encephalitozoon spp

encephalitozoon spp
Techne ® qPCR test
Encephalitozoon
16S ribosomal RNA (18S) gene
150 tests
For general laboratory and research use only
Quantification of Encephalitozoon genomes.
Advanced kit handbook HB10.03.07
1
Introduction to Encephalitozoon
Encephalitozoon is a genus of fungi of the microsporidium phylum containing species with
very small genomes of just over 2.4Mbp in length. Species within this genus form spores of
1-3um in diameter containing a posterior vacuole, anterior anchoring disc and polaroplast.
Microsporidia species infect many animals including humans and can cause
microsporidiosis and keratoconjunctivitis, most commonly in immunocompromised
individuals.
Spores of the encephalitozoon species are hardy and can survive in the environment for
long periods of time. Spores are inhaled or ingested and once within the host, extends a
polar tubule which is used to inject the sporoplasm into the host cell. The sporoplasm
replicates by merogony or sporogony and does so in parasitophorous vacules within the
cytoplasm. Meronts are formed around the edge of the vacuole and differentiate into
sporoblast and spores as they migrate to the center of the vacuole. As the spores multiply
and fill the host cell, the cell membrane is disrupted, releasing the spores to infect other
cells and shed in excrement.
There are four species which commonly infect humans with some resulting clinical
symptoms including diarrhea and cholecystits (E. intestinalis) while others causes
encephalitis (E. cuniculi) and Keratoconjunctivitis, an inflammation of the cornea and
conjunctiva (E. hellum). Treatment with antimicrobial agents such as albendazole and
fumagillin usually clears the infection.
Quantification of Encephalitozoon genomes.
Advanced kit handbook HB10.03.07
2
Specificity
MAX MIN
The Techne qPCR Kit for Encephalitozoon (Encephalitozoon) genomes is designed for the
in vitro quantification of Encephalitozoon genomes. The kit is designed to have the
broadest detection profile possible whilst remaining specific to the Encephalitozoon
genome.
The primers and probe sequences in this kit have 100% homology with a broad range of
Encephalitozoon sequences based on a comprehensive bioinformatics analysis.
The PrimerDesign™Quantification Kit for Encephalitozoon has been designed for the
specific and exclusive in vitro quantification of this genus . The 16S ribosomal gene, is
the ideal target to achieve a broad based detection profile for all species within this
genus. The primers and probe sequences in this kit have 100% homology with a broad
range of clinically relevant reference sequences based on a comprehensive
bioinformatics analysis. Other closely related species within the Unikaryonidae family are
not detected.
If you require further information, or have a specific question about the detection profile of
this kit then please send an e.mail to [email protected] and our
bioinformatics team will answer your question.
Quantification of Encephalitozoon genomes.
Advanced kit handbook HB10.03.07
3
Kit Contents
• Encephalitozoon specific primer/probe mix (150 reactions BROWN)
FAM labelled
• Encephalitozoon 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 Encephalitozoon genomes.
Advanced kit handbook HB10.03.07
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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 Encephalitozoon 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 Encephalitozoon genomes.
Advanced kit handbook HB10.03.07
5
Principles of the test
Real-time PCR
A Encephalitozoon 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 Encephalitozoon 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
Encephalitozoon 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 Encephalitozoon 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 Encephalitozoon genomes.
Advanced kit handbook HB10.03.07
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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 Encephalitozoon 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 Encephalitozoon
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 Encephalitozoon 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
Encephalitozoon 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 Encephalitozoon 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
Encephalitozoon 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
15 µl
Final Volume
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
Encephalitozoon primer/probe mix (BROWN)
1 µl
RNAse/DNAse free water (WHITE)
4 µl
15 µl
Final Volume
Quantification of Encephalitozoon genomes.
Advanced kit handbook HB10.03.07
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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 Encephalitozoon 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
Encephalitozoon 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 Encephalitozoon genomes.
Advanced kit handbook HB10.03.07
11
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