eiav

eiav
Techne ® qPCR test
Equine infectious anemia
virus
Polymerase (pol) gene
150 tests
For general laboratory and research use only
Quantification of Equine infectious anemia virus genomes
Advanced kit handbook HB10.01.08
1
Introduction to Equine infectious anemia virus
Equine infectious anaemia virus (EIAV) is a retrovirus of the lentivirus subfamily.
Compared to the other lentiviruses the EIAV genome is the smallest, with a size of
approximately 8.2 kb and genetically the simplest. They cause Equine infectious anaemia
or Swamp fever in all members of equidae, including horses, mules and donkeys.
The EIA virus can be transmitted by blood sucking insect vectors like horse flies, deer flies
(Tabanids), stable flies (Stomoxys spp) and mosquitoes. The viruses can also cross the
placental barrier to cause foetal infections. EIA viruses localize and multiply in
macrophages of many organs, especially in spleen, liver, kidney and lymph nodes. On
activation these viruses start reproducing eventually lysing the host cell, allowing spread
and further infection of other cells. This causes recurring cycles, producing intermittent
attacks of illness followed by periods when the horse is free of any symptoms and appears
normal.
The incubation period of the virus can be from a week to 45 days. The Clinical signs of
Equine infectious anaemia includes fever, loss of appetite, frequent urination, weight loss,
diarrhea, yellowish discoloration of the conjunctiva, , progressive weakness,
haemorrhaging and also abortion of pregnant mares.
Quantification of Equine infectious anemia virus genomes
Advanced kit handbook HB10.01.08
2
Specificity
The Techne® qPCR Kit for Equine infectious anemia virus (EIAV) genomes is designed for
the in vitro quantification of EIAV genomes. The kit is designed to have the broadest
detection profile possible whilst remaining specific to the EIAV genome.
The primers and probe sequences in this kit have 100% homology with a broad range of
EIAV sequences based on a comprehensive bioinformatics analysis.
If you require further information, or have a specific question about the detection profile of
this kit then please send an e.mail to technehelp@bibby-scientific.com and our
bioinformatics team will answer your question.
Quantification of Equine infectious anemia virus genomes
Advanced kit handbook HB10.01.08
3
Kit Contents
• EIAV specific primer/probe mix (150 reactions BROWN)
FAM labelled
• EIAV positive control template (for Standard curve RED)
• Internal extraction control primer/probe mix (150 reactions BROWN)
VIC labelled as standard
• Internal extraction control RNA (150 reactions BLUE)
• Endogenous control primer/probe mix (150 reactions BROWN)
FAM labelled
• EIAV/Internal extraction control/endogenous control RT primer mix (150 reactions
GREEN)
Required for two step protocol only
• RNAse/DNAse free water (WHITE)
for resuspension of primer/probe mixes and internal extraction control RNA
• Template preparation buffer (YELLOW)
for resuspension of positive control template and standard curve preparation
Reagents and equipment to be supplied by the
user
Real-Time PCR Instrument
RNA extraction kit
This kit is rdesigned to work well with all processes that yield high quality RNA
with minimal PCR inhibitors.
Lyophlised OneStep 2x qRT-PCR MasterMix
Contains complete one step qRT-PCR MasterMix
Pipettors and Tips
Vortex and centrifuge
1.5 ml tubes
Quantification of Equine infectious anemia virus genomes
Advanced kit handbook HB10.01.08
4
Kit storage and stability
This kit is stable at room temperature but should be stored at -20ºC on arrival. Techne
does not recommend using the kit after the expiry date stated on the pack. Once the
lyophilized components have been re-suspended, unnecessary repeated freeze/thawing
should be avoided. The kit is stable for six months from the date of resuspension under
these circumstances.
If a standard curve dilution series is prepared this can be stored frozen for an extended
period. If you see any degradation in this serial dilution a fresh standard curve can be
prepared from the positive control.
Suitable sample material
All kinds of sample material suited for PCR amplification can be used. Please ensure the
samples are suitable in terms of purity, concentration, and RNA/DNA integrity (An internal
PCR control is supplied to test for non specific PCR inhibitors). Always run at least one
negative control with the samples. To prepare a negative-control, replace the template
RNA sample with RNAse/DNAse free water.
