Thermo Fisher Scientific VetMAX PRRSV EU and NA 2.0 Kit Operating instructions
- Type
- Operating instructions
VetMAX™ PRRSV EU & NA 2.0 Kit
Real-time RT-PCR detection of the virus responsible for porcine reproductive and respiratory syndrome
Catalog Number A35751
Doc. Part No. 100073374 Pub. No. MAN0016810 Rev. D.0
Technology Species Sample matrices Test type
Real-time RT-PCR (RNA)
• Triplex assay
• Exogenous IPC
Porcine
• Serum
• Whole blood
• Semen
• Tissue
• Oral fluid
• Cell culture supernatant
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 thermosher.com/support.
Product description
The Applied Biosystems™ VetMAX™ PRRSV EU & NA 2.0 Kit
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 PRRSV 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 uorescent hydrolysis probe chemistry.
The kit has been validated for use on RNA extracted from serum,
whole blood, semen, tissue, oral uid, and cell culture
supernatant. A validation report is available by request from your
sales representative or technical specialist.
PRRSV causes signicant 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 virus, and is a powerful tool for herd
health management.
The kit includes the following assays and reagents.
•3 – Mix PRRS EU/NA 2.0: Contains primers, probes, buer,
and enzyme for optimized triplex real-time RT-PCR detection
of PRRSV EU, PRRSV NA, and IPC targets.
•4a – EPC PRRS EU/NA 2.0: RNA template with PRRSV EU
and PRRSV NA 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 PRRS: Added to each test and control sample at the
lysis step of the RNA isolation procedure. It serves as an
exogenous internal positive control for the RNA isolation
procedure, and is used to monitor for the presence of PCR
inhibitors.
Contents and storage
Component Amount[1] Storage
3 – Mix PRRS EU/NA 2.0 2 × 600 µL
–30°C to –10°C4a – EPC PRRS EU/NA 2.0 2 × 150 µL
5 – IPC PRRS 2 × 200 µL
[1] Volumes are sufficient for 100 real-time RT-PCR reactions (12 µL).
Required materials not supplied
MLS: Fisher Scientic (sherscientic.com) or other major
laboratory supplier.
Item Source
Real‑time PCR instrument, one of the following:
Applied Biosystems™ 7500 Real-Time PCR
System
• Precision Plate Holder for 7500 Real-
Time PCR Systems (A24820)
• Precision Plate Holder for 0.2 mL Tubes
and Strips (4367033)
Contact your local
sales office.
Applied Biosystems™ 7500 Fast Real-Time
PCR System
• Precision Plate Holder for 7500 Fast
Real-Time PCR Systems ( 4359652)
• 7500 Fast Precision Plate Holder, for
0.1 mL Tube Strips (A29252)
Applied Biosystems™ QuantStudio™ 5 Real-
Time PCR System
INSTRUCTIONS FOR USE
For Veterinary Use Only. For In Vitro Use Only.
Item Source
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 Buffer MLS
Procedural guidelines
• Include the following control reactions for each real-time RT-
PCR run.
– Positive control—use 4a – EPC PRRS EU/NA 2.0.
– Extraction control—use mock-puried samples that
have been prepared in the same RNA isolation
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 4).
Guidelines for input RNA
Step, process, or parameter Recommendation
RNA isolation method Applied Biosystems™ MagMAX™
CORE Nucleic Acid Purification
Kit (Cat. No. A32700)
Change to the RNA isolation
method for test samples and
mock-purified samples
Add 4 µL of 5 – IPC PRRS to the
lysis solution used for RNA
isolation.
Preparation of mock-purified
samples, for use in extraction
control PCRs
Prepare at least one mock-
purified sample, using Nuclease-
free Water (not DEPC-Treated) as
the starting material.
Process the mock-purified
sample concurrently in the same
RNA isolation procedure that is
used for test samples.
Before you begin
1. Thaw reagents and samples:
a. Thaw 3 – Mix PRRS EU/NA 2.0 in an ice bucket or
refrigerated rack.
b. Thaw 4a – EPC PRRS EU/NA 2.0, 5 – IPC PRRS, and
RNA samples in a separate ice bucket or refrigerated
rack.
2. Thoroughly mix the contents of each tube by vortexing, then
centrifuge briey to bring the contents to the boom of the
tube.
Store thawed reagents and samples at 2–8°C until use.
Set up the PCR reactions
1. Dispense 12 µL of 3 – Mix PRRS EU/NA 2.0 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 PRRS EU/NA 2.0 8.0 µL
Extraction control Mock-purified sample 8.0 µL
No-template
control (NTC)
Nuclease-free water 8.0 µL
3. Seal each plate or tube, mix, then centrifuge briey to bring
the contents to the boom of the plate wells or tubes.
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 3 – Mix PRRS
EU/NA 2.0)
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 aecting
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
2
VetMAX
™
PRRSV EU & NA 2.0 Kit Instructions For Use
– 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 PRRS.
3. Run the appropriate PCR instrument program, collecting
real-time amplication data during the 60°C incubation.
Guidelines for data analysis
• Follow the instrument user guide for raw data analysis.
• Analyze each uorescent dye channel independently.
• Manually set the baseline and threshold seings for data
analysis.
a. For each reporter dye, congure the analysis seings for
manual Ct seing.
b. Save the analysis seings, then export the results.
• Ensure that samples showing amplication in the
normalization data also show amplication in the raw
uorescence data.
• See the Certicate of Analysis for the manufacturing batch of
the kit to validate the run and interpret the results.
Validation criteria
Refer to the CtQC values in the Certicate 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
Positive control Ct = CtQC EU of 4a – EPC
PRRS EU/NA 2.0 ± 3 Ct[1] Ct = CtQC NA of 4a – EPC
PRRS EU/NA 2.0 ± 3 Ct[1] Any value[2] PCR is validated.
Extraction control Ct > 40 Ct > 40 Ct = CtQC 5 – IPC PRRS ± 3 Ct[3] RNA isolation 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] The IPC Ct value of the positive control reaction is not used for validation.
[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.
VetMAX
™
PRRSV EU & NA 2.0 Kit Instructions For Use
3
Retest samples with invalid results
1. Dilute the RNA samples 1:10 in 1X TE buer.
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), but the IPC result is non-compliant. The result is invalid.
3. For diluted samples with invalid results, repeat the RNA isolation 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 amplied 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.
–Amplication and analysis of products.
• Do not bring amplied 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 pipeor or aerosol-resistant
barrier pipee tips.
• Clean lab benches and equipment periodically with
10% bleach solution or DNA decontamination solution.
Customer and technical support
Visit thermosher.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
–Certicates 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 aliate(s) warrant their
products as set forth in the Life Technologies' General Terms and
Conditions of Sale at www.thermosher.com/us/en/home/global/
terms-and-conditions.html. If you have any questions, please
contact Life Technologies at www.thermosher.com/support.
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.
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.
Revision history: Pub. No. MAN0016810
Revision Date Description
D.0 25 February 2019 Corrected the acronym for the virus.
C.0 27 December 2018 Add instructions to manually apply the baseline settings.
B.0 30 October 2018 Correct volume of 5 – IPC PRRS.
A.0 21 August 2018 New document.
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.
©2019 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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25 February 2019
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Thermo Fisher Scientific VetMAX PRRSV EU and NA 2.0 Kit Operating instructions
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- Operating instructions
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