b dendrobatidis

Techne ® qPCR test
Batrachochytrium
dendrobatidis
5.8S ribosomal RNA (5.8S)
150 tests
For general laboratory and research use only
Quantification of Batrachochytrium dendrobatidis genomes.
Advanced kit handbook HB10.03.07
1
Introduction to Batrachochytrium dendrobatidis
Batrachochytrium dendrobatidis (B. dendrobatidis) is a fungus responsible for causing
chytridiomycosis in amphibians. The disease has devastated amphibian populations
around the world, in a global decline towards multiple extinctions. The DNA genome is 23.7
Mb in size and it is estimated to consist of approximately 8,800 genes. B. dendrobatidis
exists as a reproductive sporangium and a motile, uniflagellated zoospore released from
the zoosporangium. Zoospores of B. dendrobatidis are typically 3-5 µm in size, have an
elongate–ovoidal body with a single, posterior flagellum 19-20 µm long.
Chytridiomycosis has been linked to dramatic population declines or even extinctions of
amphibian species all over the world and the fungus is responsible for a 100% mortality in
some amphibian populations. Zoospores first encounter amphibian skin and give rise to
sporangia, which produce new zoospores. The disease then progresses as these new
zoospores re-infect the host. The zoospores are capable of chemotaxis, and can move
towards a variety of molecules that are present on the amphibian surface, such as sugars,
proteins and amino acids. B. dendrobatidis also contains a variety of proteolytic enzymes
and esterases that help it digest amphibian cells and use amphibian skin as a nutrient
source. Once the zoospore reaches its host, it forms a cyst underneath the surface of the
skin, and initiates the reproductive portion of its life cycle. The encysted zoospores develop
into zoosporangia, which may produce more zoospores that can reinfect the host, or be
released into the surrounding aquatic environment. The amphibians infected with these
zoospores are shown to die from cardiac arrest.
Morphological changes in amphibians infected with the fungus include a reddening of the
ventral skin, convulsions with extension of hind limbs, accumulations of sloughed skin over
the body, sloughing of the superficial epidermis of the feet and other areas, slight
roughening of the surface with minute skin tags, and occasional small ulcers or
haemorrhage. Behavioural changes can include lethargy, a failure to seek shelter, a failure
to flee, a loss of righting reflex, and abnormal posture (e.g. sitting with the hind legs away
from the body). B. dendrobatidis can grow within temperature range of 4-25°C. The ability
to survive at low temperatures, gives the fungus the ability to overwinter in its hosts, even
in aquatic environments.
Quantification of Batrachochytrium dendrobatidis genomes.
Advanced kit handbook HB10.03.07
2
Specificity
MAX MIN
The Techne qPCR Kit for Batrachochytrium dendrobatidis (B.dendrobatidis) genomes is
designed for the in vitro quantification of B.dendrobatidis genomes. The kit is designed to
have the broadest detection profile possible whilst remaining specific to the B.
dendrobatidis genome.
The primers and probe sequences in this kit have 100% homology with a broad range of
B.dendrobatidis 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 Batrachochytrium dendrobatidis genomes.
Advanced kit handbook HB10.03.07
3
Kit Contents
• B.dendrobatidis specific primer/probe mix (150 reactions BROWN)
FAM labelled
• B.dendrobatidis positive control template (for Standard curve RED)
• Internal extraction control primer/probe mix (150 reactions BROWN)
VIC labelled as standard
• Internal extraction control DNA (150 reactions BLUE)
• Endogenous control primer/probe mix (150 reactions BROWN)
FAM labelled
• RNAse/DNAse free water (WHITE)
for resuspension of primer/probe mixes and internal extraction control DNA
• 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
DNA extraction kit
This kit designed to work well with all processes that yield high quality DNA with minimal
PCR inhibitors.
Lyophilised 2x qPCR Mastermix
This kit is designed to work well with all commercially available Mastermixes.
Pipettors and Tips
Vortex and centrifuge
Thin walled 1.5 ml PCR reaction tubes
Quantification of Batrachochytrium dendrobatidis genomes.
Advanced kit handbook HB10.03.07
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 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 DNA sample
with RNAse/DNAse free water.
Dynamic range of test
Under optimal PCR conditions Techne B.dendrobatidis 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® 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 cannot be construed as an authorization or implicit license to practice PCR under any patents
held by Hoffmann-LaRoche Inc.
Quantification of Batrachochytrium dendrobatidis genomes.
Advanced kit handbook HB10.03.07
5
Principles of the test
Real-time PCR
A B.dendrobatidis 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 B.dendrobatidis DNA. 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 B.
dendrobatidis 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 B.dendrobatidis 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 falsepositive 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.
Quantification of Batrachochytrium dendrobatidis genomes.
Advanced kit handbook HB10.03.07
6
Internal DNA extraction control
When performing DNA extraction, it is often advantageous to have an exogenous source
of DNA template that is spiked into the lysis buffer. This control DNA is then co-purified
with the sample DNA and can be detected as a positive control for the extraction process.
Successful co-purification and real-time PCR for the control DNA also indicates that PCR
inhibitors are not present at a high concentration.