Dynamic range of test
Under optimal PCR conditions Techne EIAV detection kits have very high priming
efficiencies of >95% and can detect less than 100 copies of target template.
Notices and disclaimers
This product is developed, designed and sold for research purposes only. It is not intended for human diagnostic or
drug purposes or to be administered to humans unless clearly expressed for that purpose by the Food and Drug
Administration in the USA or the appropriate regulatory authorities in the country of use. During the warranty period
Techne® Prime Pro detection kits detection kits allow precise and reproducible data recovery combined with excellent
sensitivity. For data obtained by violation to the general GLP guidelines and the manufacturer’s recommendations the
right to claim under guarantee is expired. PCR is a proprietary technology covered by several US and foreign patents.
These patents are owned by Roche Molecular Systems Inc. and have been sub-licensed by PE Corporation in certain
fields. Depending on your specific application you may need a license from Roche or PE to practice PCR. Additional
information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of
Licensing at Roche Molecular Systems, 1145 Atlantic Avenue, Alameda, CA 94501 or Applied Biosystems business
group of the Applera Corporation, 850 Lincoln Centre Drive, Foster City, CA 94404. In addition, the 5' nuclease assay
and other homogeneous amplification methods used in connection with the PCR process may be covered by U.S.
Patents 5,210,015 and 5,487,972, owned by Roche Molecular Systems, Inc, and by U.S. Patent 5,538,848, owned by The
Perkin-Elmer Corporation.
Trademarks
Techne™ is a trademark of Bibby Scientific Ltd.
The PCR process is covered by US Patents 4,683,195, and 4,683,202 and foreign equivalents owned by Hoffmann-La
Roche AG. BI, ABI PRISM® GeneAmp® and MicroAmp® are registered trademarks of the Applera Genomics (Applied
Biosystems Corporation). BIOMEK® is a registered trademark of Beckman Instruments, Inc.; iCycler™ is a registered
trademark of Bio-Rad Laboratories, Rotor-Gene is a trademark of Corbett Research. LightCycler™ is a registered
trademark of the Idaho Technology Inc. GeneAmp®, TaqMan® and AmpliTaqGold® are registered trademarks of Roche
Molecular Systems, Inc., The purchase of the Techne® Prime Pro reagents ™ reagents cannot be construed as an
authorization or implicit license to practice PCR under any patents held by Hoffmann-LaRoche Inc.
Quantification of Equine infectious anemia virus genomes
Advanced kit handbook HB10.01.08
5
Principles of the test
Real-time PCR
A EIAV specific primer and probe mix is provided and this can be detected through the
FAM channel.
The primer and probe mix provided exploits the so-called TaqMan® principle. During PCR
amplification, forward and reverse primers hybridize to the EIAV cDNA. A fluorogenic
probe is included in the same reaction mixture which consists of a DNA probe labeled with
a 5`-dye and a 3`-quencher. During PCR amplification, the probe is cleaved and the
reporter dye and quencher are separated. The resulting increase in fluorescence can be
detected on a range of real-time PCR platforms.
Positive control
For copy number determination and as a positive control for the PCR set up, the kit
contains a positive control template.
This can be used to generate a standard curve of EIAV copy number / CT value.
Alternatively the positive control can be used at a single dilution where full quantitative
analysis of the samples is not required. Each time the kit is used, at least one positive
control reaction must be included in the run. A positive result indicates that the primers
and probes for detecting the target EIAV gene worked properly in that particular
experimental scenario. If a negative result is obtained the test results are invalid and must
be repeated. Care should be taken to ensure that the positive control does not
contaminate any other kit component which would lead to false-positive results. This can
be achieved by handling this component in a Post PCR environment. Care should also be
taken to avoid cross-contamination of other samples when adding the positive control to
the run. This can be avoided by sealing all other samples and negative controls before
pipetting the positive control into the positive control well.
Negative control
To validate any positive findings a negative control reaction should be included every time
the kit is used. For this reaction the RNAse/DNAse free water should be used instead of
template. A negative result indicates that the reagents have not become contaminated
while setting up the run. If a positive result is obtained the results should be ignored and
the test samples repeated. Possible sources of contamination should first be explored and
removed.
