PCR Instrument with Thermo Scientific™ RapidFinder™ Analysis Software v1.2 or
later was added.
A.0 30 March 2015 New document converted from Imegen document for the RapidFinder™ Equine ID Kit.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a
trademark of Roche Molecular Systems, Inc., used under permission and license. Imegen agro is a trademark of Health in Code SL.
©2015-2023 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■CHAPTER1Productinformation .................................................. 5
Productdescription ............................................................. 5
Kit contents and storage ......................................................... 5
Materials required but not provided ................................................ 6
■CHAPTER2Methods ............................................................... 8
Input DNA requirements ......................................................... 8
Determine the number of reactions, then thaw the reagents .......................... 8
Set up the PCR reactions ........................................................ 8
Set up and run the real-time PCRinstrument ....................................... 9
Analyze results .................................................................. 9
Interpretation of results ......................................................... 10
Quantitativeanalysis ........................................................... 10
■APPENDIXATroubleshooting .................................................... 12
■APPENDIXBSupplementalinformation ........................................ 14
Kit sensitivity andspecificity ..................................................... 14
UNE‑EN ISO 9001certification ................................................... 15
UNE‑EN ISO 14001certification ................................................. 15
■APPENDIXCGood laboratory practices forPCR .............................. 16
Plate layoutsuggestions ........................................................ 16
■APPENDIXDSafety ............................................................... 17
Chemicalsafety ................................................................ 18
Biological hazardsafety ......................................................... 19
RapidFinder™ Equine ID Kit User Guide 3
■APPENDIXEDocumentation and support ...................................... 20
Food safety support ............................................................ 20
Customer and technical support ................................................. 20
Relateddocumentation ......................................................... 21
Limited product warranty ........................................................ 21
Contents
4RapidFinder™ Equine ID Kit User Guide
Product information
Product description
Identification of meat species present in food samples is an essential step for verification of origin
and traceability of raw materials, as well as for quality control of handling and cleaning processes in
production lines. The Thermo Scientific™ RapidFinder™ Equine ID Kit enables real-time PCR detection
of equine (Equus caballus) DNA that is present in food and feed samples. The kit detects blends
containing ≥0.01% of equine DNA.
The kit includes:
•All reagents necessary for the real‐time PCR reaction—specific FAM™-labeled probe and primers
for equine mitochondrial DNA, DNA polymerase enzyme, and other buer components.
•An internal positive control (IPC)—VIC™-labeled probe, primers, and template, to monitor for PCR
inhibition.
•A Positive Control, to confirm equine DNA detection.
Unknown samples and control samples are provided by the investigator.
Note: The qPCR instrument must be calibrated with the following dyes before use: FAM™ and VIC™.
Kit contents and storage
Table1RapidFinder™ Equine ID Kit (Cat. No.A15570)
Component Amount (48 reactions) Storage[1]
Equine Master Mix (red pad) 360 µL –20°C
General Master Mix (white pad) 600 µL –20°C upon receipt. 2–8°C after initial use. Store
protected from light.
Positive Control (orange cap) 60µL –20°C
[1] See the expiration date on the box.
1
RapidFinder™ Equine ID Kit User Guide 5
Materials required but not provided
Unless otherwise indicated, all materials are available through the Thermo Fisher Microbiology
ordering process or thermofisher.com. They may also be available through Fisher Scientific
(fisherscientific.com), MLS, or another major laboratory supplier.
Catalog numbers that appear as links open the web pages for those products.
Item Source
Real-time PCR instrument, one of the following:
Applied Biosystems™ QuantStudio™ 5 Real‑Time
PCR System
Contact your local microbiology sales representative.
Applied Biosystems™ 7500 Fast Real-Time PCR
System
Recommended equipment for automated DNA isolation, one of the following:
KingFisher™ Flex Purification System with 96 Deep-
Well Head
A32681
Other equipment
Adjustable micropipettors (10µL, 20µL, 200µL)
Available through the Thermo Fisher Microbiology
ordering process. See thermofisher.com/plastics for
more information.
Benchtop microcentrifuge with adaptors for PCR
plates and/or tubes
Laboratory mixer (Vortex mixer or equivalent)
Optical reaction plates and covers, or optical PCR tubes and caps
MicroAmp™ Fast Optical 96-Well Reaction Plate, 0.1
mL
4346907
MicroAmp™ Optical Adhesive Film, 100 covers 4311971
MicroAmp™ Fast 8-Tube Strip, 0.1mL (See below
for caps.)
