Thermo Fisher Scientific VetMAX Instruction Manual
INSTRUCTIONS FOR USE
VetMAX™ African Swine Fever Virus Detection Kit
T
aqMan® real‑time PCR detection of African swine fever virus
Catalog Number A28809
Doc. Part No. 100027918 Pub. No. MAN0010783 Rev. C.0
Technology Species Samples Test type
Real-time PCR (DNA)
Duplex assay
•
• Exogenous IPC
Swine
WARNING! Read the Safety Data Sheets (SDSs) and
follow the handling instructions. W
protective eyewear, clothing, and gloves. Safety Data
Sheets (SDSs) are available from thermofisher.com/
support.
ear appropriate
Product description
The Applied Biosystems™ V
Detection Kit (Cat. No. A28809) enables detection of the African
swine fever virus (ASFV) in swine blood, serum, or tissues by
real‑time PCR amplication of the ASFV P72 gene.
The assay is a single‑well real‑time PCR in which ASFV and
exogenous Internal Positive Control (IPC) targets are amplied
and detected using uorescent TaqMan® probes.
The kit includes:
• 3 - Mix ASFV: Contains primers, TaqMan® probes, buffer, and
enzyme for optimized duplex real-time PCR amplication of
ASFV and IPC targets.
• 4a - EPC ASFV: Nucleic acid template for P72 target
amplication. It serves as an external positive control for the
real-time PCR reaction, and it is used to set the cycle
threshold (Ct) for evaluating test results.
• 5 - IPC ASFV: Internal positive control added to each sample
and control at the lysis step of the DNA extraction procedure.
It serves as a control for the DNA purication process, and it
is used to monitor for the presence of PCR inhibitors.
etMAX™ African Swine Fever Virus
Blood
Serum
Tissues Individual
Pooled samples (5 or 10 samples)
Individual
Required materials not supplied
Unless otherwise indicated, all materials ar
thermofisher.com. MLS: Fisher Scientic (fisherscientific.com)
or other major laboratory supplier.
Item Source
Applied Biosystems™ 7500 Real-T
System
96-well plate, strip tubes (8- or 12-wells),
microtubes or capillaries compatible with
thermal cycler used
Nuclease-free pipettes and filtered pipette tips 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 DNA samples
and controls are prepared
Plate covers or caps compatible with the
plates, strip tubes, micr
Nuclease-free reagent tubes for preparing
master mix
Nuclease-free Water (not DEPC-Treated) AM9939
1X TE Buffer MLS
otubes, or capillaries
ime PCR
e available through
Contact your local
sales office.
MLS
MLS
MLS
MLS
Contents and storage
Component Amount
3 – Mix ASFV 2 × 1000 µL
5 – IPC ASFV 1 × 500 µL
[1]
ufficient for 100 25‑µL real‑time PCR reactions.
S
[2]
See packaging for expiration date.
For Veterinary Use Only. For In Vitro Use Only.
[1]
Storage
–30°C to –10°C4a – EPC ASFV 2 × 90 µL
Procedural guidelines
•
For each real‑time PCR run, include the controls indicated in
[2]
“Set up the PCR reactions” on page 2.
• Follow “Good laboratory practices for PCR and RT-PCR” on
page 4 to prevent false positives and contamination of test
samples with PCR products.
Requirements for input DNA
We recommend using the MagMAX™ Pathogen RNA/DNA Kit (Cat.
No. 4462359) for DNA extraction from biological samples, but you
can also use other high quality DNA extraction methods after
proper validation in your laboratory. In addition, prepare mock-
puried sample using nuclease‑free water as the starting material
and the same DNA isolation method used for test samples.
IMPORTANT! Add 5 µL of 5 - IPC ASFV to the lysis solution
used for DNA isolation for each sample and extraction contr
ol.
Before you begin
1.
Thaw reagents and samples:
a.
Thaw 3 – Mix ASFV in an ice bucket or refrigerated
rack.
b.
Thaw 4a – EPC ASFV, 5 – IPC ASFV, and DNA samples
in a separate ice bucket or refrigerated rack.
2.
Thoroughly mix the contents of each tube by vortexing, then
briey centrifuge.
Store thawed reagents, controls, and samples at 2–8°C until use.
Set up the PCR reactions
1.
Dispense 20 µL of 3 – Mix ASFV to the appropriate number
of PCR plate wells, strip tubes, or capillaries.
2.
Add sample or control according to the following table:
Sample type Component
Test sample Sample DNA 5.0 µL
Volume per
eaction
r
Set up and run the real-time PCR instrument
1.
Following the manufacturer's instructions, set up the
real‑time PCR run using the following parameters.
• Reaction volume: 25 µL
• Passive reference: ROX™ dye (included in 3 – Mix ASFV)
Note: ROX™ dye must be set up if the instrument is
capable of detecting it. Real‑time PCR instruments that
do not detect ROX™ dye can be used without affecting
the accuracy of the reading.
• Select detectors and assign TaqMan® probe reporter
dyes and quenchers for each well, tube, or capillary
used in the analysis.
Target Reporter Quencher
ASFV FAM™ dye Non-fluorescent
IPC VIC™ dye TAMRA™ dye
[1]
AMRA™ dye must be set up for real‑time PCR analysis if the
T
instrument is capable of detecting it. Real‑time PCR instruments that
do not detect TAMRA™ dye can be used without affecting the
accuracy of the reading.
• Thermal cycling program:
Stage Repetitions Temperature Time
1 1 50°C 2 minutes
2 1 95°C 10 minutes
3 45 95°C 15 seconds
2.
Run the thermal cycler pr
amplication data during stage 3.
60°C 1 minute
ogram, collecting real-time
quencher (NFQ)
[1]
Positive control 4a – EPC ASFV 5.0 µL
Extraction control Mock-purified sample 5.0 µL
No-template
ol (NTC)
contr
3.
Seal each plate or tube, mix, then centrifuge briey to bring
Nuclease-free Water 5.0 µL
the contents to the bottom of the plate wells or tubes.
2 V
etMAX™ African Swine Fever Virus Detection Kit Instructions for Use
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