Internal RNA extraction control
When performing RNA extraction, it is often advantageous to have an exogenous source
of RNA template that is spiked into the lysis buffer. This control RNA is then co-purified
with the sample RNA and can be detected as a positive control for the extraction process.
Successful co-purification and real-time PCR for the control RNA also indicates that PCR
inhibitors are not present at a high concentration.
A separate RT primer mix and a real-time PCR primer/probe mix are supplied with this kit
to detect the exogenous RNA using real-time PCR. The PCR primers are present at PCR
limiting concentrations which allows multiplexing with the target sequence primers.
Amplification of the control cDNA does not interfere with detection of the EIAV target
cDNA even when present at low copy number. The Internal control is detected through the
VIC channel and gives a CT value of 28+/-3 depending on the level of sample dilution.
Endogenous control
To confirm extraction of a valid biological template, a primer and probe mix is included to
detect an endogenous gene. Detection of the endogenous control is through the FAM
channel and it is NOT therefore possible to perform a multiplex with the EIAV primers. A
poor endogenous control signal may indicate that the sample did not contain sufficient
biological material.
Carry-over prevention using UNG (unsuitable for onestep procedure and optional for
two step procedure)
Carry over contamination between PCR reactions can be prevented by including uracil-NQuantification of Equine infectious anemia virus genomes
Advanced kit handbook HB10.01.08
6
glycosylase (UNG) in the reaction mix. Some commercial mastermix preparations contain
UNG or alternatively it can be added as a separate component. UNG can only prevent
carry over from PCR reactions that include deoxyuridine triphosphate (dUTP) in the
original PCR reaction. Techne recommend the application of 0.2U UNG per assay with a
15 minute incubation step at 37°C prior to amplification. The heat-labile UNG is then
inactivated during the Taq polymerase activation step.
Quantification of Equine infectious anemia virus genomes
Advanced kit handbook HB10.01.08
7
Reconstitution Protocol
To minimize the risk of contamination with foreign DNA, we recommend that all
pipetting be performed in a PCR clean environment. Ideally this would be a
designated PCR lab or PCR cabinet. Filter tips are recommended for all pipetting
steps.
1.
Pulse-spin each tube in a centrifuge before opening.
This will ensure lyophilised primer and probe mix is in the base of the tube and
is not spilt upon opening the tube.
2.
Reconstitute the kit components in the RNase/DNase free water supplied,
according to the table below:
To ensure complete resuspension, vortex each tube thoroughly.
Component - resuspend in water
Pre-PCR pack
Volume
EIAV primer/probe mix (BROWN)
165 µl
Internal extraction control primer/probe mix (BROWN)
165 µl
RT primer mix (GREEN)
165 µl
Endogenous control primer/probe mix (BROWN)
165 µl
Pre-PCR heat-sealed foil
600 µl
Internal extraction control RNA (BLUE)
3.
Reconstitute the positive control template in the template preparation
buffer supplied, according to the table below:
To ensure complete resuspension, vortex this tube thoroughly.
Component - resuspend in template preparation buffer
Post-PCR heat-sealed foil
Positive Control Template (RED) *
Volume
500 µl
* This component contains high copy number template and is a VERY significant contamination
risk. It must be opened and handled in a separate laboratory environment, away from the other
components.
RNA extraction
The internal extraction control RNA can be added either to the RNA lysis/extraction
buffer or to the RNA sample once it has been resuspended in lysis buffer.
DO NOT add the internal extraction control RNA directly to the unprocessed biological
sample as this will lead to degradation and a loss in signal.
1.
Add 4µl of the Internal extraction control RNA (BLUE) to each sample in RNA
lysis/extraction buffer per sample.
2.
Complete RNA extraction according to the manufacturers protocols.
Quantification of Equine infectious anemia virus genomes
Advanced kit handbook HB10.01.08
8
One Step RT-PCR detection protocol
A one step approach combining the reverse transcription and amplification in a
single closed tube is the preferred method.
For optimum performance and sensitivity.