4358293
MicroAmp™ Optical 8-Cap Strips 4323032
Other plastics and consumables
Aerosol-resistant pipette tips Available through the Thermo Fisher Microbiology
ordering process. See thermofisher.com/plastics for
more information.
1.5-mL nuclease-free microcentrifuge tubes
Powder-free disposable gloves
Reagents
Nuclease-free water (not DEPC-Treated) AM9938
Recommended kits for DNA isolation, one of the following:
Chapter1Product information
Materials required but not provided
1
6RapidFinder™ Equine ID Kit User Guide
(continued)
Item Source
GMO Extraction Kit 4466336
For high-throughput isolation:
Lysis Buer 1 + RNase GMO Extraction Kit
PrepSEQ™ Nucleic Acid Extraction Kit
A24401
4428176, 4480466
Chapter1Product information
Materials required but not provided 1
RapidFinder™ Equine ID Kit User Guide 7
Methods
Input DNA requirements
•Prepare the DNA sample with a method that allows processing of 10–20g of food sample.
–For low-throughput, manual processing, use the GMO Extraction Kit.
–For automated processing, it is recommended to use the Lysis Buer1+RNase GMO
Extraction Kit and the PrepSEQ™ Nucleic Acid Extraction Kit with the KingFisher™ Flex
Purification System with 96 Deep-Well Head.
•Prepare at least one mock-purified sample as a negative extraction control, processed with the
same DNA isolation method that is used for test samples.
•Dilute the final DNA sample to 10ng/µL for the PCR.
Determine the number of reactions, then thaw the reagents
1. Plan to include the following reactions.
•Single reaction for each test sample
•Single reaction for each of the following controls:
–Positive Control (included in the kit).
–Negative extraction control (mock-purified samples).
–No-template control reactions; use nuclease-free water in place of sample DNA.
2. Thaw all reagents, vortex to mix thoroughly, then place on ice.
Set up the PCR reactions
1. Combine the following components for the number of reactions required plus 10% overage.
Component Volume per reaction
Equine Master Mix (red pad) 7.5 µL
General Master Mix (white pad) 12.5 µL
2. Mix thoroughly by vortexing, then distribute 20 µL to each reaction well or tube.
3. Add 5µL of DNA sample (10ng/µL), mock-purified sample (negative extraction control),
nuclease-free water (no-template control), or Positive Control to the appropriate wells.
4. Seal each plate or tube, mix, then centrifuge briefly to bring the contents to the bottom.
2
8RapidFinder™ Equine ID Kit User Guide
Set up and run the real-time PCR instrument
See the appropriate instrument user guide for detailed instructions to set up and run the real-time PCR
instrument.
1. Set up the real-time PCR instrument using the following settings:
•Reaction volume: 25 µL
•Passive reference dye: ROX™ dye included
•TaqMan™ probe reporter dyes and quenchers:
Target Reporter Quencher
Equine DNA FAM™ dye NFQ-MGB
IPC VIC™ dye NFQ-MGB
•Thermal cycler settings:
Setting Stage 1
Enzyme activation
Stage 2
PCR
Number of cycles 1 (Hold) 36
Denature Anneal/extend[1]
Temperature 95°C 95°C 60°C
Time 10 minutes 15 seconds 1 minute
[1] Fluorescence is acquired during the annealing/extension stage.
2. Load the reactions, run the thermal cycler program and collect real-time amplification data.
Analyze results
The general process for analyzing results is described in this section. The details of data analysis
depend on the real-time PCR instrument that you use; see the appropriate user guide for instructions
on how to analyze your data.
1. View the amplification plots for all reactions to make sure that they appear normal.
2. Use the Auto instrument setting to set the baseline.
3. Set the FAM™ and VIC™ threshold to 0.1.
Chapter2Methods
Set up and run the real-time PCR instrument 2
RapidFinder™ Equine ID Kit User Guide 9
4. Check that the results obtained in all control wells are as expected. For unexpected control results,
see Appendix A, “Troubleshooting”.
Reaction type FAM™ channel
(Equine DNA)
VIC™ channel
(IPC)[1]
Positive Control + +
Negative extraction control — +
No-template control — +
[1] In all reactions, the Ct of the IPC should be close to the Ct of the Positive Control.
