Thermo Fisher Scientific VetMAX PRRSV EU & NA 3.0 Kit Operating instructions

Brand
Thermo Fisher Scientific
Model
VetMAX PRRSV EU & NA 3.0 Kit
Type
Operating instructions
VetMAX™ PRRSV EU & NA 3.0 Kit
Real-time RT-PCR detection of the virus responsible for porcine reproductive and respiratory syndrome
Catalog NumberA57016
Doc. Part No. 100119740Pub.No. MAN0028563 Rev. B.0
Technology Species Sample matrices Test type
Real-time RT-PCR (RNA)
• Triplex assay
• Exogenous IPC
Porcine
• Serum
• Whole blood
• Semen
• Tissue
• Oral fluid
• Processing
fluid
Individual or
pooled[1]
[1] Pool up to five samples for serum, whole blood, or semen sample matrices.
WARNING! Read the Safety Data Sheets (SDSs) and
follow the handling instructions. Wear appropriate protective
eyewear, clothing, and gloves. Safety Data Sheets (SDSs) are
available from thermofisher.com/support.
Product description
The Applied Biosystems™ VetMAX™ PRRSV EU & NA 3.0 Kit
(Cat.No.A57016) enables real-time RT-PCR detection of the porcine
reproductive and respiratory syndrome virus (PRRSV). The kit has
an updated design with the ability to detect circulating PRRS virus
strains. The kit provides assays and reagents for single-well, real-time
RT-PCR detection of PRRSV EU, PRRSV NA, and internal positive
control (IPC) targets using fluorescent hydrolysis probe chemistry.
The kit has been validated for use on RNA extracted from serum,
whole blood, semen, tissue, oral fluid, and processing fluid. A
validation report is available by request from your sales representative
or technical specialist.
PRRSV causes significant losses in the swine industry due to
reproductive disorders, respiratory conditions, and growth retardation.
PRRSV RNA can be detected in blood and semen 24–48 hours
after infection. Real-time RT-PCR is the most sensitive method for
detecting PRRSV, and is a powerful tool for herd health management.
The kit includes the following assays and reagents.
•1–Sequences PRRSV3.0: Contains primers and probes that are
optimized for triplex real-time RT-PCR detection of PRRSV EU,
PRRSV NA, and IPC targets.
•2 – Master Mix PRRSV 3.0: Contains enzymes and buers that
are necessary for the triplex real-time RT-PCR reaction.
•4a – EPC PRRSV EU/NA 3.0: RNA template with PRRSV EU,
PRRSV NA, and Xeno™ (IPC) target sequences. It serves as an
external positive control for the real-time RT-PCR components,
and is used to set validation criteria for test results.
•5 – IPC PRRSV 3.0: Added to each sample and extraction control
during RNA extraction. It serves as an extraction control for
the RNA extraction procedure, and is used to monitor for the
presence of PCR inhibitors.
Contents and storage
Component Amount[1] Storage
1–Sequences PRRSV3.0 220µL
–30°C to –10°C
2 – Master Mix PRRSV3.0 1,100µL
4a–EPC PRRSV EU/NA3.0 2×80µL
5–IPC PRRSV3.0 215µL
[1] Volumes are sufficient for 100 real-time RT-PCR reactions (12µL).
Required materials not supplied
Unless otherwise indicated, all materials are available through
thermofisher.com. "MLS" indicates that the material is available from
fisherscientific.com or another major laboratory supplier.
Item Source
Real‑time PCR instrument, one of the following:
Applied Biosystems™ 7500 Real-Time PCR
System
• Precision Plate Holder for plates (A24820)
• Precision Plate Holder for 0.2‑mL tubes and
strips (4367033)
Contact your local sales
oce.
Applied Biosystems™ 7500 Fast Real-Time PCR
System
• Precision Plate Holder for plates (4359652)
• 7500 Fast Precision Plate Holder for 0.1‑mL
tube strips (A29252)
Applied Biosystems™ QuantStudio™ 5 Real-Time
PCR System
Equipment
Adjustable pipettors MLS
Microcentrifuge MLS
Centrifuge with plate adapter MLS
Laboratory mixer (vortex or equivalent) MLS
Two ice buckets or refrigerated racks:
• One for the PCR setup area where the PCR
master mix is prepared
• One for the area where RNA samples and
controls are prepared
MLS
Tubes, plates, and other consumables
Optical reaction plates and adhesive covers thermofisher.com/
plastics
Pipette tips thermofisher.com/
pipettetips
Disposable gloves MLS
Reagents
Nuclease-Free Water (not DEPC-Treated) AM9939
1X TE Buer MLS
Procedural guidelines
• Include the following control reactions for each real-time RT-PCR
run.
