c belli

c belli
Techne ® qPCR test
Cystoisospora belli
Internal transcribed spacer 1
150 tests
For general laboratory and research use only
Quantification of Cystoisospora belli genomes.
Advanced kit handbook HB10.03.07
1
Introduction to Cystoisospora belli
Cystoisospora belli, (previously known as Isospora belli) is a coccidian protozoan that
infects humans and some primates.
Coccidian protozoa are microscopic, spore-forming, single-celled obligate intracellular
parasites belonging to the class Conoidasida. As obligate intracellular parasites, they must
live and reproduce within an animal cell.
C. belli has linear DNA consisting of ~3kbp base pairs.
The oocyts are ovoid and range in size from 23–36 µm by 12–17 µm. I. belli sporocysts are
contained within the oocysts and appear circular. The general size of sporocysts is 12–14
µm by 7–9 µm. Sporozoites and merozoites are small rice shaped granules.
The number of chromosomes in C. belli has not yet been determined as the chromosomes
do not condense during mitosis. Many organisms in the phylum Apicomplexa, including the
very closely related Toxoplasma gondii, have a 35 kb circular piece of DNA in addition to
linear chromosomes called an apicoplast. It is not known whether C. belli has an
apicoplast in addition to its linear DNA.
C. belli is transmitted by faecal-oral contamination. This occurs more frequently under poor
sanitation conditions.
Because I. belli thrives under immunodepressed conditions, it is often seen in conjunction
with HIV infection. Once HIV has caused the CD4 count to drop below 300 cells/mm3, I.
belli can infect a person without effective interference from the host immune system.
In healthy individuals I. belli is usually asymptomatic or produces self limiting diarrhoea. If
the host is young or immunosuppressed the symptoms can include: fluid loss by diarrhoea,
weight loss, mild fever, vomiting, and abdominal pain.
C. belli is diagnosed by identification of the oocyst through examining a stool sample under
a microscope. The diagnostic stage is the immature oocyst that contains a spherical mass
of protoplasm. Absolute confirmation can be obtained made by extracting DNA from a
faecal sample and performing nucleic acid testing to determine if C. belli is present.
Quantification of Cystoisospora belli genomes.
Advanced kit handbook HB10.03.07
2
Specificity
MAX MIN
The Techne qPCR Kit for Cystoisospora belli (C.belli) genomes is designed for the in vitro
quantification of C.belli genomes. The kit is designed to have the broadest detection
profile possible whilst remaining specific to the C.belli genome.
The primers and probe sequences in this kit have 100% homology with a broad range of
C.belli 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 Cystoisospora belli genomes.
Advanced kit handbook HB10.03.07
3
Kit Contents
• C.belli specific primer/probe mix (150 reactions BROWN)
FAM labelled
• C.belli 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 Cystoisospora belli 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 C.belli 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 Cystoisospora belli genomes.
Advanced kit handbook HB10.03.07
5
Principles of the test
Real-time PCR
A C.belli 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 C.belli 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 C.
belli 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 C.belli 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.
Quantification of Cystoisospora belli 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 C.belli 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 C.belli 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 Cystoisospora belli 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
C.belli 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 Cystoisospora belli 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
C.belli 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
C.belli primer/probe mix (BROWN)
1 µl
RNAse/DNAse free water (WHITE)
4 µl
15 µl
Final Volume
Quantification of Cystoisospora belli 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 Cystoisospora belli 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 C.
belli 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 Cystoisospora belli genomes.
Advanced kit handbook HB10.03.07
11
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