5. Establish the positive cut-o value for the test samples and assign results:
Ct (cut-o) = Ct (Positive Control) + 3.32
Sample Ct value Sample result
Ct > Ct (cut-o) Negative
Ct ≤ Ct (cut-o) Positive[1]
[1] For fresh or minimally processed meat samples, the cut-off value corresponds to approximately 0.01% equine DNA, when the
DNA sample concentration is 10ng/µL.
Interpretation of results
Interpret unknown sample results according to the following table.
FAM™ channel
(Equine DNA)
VIC™ channel
(IPC)
Interpretation
— + Equine DNA not detected.
+ + Equine DNA detected.
— — Invalid result. See Appendix A, “Troubleshooting”.
+ — This result is expected in reactions that have a strong FAM™ signal. See
Appendix A, “Troubleshooting”.
Quantitative analysis
For samples with positive results, the percentage of equine DNA with respect to total animal DNA
can be quantified using the RapidFinder™ Equine ID Kit in combination with the RapidFinder™ Quant
Multi-Meat Set (Cat. no.A24399) or the RapidFinder™ Quant Equine Set (Cat. no.A15579).
The RapidFinder™ Quant Multi-Meat Set includes the Multi-Meat Standard, a plasmid DNA quantitation
standard that contains an equine-specific DNA target and a highly conserved animal-specific
mitochondrial genomic region (total animal DNA target). The kit also includes a TaqMan™ assay for the
total animal DNA target. Both equine and total animal DNA present in each sample can be quantified
relative to the Multi-Meat Standard using primer and probe sets from both kits. (The RapidFinder™
Quant Multi-Meat Set can also be used to quantify other animal species DNA in a similar manner.)
Chapter2Methods
Interpretation of results
2
10 RapidFinder™ Equine ID Kit User Guide
Likewise, the RapidFinder™ Quant Equine Set includes the Equine Standard, which can be used to
quantify equine and total animal DNA in a similar manner.
For detailed instructions, see the RapidFinder™ Quant Multi-Meat Set User Guide (Pub.
no.MAN0009930) or to the RapidFinder™ Quant Equine Set User Guide (Pub. no.MAN0013473).
Note: The Positive Control included in the RapidFinder™ Quant Equine Set is not intended for use as a
quantitation standard.
Chapter2Methods
Quantitative analysis 2
RapidFinder™ Equine ID Kit User Guide 11
Troubleshooting
Observation Possible cause Recommended action
In the Positive Control wells,
no target-specific and no IPC
signals are detected.
PCR amplification failed. Check that the thermal cycler settings and
amplification program are correct.
In the negative extraction
control wells, target-specific
and IPC signals are detected.
Contamination occurred during
the DNA extraction procedure.
Contamination may be due to errors in
sample handling, reagent contamination, or
environmental contamination.
•Check that the DNA extraction protocol
was performed correctly.
•Take care to avoid contamination during
sample homogenization: decontaminate
grinding equipment or homogenizer with
10%bleach or DNAZap™ Solutions (Cat.
No.AM9890).
•Decontaminate benchtop surfaces and
other equipment where the DNA
extraction process is performed with
10%bleach or DNAZap™ Solutions.
•If necessary, use fresh reagents and
repeat the DNA extraction.
In the no-template control
wells, target-specific and IPC
signals are detected.
Contamination occurred during
PCR.
Contamination may be due to errors in
sample handling, reagent contamination, or
environmental contamination.
•Decontaminate benchtop surfaces and
other equipment where PCR is performed
with 10%bleach or DNAZap™Solutions
(Cat. No. AM9890).
•Use fresh reagents and repeat the PCR.
•Set up the Positive Control PCR reactions
last to avoid cross-contamination.
In unknown wells, no IPC
signal is detected, but target-
specific signal is detected.
A high copy number of
target DNA existed in
the samples, resulting in
preferential amplification of the
target-specific DNA.
No action is required. The result is considered
positive.
In unknown wells, no IPC
or target-specific signal is
detected.
Excess sample DNA was used
in PCR; the recommended
maximum is 250ng.
Repeat the PCR with the correct amount of
DNA. If DNA quantification is not possible,
dilute the DNA sample.