– Positive control—Use 4a – EPC PRRSV EU/NA 3.0.
INSTRUCTIONS FOR USE
For Veterinary Use Only. For In Vitro Use Only.
– Extraction control—Use mock samples that have been
prepared in the same RNA extraction procedure as the test
samples.
– No-template control (NTC)—Use nuclease-free water.
• Follow good laboratory practices to prevent false positives and
contamination of test samples with PCR products (see “Good
laboratory practices for PCR and RT-PCR” on page3).
Guidelines for input RNA
Step, process, or parameter Recommendation
RNA extraction method Applied Biosystems™ MagMAX™
CORE Nucleic Acid Purification Kit
(Cat.No.A32700)
Change to the RNA extraction
method for test samples and mock
samples
Add 2µL of 5 – IPC PRRSV 3.0 to
each sample and extraction control
during the RNA extraction.
Preparation of mock samples, for use
in extraction control PCRs
Prepare at least one mock
sample, using nuclease‑free water
(not DEPC‑treated) as the starting
material.
Process the mock sample
concurrently in the same RNA
extraction procedure that is used for
test samples.
Before you begin
1. Thaw reagents and samples.
a. Thaw 1 – Sequences PRRSV 3.0 and 2 – Master Mix
PRRSV 3.0 in an ice bucket or refrigerated rack.
b. Thaw 4a–EPCPRRSVEU/NA3.0, 5–IPC PRRSV3.0, and
RNA samples in a separate ice bucket or refrigerated rack.
2. Vortex each tube to thoroughly mix, then centrifuge briefly to
collect the contents.
Store thawed reagents and samples at 2–8°C until use.
Prepare the real-time RT‑PCR reaction mix
1. Prepare the RT‑PCR reaction mix in an ice bucket or refrigerated
rack according to the following table.
Component
Volume per reaction[1]
For 1
sample
For N
samples
1–SequencesPRRSV3.0 2µL N×2µL
2 – MasterMixPRRSV3.0 10µL N×10µL
Total RT‑PCR reaction mix 12µL N×12µL
[1] Prepare sufficient volume to allow for an additional reaction with respect to the
total number of reactions to be carried out during the analysis (samples and
controls). Never mix components from different lots of kits (see Certificate of
Analysis).
2. Vortex to mix the RT‑PCR reaction mix thoroughly, then
centrifuge briefly to collect the contents.
IMPORTANT! The RT‑PCR reaction mix must be mixed
thoroughly.
Set up the RT‑PCR reactions
1. Dispense 12 µL of the RT‑PCR reaction mix to the required
number of plate wells or tubes.
2. Add the indicated component for each reaction type.
Reaction type Component Volume per
reaction
Test sample Sample RNA 8.0µL
Positive control 4a–EPCPRRSVEU/NA3.0 8.0µL
Extraction control Mock sample 8.0µL
No-template control
(NTC)
Nuclease-free water 8.0µL
3. Seal each plate or tube, mix, then centrifuge briefly to collect the
contents.
Set up and run the real-time PCR instrument
1. Following the manufacturer's instructions, set up the real-time
RT-PCR run using the following parameters.
• Reaction volume: 20µL
• Passive reference: ROX™ dye (included in 2 – Master Mix
PRRSV 3.0)
Note: ROX™ dye must be selected if the instrument is capable
of detecting it. Real-time PCR instruments that do not detect
ROX™ dye may be used without aecting the accuracy of the
reading.
• Real-time PCR instrument program:
– Standard mode (7500 Real-Time PCR System,
QuantStudio™ 5 Real-Time PCR System)
Stage Repetitions Temperature Time
1 1 50°C 5 minutes
2 1 95°C 10 minutes
3 40 95°C 15seconds
60°C 1 minute
– Fast mode (7500 Fast Real-Time PCR System,
QuantStudio™ 5 Real-Time PCR System)
Stage Repetitions Temperature Time
1 1 50°C 5 minutes
2 1 95°C 10 minutes
3 40 95°C 3 seconds
60°C 30seconds
2. Select or create dye detectors, then assign to each well or tube.
Target Reporter Quencher
PRRSV EU VIC™ dye Nonfluorescent
quencher (NFQ)
PRRSV NA FAM™ dye
IPC[1] Cy5™ dye
[1] IPC: 5 – IPC PRRSV 3.0.
