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VET-22 - Canine distemper virus
| Product number |
VET-22 |
| Product name |
Canine distemper virus |
| Quantity |
50 |
| Supplier |
Gentaur
|
Canine distemper virus - 50
REF VET-22
VER 21.04.04
CDV Vet
Key to symbols used
REF List Number
Store at 2-8°C
VET Veterinary Test
Caution!
LOT Lot Number
VER Version
Expiration Date
Consult instructions for use
Contains reagents
Manufacturer
NAME
CDV Vet
INTENDED USE
CDV Vet is an in vitro veterinary nucleic acid amplification test for qualitative detection of ?anine
Distemper Virus RNA in clinical specimens.
PRINCIPLE OF ASSAY
CDV Vet Test is based on four major processes: isolation of ?anine Distemper Virus RNA from
specimens, reverse transcription of the RNA, amplification of the cDNA of ?anine Distemper Virus,
detection of the amplified products on agarose gel.
MATERIALS PROVIDED
Part N° 1 – “Ribo-Sorb”: isolation of RNA from clinical specimens;
Part N° 2 – “Reverta-R”: reverse transcription of the RNA
Part N° 3 – “CDV Vet”: amplification kit;
Part N° 1 – “Ribo-Sorb”:
• Lysis Solution, 22,5 ml;
• Washing Solution, 20 ml;
• Sorbent, 1,25 ml.
• RNA-eluent, 5 x 0,5ml;
• Negative Control, 1,6 ml;
Contains reagents for 50 test.
Part N° 2 – “Reverta-R”:
• DTT (lyophilized), 5 tubes;
• RT-mix, 5 x 0,125 ml;
• Reverse transcriptase (M-MLV), 0,03 ml.
• TE-buffer, 1,0 ml.
Contains reagents for 60 tests
Part N° 3 – “CDV Vet”:
• PCR-mix-1 55 ready-to-use single-dose test tubes;
• PCR-mix-2, 0,6 ml;
• Mineral Oil, 2,0 ml;
• Positive Control cDNA C+, 0,2 ml;
• RNA Positive Control, 0,1 ml;
• Internal Control, 2 x 0,125 ml.
• DNA buffer, 0,5 ml;
Contains reagents for 55 tests.
MATERIALS REQUIRED BUT NOT PROVIDED
Zone 1: sample preparation:
• Biological cabinet
• Desktop microcentrifuge for “eppendorf” type tubes (RCF max. 16,000 x g); Eppendorf 5415D or
equivalent
• 60°C ± 2°C dry heat block
• Vortex mixer
• Pipettors (capacity 5-40 µl; 40-200 µl; 200-1000 µl) with aerosol barrier
• 1,5 ml polypropylene sterile tubes (Sarstedt, QSP, Eppendorf)
• 70% Ethanol (freshly prepared mixture of reagent grade 96% ethanol and distilled water)
• Acetone
Zone 2: RT and amplification:
• Thermalcycler
• Workstation
• Pipettors (capacity 0,5-10 µl; 5-40 µl) with aerosol barrier
• Tube racks
Reagents non provided
• Detection agarose kit (Precast gel 2% REF G1-1)
WARNINGS AND PRECAUTIONS
1. Lysis Solution contains guanidine thiocyanate. Guanidine thiocyanate is harmful if inhaled, or
comes into contact with skin or if swallowed. Contact with acid releases toxic gas. (Xn; R: 20/21/22-
36/37/38; S: 36/37/39).
2. Some components of this kit contain Sodium Azide as a preservative. Do not use metal tubing
for reagent transfer.
3. Wear disposable gloves, laboratory coats and eye protection when handling specimens and reagents.
Thoroughly wash hands afterward.
4. Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in laboratory work areas.
5. Do not use a kit after its expiration date.
6. Dispose of all specimens and unused reagents in accordance with local regulation.
7. Biosafety Level 2 should be used for materials that contain or are suspected of containing infectious
agents.
8. Clean and disinfect all spills of specimens or reagents using a disinfectant such as 0,5% sodium
hypochlorite, or other suitable disinfectant.
9. Avoid contact of specimens and reagents with the skin, eyes and mucous membranes. If these
solutions come into contact, rinse immediately with water and seek medical advice immediately.