A
12 RapidFinder™ Equine ID Kit User Guide
Observation Possible cause Recommended action
In unknown wells, no IPC
or target-specific signal is
detected.
(continued)
PCR inhibitors were present in
the sample DNA.
Repeat the DNA extraction. If the problem
persists, contact Technical Support.
AppendixATroubleshooting
Quantitative analysis A
RapidFinder™ Equine ID Kit User Guide 13
Supplemental information
Kit sensitivity and specificity
The detection limit was calculated with standard samples consisting of mixtures of raw equine meat
and other species. The RapidFinder™ Equine ID Kit can detect blends with as little as 0.01% (w/w) of
equine meat. The limit of detection in processed samples varies depending on the composition and
food processing.
The kit specificity was tested by comparison of the probe and primer sequences with the NCBI
database, and it was also experimentally tested on a collection of reference DNAs, with the following
results:
Table2Animal species used during the specificity assay for the RapidFinder™ Equine ID Kit
Meat species Result
Sheep (Ovis aries) Not detected
Goat (Capra aegagrus hircus) Not detected
Horse (Equus caballus) Detected
Mule (Equus asinus × Equus caballus) Detected
Donkey (Equus asinus)[1] Detected
Beef (Bos taurus) Not detected
Water bualo (Bubalus bubalis) Not detected
Fallow deer (Dama dama) Not detected
Chicken (Gallus gallus) Not detected
Turkey (Meleagris gallopavo) Not detected
Duck (genus Anas) Not detected
Ostrich (Struthio camelus) Not detected
Goose (Anser anser) Not detected
Human (Homo sapiens) Not detected
[1] Donkey is detected but with less sensitivity than horse or mule.
B
14 RapidFinder™ Equine ID Kit User Guide
UNE‑EN ISO 9001 certification
Health in Code S.L. is certified against the standard UNE-EN ISO 9001:2015 "Quality management
systems" for the design, development, manufacture, and commercialization of kits for genetic analysis.
UNE‑EN ISO 14001 certification
Health in Code S.L. is certified against the standard UNE-EN ISO 14001:2015 “Environmental
Management Systems” for the design, development, manufacture, and commercialization of kits for
genetic analysis.
AppendixBSupplemental information
UNE‑EN ISO 9001 certification B
RapidFinder™ Equine ID Kit User Guide 15
Good laboratory practices for PCR
Note: Spin tubes/plates before performing PCR. Spinning of PCR tubes is most easily accomplished
by using a centrifuge designed for PCR tubes or plates. Follow manufacturer instructions for loading
tubes/plates.
To avoid amplicon contamination of samples, follow these guidelines when preparing or handling
samples for PCR amplification:
•Wear clean gloves and a clean lab coat (not previously worn while handling amplified products or
used during sample preparation).
•Change gloves whenever you suspect that they are contaminated.
•Maintain separate areas and dedicated equipment and supplies for:
–Sample preparation and reaction setup.
–Amplification and analysis of products.
•Do not bring amplified products into the reaction setup area.
•Open and close all sample tubes carefully. Avoid splashing or spraying samples.
•Keep reactions and components capped as much as possible.
•Use a positive-displacement pipettor or aerosol-resistant barrier pipette tips.
•Do not open reaction tubes after PCR.
•Do not autoclave reaction tubes after PCR.
•Clean lab benches and equipment periodically with 10% bleach solution or DNAZap™ Solutions
(Cat. No.AM9890) according to the Thermo Fisher Scientific PCR Decontamination Protocol. After
cleaning with bleach we recommend a rinse with distilled water or an ethanol solution because
bleach will rust stainless steel. Note that minor discoloration of metal parts may occur.
For additional information, refer to EN ISO 22174:2005 or www.thermofisher.com/us/en/home/life-
science/pcr/real-time-learning-center/real-time-pcr-basics.html.
Plate layout suggestions
•Separate dierent targets by a row if enough space is available.
•Put at least one well between unknown samples and controls if possible.
•Separate negative and positive controls by one well if possible.
•Place replicates of one sample for the same target next to each other.
•Start with the unknown samples and put controls at the end of the row or column.
•Put positive controls in one of the outer rows or columns if possible.
•Consider that caps for PCR tubes come in strips of 8 or 12.
