VetqPCR-realtime Exosomes

VetqPCR-realtime PCR – Real Time

VetqPCR-realtime™ A. phagocytophilum Real Time PCR

Kit Components
Principle and use:

50 / 100 /150 tests (Ready to use kit)

• Run a positive control, a negative control, and an internal
control for each 12 samples.

• The mineral oil is necessary, only when using a thermalcycler
that employs a top heating method.

Preparation of the pathogen Mixture

1) Prepare the reaction mixture for sample, positive control,
negative control, and internal control by combining the
reagents as shown in the table 1. The final reaction volume
should be 13.5 μL
Table 1. Reaction components for PCR Sample Positive control Negative
control
control
Internal
Principles of the test
VetqPCR-realtime™ A. phagocytophilum
VetqPCR-realtime™ Internal Control
PCR Grade water
DNA isolated from the sample
A. phagocytophilum Positive control
A. phagocytophilum Negative control
VetqPCR-realtime™ A. phagocytophilum
VetqPCR-realtime™ Internal Control
PCR Grade water
A. phagocytophilum Positive control
A. phagocytophilum Negative control
Mineral Oil

STEP 1

50 test 100 test 150 test

Pulse-spin each tube in a centrifuge before opening.
Completely thaw the components of the kit prior to use.
Homogenize the solutions for 5 seconds prior to pipetting.
This amplification kit has been manufactured by Bioingentech
Ltd. Chile to detect Anaplasma phagocytophilum in real
time PCR. This is a posibility absolute quantification or
qualitative assay.
Real time PCR is based on fluorogenic dyes. Up to 36 Ct should
be taken positive. Value between 36-40 Ct should be taken as
marginal positive.
This kit needs DNA which can be isolated from blood, serum,
faeces, respiratory fluid, digestive system, tissue and others.
See (PU-A001, PU-A002 & PU-A003) included in advanced
formats.
The Anaplasma phagocytophilum specific primer and
probe mix are provided and this can be detected through
the yellow channel.
During PCR amplification, forwar and reverse primers hybridize
to the Anaplasma phagocytophilum. A fluorogenic probe is
included in the same reaction mixture which consists of a DNA
probe labeled with a 5-dye Chods ZX™ and 3-quencher kurü Zy™.
Internal control consists of a DNA probe labeled with a 5-dye
kellú ZZ™ and a 3-quencher kurü Zy™.
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 realtime PCR
platforms.

Procedure:

Instrument Compatibility in:
Please read through the entire procedure before
starting.

Total Volumen 13.5μL 13.5μL 13.5μL 13.5μL
* LightCycler 2.0
* LightCycler 480
* Mastercycler® ep realplex
* Mx3000P QPCR System
* Mx3005P QPCR System
* RotorGene 3000
* RotorGene 6000
* RorotGeneQ
* SLAN® Real-Time PCR
* Smartcycles II
* Applied 7300 and 7500
* ABI 7300
* ABI 7500FAST
* ABI 7900
* AB Step One
* AB Step One Plus
* Agilent Mx3005P
* CFX96 & CFX384
* ExiCyclerTM 96
* iQ5 & MyiQ Cycler
* Illumina Eco
* LightCycler Nano

60 seconds
x 1 cycles
x 45 cycles
PCR cycle Temp. Time
Initial Denaturation 94ºC 2 min.
Denaturation
Annealing
95°C
60°C
Table 2. Real Time cycling parameters
Once the program will be finished one can see the graphics.
The negative control should run along with the bottom and
positive control must give a curve in the software graphics. Use
your software to analyse the results.
Step 3
Place the tubes in a Instrument and perform amplification
according to the program outlined in Table 2.
2μl DNA isolated from the sample
6μl PCR Grade water
5.5μl VetqPCR-realtime™ A. phagocytophilum
2μl DNA isolated from the sample
6μl PCR Grade water
5.5μl VetqPCR-realtime™ Internal Control
2μl A. phagocytophilum Positive control
6μl PCR Grade water
5.5μl VetqPCR-realtime™ A. phagocytophilum
2μl A. phagocytophilum Negative control
6μl PCR Grade water
5.5μl VetqPCR-realtime™ A. phagocytophilum
Visual explanation:
Negative Control:
Positive Control:
Sample:
Internal Control:
Observation:
homogenize solution in each tube during 10 seconds.
1) Qualitative analysis:
Ct (Threshold cycle) value of each sample can be read as follows.
Ct value result
> 40 negative ≤ 40 positive
Interpretation of the test
Total: 13,5 μL
Total: 13,5 μL
Total: 13,5 μL
Total: 13,5 μL
Homogenize tube.
2) Quantitative analysis:
Table 3. Preparation of standard curve dilution
series. A. phagocytophilum positive control:
Standar
curve Concentration Copy
Number

Preparation seriesa fresh dilution

Positive Control (0,3 ng/µl )
+ 18uL PCR Grade Water
2uL Tube N°1
+
18uL PCR Grade Water
2uL Tube N°2
+
18uL PCR Grade Water
2uL Tube N°3
+
18uL PCR Grade Water
2uL Tube N°4
+
18uL PCR Grade Water
2uL Tube N°5
+
18uL PCR Grade Water
Tube N°7: 2uL Tube N°6
+
18uL PCR Grade Water
A. phagocytophilum
Average Positive Control Concentration
* We will send a Quality Control report for each purchase
** If you want to obtain 13 DNA copies you must include a
new dilution tube (Tube N° 8)

 

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