All pipetting steps and experimental plate set up should be performed on ice. After
the plate is poured proceed immediately to the One Step amplification protocol.
Prolonged incubation of reaction mixes at room temperature can lead to PCR
artifacts that reduce the sensitivity of detection.
1.
For each RNA sample prepare a reaction mix according to the table below:
Include sufficient reactions for positive and negative controls.
Component
OneStep 2x qRT-PCR MasterMix
10 µl
EIAV primer/probe mix (BROWN)
1 µl
Internal extraction control primer/probe mix (BROWN)
1 µl
RNAse/DNAse free water (WHITE)
3 µl
Final Volume
2.
Volume
15 µl
For each RNA sample prepare an endogenous control reaction according to
the table below (optional):
This control reaction will provide crucial information regarding the quality of
the biological sample.
Component
OneStep 2x qRT-PCR MasterMix
Endogenous control primer/probe mix (BROWN)
RNAse/DNAse free water (WHITE)
Final Volume
Volume
10 µl
1 µl
4 µl
15 µl
3.
Pipette 15µl of these mixes into each well according to your real-time PCR
experimental plate set up.
4.
Pipette 5µl of RNA template into each well, according to your experimental
plate set up.
For negative control wells use 5µl of RNAse/DNAse free water. The final volume in
each well is 20µl.
Quantification of Equine infectious anemia virus genomes
Advanced kit handbook HB10.01.08
9
5.
If a standard curve is included for quantitative analysis prepare a
reaction mix according to the table below:
Component
Volume
OneStep 2x qRT-PCR MasterMix
10 µl
EIAV primer/probe mix (BROWN)
1 µl
4 µl
15 µl
RNAse/DNAse free water (WHITE)
Final Volume
6.
Preparation of standard curve dilution series.
1) Pipette 90µl of template preparation buffer into 5 tubes and label 2-6
2) Pipette 10µl of Positive Control Template (RED) into tube 2
3) Vortex thoroughly
4) Change pipette tip and pipette 10 µl from tube 2 into tube 3
5) Vortex thoroughly
Repeat steps 4 and 5 to complete the dilution series
Standard Curve
Tube 1 Positive control (RED)
Tube 2
2 x 105 per µl
2 x 104 per µl
2 x 103 per µl
2 x 102 per µl
20 per µl
2 per µl
Tube 3
Tube 4
Tube 5
Tube 6
7.
Copy Number
Pipette 5µl of standard template into each well for the standard curve according
to your plate set-up
The final volume in each well is 20µl.
Quantification of Equine infectious anemia virus genomes
Advanced kit handbook HB10.01.08
10
One Step Amplification Protocol
Amplification conditions using OneStep 2x RT-qPCR MasterMix.
Step
Time
Temp
10 mins
42 oC
Enzyme activation
2 mins
95 oC
Denaturation
10 secs
95 oC
DATA COLLECTION *
60 secs
60 oC
Reverse Transcription
50 Cycles
* Fluorogenic data should be collected during this step through the FAM and VIC channels
Quantification of Equine infectious anemia virus genomes
Advanced kit handbook HB10.01.08
11
Interpretation of Results
Internal
control
Negative
control
Positive
control
+ive
+ive
-ive
+ive
+ive
+ive
-ive
-ive
+ive
+ive
-ive
+ive
-ive
+ive
-ive
-ive
-ive
-ive
-ive
Experiment fail
+ive
+ive
+ive
+ive
Experiment fail
Target
Interpretation
Internal PCR control
The CT value obtained with the internal control will vary significantly depending on the
extraction efficiency, the quantity of RNA added to the RT and PCR reaction and the
individual machine settings. CT values of 28±3 are within the normal range. When
amplifying a EIAV sample with a high genome copy number, the internal extraction control
may not produce an amplification plot. This does not invalidate the test and should be
interpreted as a positive experimental result.
Endogenous control
The signal obtained from the endogenous control primer and probe set will vary according
to the amount of biological material present in a given sample. An early signal indicates
the presence of a good yield of biological material. A late signal suggests that little
biological material is present in the sample.
Quantification of Equine infectious anemia virus genomes
Advanced kit handbook HB10.01.08
12
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