10. Material Safety Data Sheets (MSDS) are available on request.
11. This kit is designed for use with “Ribo-Sorb” extraction kit. It is the user’s responsibility if kits other
than “Ribo-Sorb” are used to perform this RNA extraction.
12. Use of this product should be limited to personnel trained in the techniques of amplification.
13. Workflow in the laboratory must proceed in a uni-directional manner, beginning in the Extraction
Area and moving to the Amplification and Detection Area. Do not return samples, equipment and
reagents in the area where you performed previous step.
STORAGE INSTRUCTIONS
CDV Vet must be stored at 2-8°C. Part N° 2 – “Reverta-R” must be stored at -20°C.
STABILITY
CDV Vet is stable up to the expiration date indicated on the kit label.
SAMPLE COLLECTION, STORAGE AND TRANSPORT
CDV Vet can analyze RNA extracted with Ribo-Sorb (REF K-2-1) from:
• mucosal swabs (conjuintival, nasal, rectal): swab area and place in “Eppendorf” tube with 0,5 ml of
saline water or PBS sterile. Agitate vigorously. Repeat the swab and agitate in the same tube.
Centrifuge at 10000g/min for 10 min. Remove and discard the supernatant. Resuspend the pellet in
100 µl of Saline water.
• serum: collected blood in Serum Separator tubes;
• feces: prepare required quantity of 1,5 ml polypropylene tubes with 1,0 ml of Saline Solution. Add to
each tube 0,1 g of feces. Vortex vigorously to get a homogeneous suspension.. Centrifuge for 5 min
at 7-12000g and use the supernatant for the extraction of the RNA.
• nasal and conjunctival lavages: centrifuge at 2000 g/min for 10-15 min. If the pellet is not visible add
10 ml of liquid and repeat centrifugation. Remove and discard the supernatant. Resuspend the pellet
in 100 µl of Saline water.
Specimens can be stored at +2-8°C for no longer than 12 hours, or frozen at -20°C to -80°C.
Transportation of clinical specimens must comply with country, federal, state and local regulations for the
transport of etiologic agents.
SPECIMEN AND REAGENT PREPARATION
1. Lysis Solution and Washing Solution (in case of their storage at +2-8??) should be warmed up to
60–65 ?? until disappearance of ice crystals. Prepare required quantity of 1.5 ml polypropylene tubes
including one tube for Negative Control of Extraction and one tube for Positive Control of
Extraction .
2. Add to each tube 5 µl of Internal Control and 450 µl Lysis Solution.
3. Add 100 µl of serum (for samples like swabs, lavages and feces add 50 µl of C– Negative Control
and 50 µl of sample) to the appropriate tube containing Lysis Solution and Internal Control.
4. Prepare Controls as follows:
• add 100 µl of C– Negative Control to each of the two control tubes.
• add 10 µl of C+ RNA Positive Control to the tube labeled Cpos.
5. Vortex the tubes and centrifuge for 5 sec at 5000g..
6. Vortex vigorously Sorbent and add 25 µl to each tube.
7. Vortex for 5-7 sec and incubate all tubes for 10 min at room temperature. Vortex periodically.
8. Centrifuge all tubes for 30 sec at 10000g and using a micropipette with a plugged aerosol barrier tip,
carefully remove and discard supernatant from each tube without disturbing the pellet. Change tips
between tubes.
9. Add 400 µl of Washing Solution to each tube. Vortex vigorously, centrifuge for 30 sec at 10000g.
and using a micropipette with a plugged aerosol barrier tip, carefully remove and discard supernatant
from each tube without disturbing the pellet. Change tips between tubes.
10. Add 500 µl of Etanolo al 70% to each tube. Vortex vigorously, centrifuge for 30 sec at 10000g. and
using a micropipette with a plugged aerosol barrier tip, carefully remove and discard supernatant
from each tube without disturbing the pellet. Change tips between tubes.
11. Repeat step 10.
12. Add 400 µl of Acetone to each tube. Vortex vigorously , centrifuge for 30 sec at 10000g and using a
micropipette with a plugged aerosol barrier tip, carefully remove and discard supernatant from each
tube without disturbing the pellet. Change tips between tubes.
