PN: PLM-0014-2
Tetracore, Inc.
Classical Swine Fever Virus (CSFV)
Diagnostic Reagents
Diagnostic reagents for the qualitative detection of Classical Swine Fever Virus RNA
extracted from cell culture supernatant or porcine tissues and fluid.
For Research Use Only
Name and intended use
The diagnostic reagents are intended for the rapid in vitro qualitative detection of
Classical Swine Fever (CSF) viral RNA by reverse transcriptase polymerase chain
reaction (RT-PCR). The reagents should be used to detect viral RNA extracted from
swine serum or tissue samples and may be used for screening of viral culture
supernatants. The test is intended for research use by veterinary or other laboratory
scientists for the presumptive identification of CSF. The tests should be performed on the
Cepheid, Smart Cycler (version I or II) instruments.
Summary and Explanation
Classical Swine Fever (CSF) is a highly contagious and often fatal disease of swine,
affecting domestic and wild pig populations. CSF Virus (CSFV), the causative agent of
CSF, is a member of the genus Pestivirus belonging to the Flaviviridae family. Other
important animal pathogens within the genus Pestivirus are Bovine Viral Diarrhea Virus
(BVDV) of cattle and Border Disease Virus (BDV) of sheep, both of which can naturally
infect pigs. Antibodies generated during these infections cross-react with CSFV in
serologic assays making CSF diagnosis problematic. (Risatti et al, 2003)
CSFV is an enveloped virus with a 12.5 Kb single stranded RNA genome of positive
polarity. The genome comprises a single open reading frame encoding for a 4000 amino
acid polyprotein that is co- and post-translationally processed by viral and cellular
proteases into 12 polypeptides. Both ends of the genome contain untranslated regions,
which are highly conserved among all virus isolates. (Risatti et al, 2003)
Clinical signs of CSF can remain undetected particularly during infections with CSFV
strains of low virulence. Moreover, gross lesions observed at necropsy are diverse and
often not pathognomonic. Rapid and precise detection of CSFV is critical for disease
containment. Current diagnostic methods including detection of viral antigen in tonsils
using fluorescent antibodies (FAT), or antigen capture enzyme linked immunosorbant
assay (ELISA), and detection of genomic RNA by reverse-transcription polymerase chain
reaction (RT-PCR) are relatively rapid diagnostic tests; however, these techniques require
centralized laboratory facilities, and clinical specimen submissions that might delay
disease diagnosis, thus affecting the efficiency of emergency disease management
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PN: PLM-0014-2
measures. (Risatti et al, 2003). The Tetracore CSF RT-PCR test was designed in a realtime format to overcome the limitations of classical methods of identification including
virus isolation and gel-based RT-PCR methods.
1. Risatti GR, Callahan JD, Nelson WM, Borca MV. Rapid detection of classical
swine fever virus by a portable real-time reverse transcriptase PCR assay. J Clin
Microbiol. 2003 Jan;41(1):500-5.
Principles of the procedure
The assay to detect CSF viral RNA is a single tube assay method that is based on
fluorogenic probe hydrolysis (TaqMan®) chemistry. The assay has a specific set of
oligonucleotide primers and a FAM/TAMRA probe for the detection of CSF viral RNA.
Product Description
Materials provided
Component
Smart Cycler Tube with Dried (CSF) RTPCR Assay
RT-PCR Rehydration Buffer
Part # SUB-0005-1
Synthetic Positive Control
Part # BLM-0003-1
1X – TE
Part # SUB-0004-1
Contents
RT-PCR diagnostic reagents
Buffer and dH2O
Double stranded oligonucleotide
Tris EDTA
Materials and Equipment Required, but not provided
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•
•
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RNA extraction materials
Cepheid, Smart Cycler, Version I or II.
Micropipettes with tips
Microfuge with adaptor for Smart Cycler tubes
Storage and Stability
The dried reagents and 1X-TE should be stored between (15ºC to 30ºC). The rehydration
buffer should be stored at (-15ºC to -25ºC), the synthetic positive control should be stored
at (2ºC to 8ºC). Stability studies of this product are ongoing and no claims are made at
this time.
PRECAUTIONS
Analysis of specimens suspected of containing foreign animal disease should be handled
by representatives of the USDA and only at designated facilities.
Limitations
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For Research Use Only.
This formulation is specially prepared for use on the Cepheid, Smart Cycler, Version I or
II. Formulations for other instruments are available under a separate catalogue number.
Please contact Tetracore, Inc. for further information (301) 258-7553.
Specimen Collection and Storage
Specimens should be processed quickly and maintained as cold as possible during
processing to prevent loss of any RNA that might be present. RNA must be extracted
using standard methods before testing.
Procedure
1.
2.
3.
4.
5.
Remove desired number of tubes from pouch and place into racks.
Add 22.5ul of rehydration buffer to each tube.
Add 2.5ul of extracted material or control to each tube.
Cap each tube until the snap is heard.
Briefly centrifuge each tube in the special microfuge that accommodates the
Cepheid, Smart Cycler tubes.
6. Pinch each tube 8-10 times to assure that the material is well mixed, as you pinch
the tube you will note the rise and fall of the meniscus of liquid within the tube.
7. Place into the Cephied Smart Cycler.
Cycling Conditions
1. RT step: 10 minutes @ 60ºC
2. 2-step PCR: (95ºC x 2 seconds, 60ºC x 30 seconds) for 55 cycles
Smart Cycler Settings
Smart Cycler I - FTTR25-Dye Set, 25µL reaction with channel 1 as FAM dye,
channel 2 as Tet dye, channel 3 as Tamra dye, channel 4 as Rox dye
Smart Cycler II - FTTC25-Dye Set, 25µL reaction with channel 1 as FAM dye,
channel 2 as Tet dye, channel 3 as Texas Red (TxR) dye, channel 4 as
Cy5 dye
Controls
One positive control (PC) and one “no template” control (NTC) should be included with
each run. For a NTC, we use 1X-TE. If a different positive control other than the kit
control is used, it is recommended that the new control be run in parallel with the kit
control to assure that the reagents perform as specified.
Performance Characteristics
No claims are made
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Interpretation
The ideal shape of a positive test is visualized on a linear scale as a sigmoidal
curve with two inflection points on a two-dimentional linear grid where the Xaxis represents the PCR cycle number and the Y-axis represents the relative
fluorescence of the signal. The inflection point is defined as the area of the curve
with the sharpest rate of change (see Fig. 1).
For a sample to be considered positive, a signal must cross the threshold level by
at least cycle 45. The positive signal must have at least one positive inflection
point that curves upward as visualized on a linear scale on either the Cepheid
Smart Cycler or ABI 7700 platforms. For a run to be valid the negative control
must remain negative until the assay endpoint at cycle 45. A positive control is
provided as an assay reference to assess PCR cyling performance.
The CSF positive control should cross the threshold at cycle (Ct) 20-25 and
should have a minimum fluorescence value of 400 on the Cepheid Smart Cycler.
The negative control should remain negative (not cross the threshold) through
cycle 45.
Figure 1. Typical positive and negative curves generated by the test on Cepheid
Smart Cycler instrument. Below two curves can be seen, each with two inflection
points. The ideal curve has a sigmoidal shape on a linear graph, and has one
positive and one negative inflection point. The red vertical lines indicate the cycle
number at which the curve crossed the positive threshold (default setting is 30).
The two overlapping flat lines are negative signals that did not cross the positive
threshold.
Ct value
Where a positive curve
crosses the threshold
Positive
inflection points
Negative
inflection
points
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