A separate primer and probe mix are supplied with this kit to detect the exogenous DNA
using real-time PCR. The primers are present at PCR limiting concentrations which allows
multiplexing with the target sequence primers. Amplification of the control DNA does not
interfere with detection of the B.dendrobatidis target DNA even when present at low copy
number. The Internal control is detected through the VIC channel and gives a CT value of
28+/-3.
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 B.dendrobatidis
primers. A poor endogenous control signal may indicate that the sample did not contain
sufficient biological material.
Carry-over prevention using UNG (optional)
Carry over contamination between PCR reactions can be prevented by including uracil-Nglycosylase (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 Batrachochytrium dendrobatidis genomes.
Advanced kit handbook HB10.03.07
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
B.dendrobatidis primer/probe mix (BROWN)
165 µl
Internal extraction control primer/probe mix (BROWN)
165 µl
165 µl
Endogenous control primer/probe mix (BROWN)
Pre-PCR heat-sealed foil
Internal extraction control DNA (BLUE)
3.
600 µl
Reconstitute the positive control template in the template preparation
buffer supplied, according to the table below:
To ensure complete resuspension, vortex the 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.
DNA extraction
The internal extraction control DNA can be added either to the DNA lysis/extraction buffer
or to the DNA sample once it has been resuspended in lysis buffer.
DO NOT add the internal extraction control DNA 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 DNA (BLUE) to each sample in DNA
lysis/extraction buffer per sample.
2.
Complete DNA extraction according to the manufacturers protocols.
Quantification of Batrachochytrium dendrobatidis genomes.
Advanced kit handbook HB10.03.07
8
Real-time PCR detection protocol
1.
For each DNA sample prepare a reaction mix according to the table below:
Include sufficient reactions for positive and negative controls.
Component
2x qPCR MasterMix
B.dendrobatidis primer/probe mix (BROWN)
Internal extraction control primer/probe mix (BROWN)
RNAse/DNAse free water (WHITE)
Final Volume
2.
Volume
10 µl
1 µl
1 µl
3 µl
15 µl
For each DNA 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
2x qPCR MasterMix
Volume
10 µl
Endogenous control primer/probe mix (BROWN)
1 µl
RNAse/DNAse free water (WHITE)
4 µl
Final Volume
15 µl
3.
Pipette 15µl of each mix into individual wells according to your real-time PCR
experimental plate set up.
4.
Prepare sample DNA templates for each of your samples.
5.
Pipette 5µl of DNA 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.
6.
If a standard curve is included for quantitative analysis prepare a reaction mix
according to the table below:
Component
2x qPCR MasterMix
Volume
10 µl
B.dendrobatidis primer/probe mix (BROWN)
1 µl
RNAse/DNAse free water (WHITE)
4 µl
Final Volume
Quantification of Batrachochytrium dendrobatidis genomes.
Advanced kit handbook HB10.03.07
15 µl
9
7.
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 10µl from tube 2 into tube 3
5) Vortex thoroughly
Repeat steps 4 and 5 to complete the dilution series
Standard Curve
Copy Number
Tube 1 Positive control (RED)
8.
Tube 2
2 x 105 per µl
2 x 104 per µl
Tube 3
2 x 103 per µl
Tube 4
2 x 102 per µl
Tube 5
20 per µl
Tube 6
2 per µl
Pipette 5µl of standard template into each well for the standard curve according
to your experimental plate set up.
The final volume in each well is 20µl.
Amplification Protocol
Amplification conditions using Lyophilsed 2x qPCR MasterMix.
Step
50 Cycles
Time
Temp
UNG treatment (if required) **
15 mins
37 oC
Enzyme activation
2 mins
95 oC
Denaturation
10s
95 oC
DATA COLLECTION *
60s
60 oC
* Fluorogenic data for the control DNA should be collected during this step through the FAM and VIC channels
** Required if your Mastermix includes UNG to prevent PCR carryover contamination
Quantification of Batrachochytrium dendrobatidis genomes.
Advanced kit handbook HB10.03.07
10
YES NO
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
-ive
+ive
-ive
-ive or +ive
-ive or +ive
+ive
-ive
*
-ive
Experiment fail
-ive
+ive
-ive or +ive
-ive
Experiment fail
Target
Interpretation
* Where the test sample is positive and the negative control is also positive the
interpretation of the result depends on the relative signal strength of the two results. This
is calculated using the delta CT method by subtracting the target CT value from the
negative control CT value (NC CT value – sample CT value). Where the test sample is
positive and the NC is detected much later (delta CT ≥ 5) then the positive test result is
reliable. Where the NC detection is at a similar level to the test sample (delta CT<5) then
the positive test result is invalidated and a negative call is the correct result.
Internal PCR control
The CT value obtained with the internal control will vary significantly depending on the
extraction efficiency, the quantity of DNA added to the PCR reaction and the individual
machine settings. CT values of 28±3 are within the normal range. When amplifying a B.
dendrobatidis 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 Batrachochytrium dendrobatidis genomes.
Advanced kit handbook HB10.03.07
11
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