3. Run the appropriate PCR instrument program, collecting real-
time amplification data during the 60°C incubation.
Guidelines for data analysis
• Follow the instrument user guide for raw data analysis.
• Set the thresholds for each target separately.
• See the Certificate of Analysis for the manufacturing batch of the kit to validate the run and interpret the results.
2 VetMAX™ PRRSV EU & NA 3.0 Kit Instructions For Use
Validation criteria
Refer to the CtQC values in the Certificate of Analysis for the manufacturing lot of the kit. The test is validated if the following criteria are met:
Reaction type PRRSV EU target
(VIC™ dye)
PRRSV NA target
(FAM™ dye)
IPC target
(Cy5™ dye) Interpretation
Amplification-positive
control
Ct=CtQC EU of
4a–EPCPRRSVEU/NA3.0
±3Ct[1]
Ct=CtQC NA of
4a–EPCPRRSVEU/NA3.0
±3Ct[1] Ct<40[2] PCR is validated.
Extraction control Ct>40 Ct>40 Ct=CtQC 5–IPCPRRSV3.0
±3Ct[3] RNA extraction is validated.
No-template control Ct>40 Ct>40 Ct>40 PCR reagents are validated.
[1] See the EPC table in the Certificate of Analysis.
[2] A positive signal is expected. The IPC Ct value of the EPC is not used for sample results interpretation.
[3] See the IPC table in the Certificate of Analysis.
Interpretation of results
PRRSV EU target
(VIC™ dye)
PRRSV NA target
(FAM™ dye)
IPC target
(Cy5™ dye) Interpretation
Ct<40 Ct>40 Any value PRRSV EU is detected.
Ct>40 Ct<40 Any value PRRSV NA is detected.
Ct<40 Ct<40 Any value PRRSV EU and NA are detected.
Ct>40 Ct>40 Ct≤Ct of extraction control +3Ct [1] PRRSV EU and NA are not detected.
Ct>40 Ct>40 Ct>Ct of extraction control +3Ct[1] Invalid result.
[1] The Ct of the extraction control must be validated as described in “Validation criteria” on page3.
Retest samples with invalid results
1. Dilute the RNA samples 1:10 in 1XTE buer.
2. Repeat the real-time RT-PCR procedure with 8 µL of the diluted RNA, then interpret the results as follows.
Result Interpretation
The diluted RNA is positive for at least one PRRSV target (NA or EU) The result is validated.
The diluted RNA is negative for both PRRSV targets (NA and EU), and the IPC result is compliant.
The diluted RNA is negative for both PRRSV targets (NA and EU), and the IPC result is non-compliant. The result is invalid.
3. For diluted samples with invalid results, repeat the RNA extraction procedure on a new aliquot of the original sample lysate.
Good laboratory practices for PCR and RT-PCR
• Wear clean gloves and a clean lab coat.
– Do not wear the same gloves and lab coat that you have
previously used when handling amplified products or preparing
samples.
• Change gloves if 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.
• Clean lab benches and equipment periodically with 10%bleach
solution or DNA decontamination solution.
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.
Limited product warranty
Life Technologies Corporation and/or its aliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of
Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at
www.thermofisher.com/support.
VetMAX™ PRRSV EU & NA 3.0 Kit Instructions For Use 3
Laboratoire Service International (LSI) | 6 Allée des Ecureuils – Parc Tertiaire du Bois-Dieu | 69380 Lissieu – France
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
Revision history:Pub.No. MAN0028563
Revision Date Description
B.0 7 June 2023 • Corrected the recommended volume of 5 – IPC PRRSV 3.0 from 4μL to 2μL (see Guidelines for input RNA).
• All instances of isolation were replaced with extraction in the document.
A.0 16 January 2023 New document for VetMAX™ PRRSV EU & NA 3.0 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.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
©2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
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7 June 2023
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Thermo Fisher Scientific VetMAX PRRSV EU & NA 3.0 Kit Operating instructions

Brand
Thermo Fisher Scientific
Model
VetMAX PRRSV EU & NA 3.0 Kit
Type
Operating instructions
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