C
16 RapidFinder™ Equine ID Kit User Guide
Safety
WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user
documentation may result in personal injury or damage to the instrument or device. Ensure that
anyone using this product has received instructions in general safety practices for laboratories and
the safety information provided in this document.
·Before using an instrument or device, read and understand the safety information provided in the
user documentation provided by the manufacturer of the instrument or device.
·Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use
appropriate personal protective equipment (gloves, gowns, eye protection, and so on). To obtain
SDSs, visit thermofisher.com/support.
D
RapidFinder™ Equine ID Kit User Guide 17
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel
read and practice the general safety guidelines for chemical usage, storage, and waste provided
below. Consult the relevant SDS for specific precautions and instructions:
·Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer
before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see
the "Documentation and Support" section in this document.
·Minimize contact with chemicals. Wear appropriate personal protective equipment when handling
chemicals (for example, safety glasses, gloves, or protective clothing).
·Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with
sucient ventilation (for example, fume hood).
·Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer
cleanup procedures as recommended in the SDS.
·Handle chemical wastes in a fume hood.
·Ensure use of primary and secondary waste containers. (A primary waste container holds the
immediate waste. A secondary container contains spills or leaks from the primary container.
Both containers must be compatible with the waste material and meet federal, state, and local
requirements for container storage.)
·After emptying a waste container, seal it with the cap provided.
·Characterize (by analysis if needed) the waste generated by the particular applications, reagents,
and substrates used in your laboratory.
·Ensure that the waste is stored, transferred, transported, and disposed of according to all local,
state/provincial, and/or national regulations.
·IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal
limitations may apply.
WARNING! HAZARDOUS WASTE (from instruments). Waste produced by the instrument is
potentially hazardous. Follow the guidelines noted in the preceding General Chemical Handling
warning.
WARNING! 4L Reagent and Waste Bottle Safety. Four-liter reagent and waste bottles can crack
and leak. Each 4-liter bottle should be secured in a low-density polyethylene safety container with the
cover fastened and the handles locked in the upright position.
AppendixDSafety
Chemical safety
D
18 RapidFinder™ Equine ID Kit User Guide
Biological hazard safety
WARNING! Potential Biohazard. Depending on the samples used on this instrument, the surface
may be considered a biohazard. Use appropriate decontamination methods when working with
biohazards.
WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents,
and blood of humans and other animals have the potential to transmit infectious diseases.
Conduct all work in properly equipped facilities with the appropriate safety equipment (for example,
physical containment devices). Safety equipment can also include items for personal protection,
such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or
goggles. Individuals should be trained according to applicable regulatory and company/ institution
requirements before working with potentially biohazardous materials. Follow all applicable local,
state/provincial, and/or national regulations. The following references provide general guidelines when
handling biological samples in laboratory environment.
·U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical
Laboratories (BMBL), 6th Edition, HHS Publication No. (CDC) 300859, Revised June 2020
www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2020-P.pdf
·Laboratory biosafety manual, fourth edition. Geneva: World Health Organization; 2020 (Laboratory
biosafety manual, fourth edition and associated monographs)
www.who.int/publications/i/item/9789240011311
AppendixDSafety
Biological hazard safety D
RapidFinder™ Equine ID Kit User Guide 19
Documentation and support
Food safety support
Website: https://www.thermofisher.com/us/en/home/industrial/food-beverage/food-
microbiology-testing.html
Health in Code website for Certificates of Analysis and other product documentation: https://
portal.imegen.es/en/certificate-of-analysis/
Support email:
•Europe, Middle East, Africa: microbiology.techsupport.uk@thermofisher.com
•North America: microbiology@thermofisher.com
Phone: Visit thermofisher.com/support, select the link for phone support, then select the appropriate
country from the dropdown list.
Customer and technical support
Visit thermofisher.com/support for the latest service and support information.
•Worldwide contact telephone numbers
•Product support information
–Product FAQs
–Software, patches, and updates
–Training for many applications and instruments
•Order and web support
•Product documentation
–User guides, manuals, and protocols
–Certificates of Analysis
–Safety Data Sheets (SDSs; also known as MSDSs)
Note: For SDSs for reagents and chemicals from other manufacturers, contact the
manufacturer.
E
20 RapidFinder™ Equine ID Kit User Guide
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Thermo Fisher Scientific RapidFinder Equine ID Kit Mode d'emploi

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