13. Incubate all tubes with open cap for 10 min at 60°C.
14. Resuspend the pellet in 50 µl of RNA-eluent. Incubate for 10 min at 60°C and vortex periodically.
The supernatant contains RNA ready for use. The RT-PCR can be performed the same day. If this is not
possible, the RNA preparations can be stored at -80°C for up to one month.
RT AND AMPLIFICATION
Reverse Transcription:
1) Prepare Reagent Mix: for 12 reactions, add 125 µl RT-mix into the tube containing lyophilized
DTT and vortex for at least 5-10 seconds.
2) Add 6 µl M-MLV into the tube with Reagent Mix, vortex for 3 sec, centrifuge for 5-7 sec and
dispense 10 µl to each tube.
3) Pipette 10 µl RNA samples to the appropriate tube. (ATTENTION: The sorbent must not be
present in the tube).Carefully mix, using the pipette.
4) Place tubes into Thermocycler and incubate at 37 o? for 30 minutes.
5) Dilute 1: 2 each obtained cDNA sample with TE-buffer (add 20 µl TE-buffer to each tube).
cDNA specimens could be stored at -20 o? for a week or at -70o? during a year
Amplification:
1) Prepare required quantity of tubes PCR-mix-1, including two additional tubes - one for Negative
Control of Amplification and one for Positive Control of Amplification.
2) Add to the each tube 10 µl PCR-mix-2 and a drop of Mineral oil (approximately 25 µl).
3) Add to appropriate tube 10 µl cDNA sample obtained after reverse transcription step.
4) Add 10 µl DNA-buffer to the tube for Negative Control of Amplification.
5) Add 10 µl Positive Control cDNA to the tube for Positive Control of Amplification.
6) Close PCR-mix-1 tubes and transfer them into the Thermalcycler only when temperature reaches
95°C and start the following program:
RESULTS ANALYSIS
Analysis of results is based on the presence or absence of specific bands of amplified DNA in Agarose
gel (2%). The length of specific amplified DNA fragments is:
• ?anine Distemper Virus – 270 bp
• Internal Control – 670 bp.
RESULTS INTERPRETATION
Table 2. Results for controls
Control Which step of
test is controlled
Specific bands
in the gel 270 bp
Specific bands
in the gel 670 bp
Interpretation
NCS RNA isolation No Yes Valid result
PCS RNA isolation Yes Yes Valid result
DNA-buffer Amplification No No Valid result
cDNA Pos Amplification Yes No Valid result
• Ensure that the Negative Controls (NCS and DNA-buffer) values for the run are valid. If the run is
invalid (contamination – detection of the band of 270 bp or defective extraction/amplification – absence
of the band of 670 bp corresponding to Internal Control), the entire test protocol (sample preparation,
amplification and detection) should be repeated. Discard any reagents that may be suspect.
• The sample is considered to be positive for ?anine Distemper Virus RNA if the band of 270 bp is
observed on agarose gel.
Sacace Biotechnologies Srl
18 San Carlo str. 81100 Caserta, Italy
*PCR: The Polymerase Chain Reaction (PCR) process is covered by patents owned by Hoffmann-La Roche and applicable in certain countries. Sacace does not encourage or support the
unauthorized or unlicensed use of the PCR process. Use of this kit is recommended for persons that either have a license to perform PCR or are not required to obtain a license.
Thermocyclers with block
temperature adjustment:
“PTC-100”(MJ Research )
BioRad , Biometra
Thermocyclers with active
temperature adjustment.
“ PE 2400” (Perkin Elmer),
Omn-E (Hibaid) and other.
Step t°C Time Cycles t°C Time Cycles
1 95 ?? Pause 95?? Pause
2 95 ?? 2 min 1 95?? 2 min 1
95 ?? 1 min 95?? 10 sec
65 ?? 1 min 65?? 10 sec
3
72 ?? 1 min
42
72 ?? 10 sec
42
4 72 ?? 1 min 1 72?? 1 min 1
5 10 ?? Storage 10?? Storage |
| EUR | 427 | | USD | 534 | | GBP | 342 | | DKK | 3.182 | | JPY | 70.265 | | PLZ | 1.456 | | SEK | 3.991 | | NOK | 3.389 |
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