Pathogen sp – Real Time DNA amplification

Biotechnology

This amplifcation kit has been manufactured by Gentaur Bioingeneering to detect pathogen sp in real time PCR.

This is apossibility absolute quantifcation or qualitative assay.
Real time PCR is based on fuorogenic dyes. Ct value between
12 – 36 should be taken positive. Value between 36 – 40 Ct
should be taken as marginal positive. Ct above 40 must be
considered as negative.

This kit needs DNA as a template which can be isolated from:

Blood, serum, faeces, respiratory fuid, cerebrospinal fuid,
digestive system, tissue, Heptopancreas, Gills, Pleopods,
Cloacal, Egg Yolk, Milk, swabs, Lee, bacterial culture, cell lines
and others.

contact Lieven Gevaert, Bioingeneer for more extraction protocols and advice

Zeptometrix NatTrol DNA controls can be used as positive and negative run control.

All our kits contain reagents for a really good
quality DNA extraction. We discarded use of affnity columns
because a lot report that indicate purifcation problems due
to the lipids present in the biological samples quickly clog
the column decreasing its performance.

OneAgroq PCR-realtime Pathogen sp DNA Real Time PCR
Kit is a screening assay for a rapid and accurate detection of
154 Pathogens sp.

Content of the test kit:

Universal qPCR Master Mix
Primer, Probes and Internal
Control Universal Mix
Pathogen sp Positive Control
Pathogen sp Negative Control
PCR grade Water
Zeptometrix NatTrol Control

Compatibile with the following qPcr Cyclers

* 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

Procedure Principle and use:

50 / 100 /150 tests (Ready to use kit)
50 test 100 test 150 test
50 x 1 vial Reaction Tubes

Instrument Compatibility:

Prepare a Master mix according to the reaction qPCR cycler

One Step Bioingentech® PCR Kits provide components for
“onestep” real time PCR detection in a convenient format
that is compatible with both rapid and standard qPCR cycling
conditions.

The One Universal qPCR DNA Master Mix includes Gentaur Bioingeneer
all reagents for an optimized qPCR.

The Pathogen sp specific primer and probe mix are provided
in the kit and these can be detected through your real time
Platform by the 5’ nuclease PCR detection method. During PCR
amplifcation, forward and reverse primers hybridize to the
Acetobacter sp target genomic DNA generated.
Fluorogenic probe is included in the same reaction mixture which
consists of a DNA probe labeled with a 5-reporter kellú ZZ™
and 3-quencher kurü Zy™ which can be detected through
green channel.
To confirm extraction of a valid biological template an Internal
control primer and probe mix is included, consists of a DNA
probe labeled with a 5-reporter Chods ZX™ and a 3-quencher
kurü Zy™ which hybridize inside a specific housekeeping
endogenous target gene.
During PCR amplifcation, the probe is cleaved and the reporter dye and quencher are
separated. As a result, a fuorescence increase can be detected
on a range of real time PCR platforms through yellow channel.
Our kits also include Positive and Negative Control which are
details in FAQ section.

• Pulse-spin each tube in a centrifuge before opening.
• Homogenize the solutions for 5 seconds prior to pipetting
• You must consider use different tips in order to avoid cross
contamination.
• Use only sterile, RNAses, DNAases and pyrogens free tips.

Before Starting

For more details you can download a complete compatibility
panel from our web site: gentaur.fr

* Remember that all our OneAgroqPCR-realtime™ Pathogen
sp DNA Real Time PCR Kits include reagents and procedures
for DNA extraction. Also we always can offer you a complete
technical support for your different sample type.

Kit Components:

Ct (Threshold cycle) value of each sample can be read as
follows.

* Is important mentioned that Ct value over 40 is considered
Negative result. If Ct value is in a 12 – 36 range, it must be
considered as Positive result. This is depending of the
sample initial concentration used for each reaction.

You should consider that sample real concentration could be modify by the sample purity when this is quantifier

* For more technical information you must request the quality
control for each kits or use the Zeptometrix NatTrol qPcr Controls

Reaction Tubes

Universal qPCR Master Mix
Primer, Probes and Internal Control
Universal Mix
PCR grade Water
DNA Sample
Pathogen sp Positive Control
Pathogen sp Negative Control

Total Volume
Step 2
See quality NatTrol
control

Important Note: Don’t forget Homogenize the tubes.

Quantitative analysis:

Preparation of standard curve dilution
series. Pathogen sp positive control:
Standard curve | Concentration Copy Number | Preparation series
in a fresh dilution:

Tube N°1:
Tube N°2:
Tube N°3:
Tube N°4:
Tube N°5:
Tube N°6:

2uL Pathogen sp
Positive Control (0,1 ng/µL)
+18 µL de PCR grade Water
2uL Tube N°1
+
18 µL de PCR grade Water
2uL Tube N°2
+
18 µL de PCR grade Water
2uL Tube N°3
+
18 µL de PCR grade Water
2uL Tube N°4
+
18 µL de PCR grade Water
2uL Tube N°5
+
18 µL de PCR grade Water
Tube N°7: 2uL Tube N°6
+
18 µL de PCR grade Water
pathogen sp

Average Positive Control Concentration

* We will send a Quality Control report for each purchase.
** For reaction mix you must use Universal qPCR Master Mix.
*** If you want to obtain less DNA copies you must include a
new dilution tube (Tube N° 8). Note: Final DNA copy number
will depend of the DNA concentration (you can see it in Quality
Control Report).

**** Zeptometrix Natrol Positive and Negative controls can be used for Iso13485 and FDA accreditation of ypour lab

Reaction components for PCR
Recommended PCR Cycling table Sample and Internal Control Positive
Control
Negative
Control

This Bioingeneer kit contains

Universal qPCR
Master Mix
Primer, Probes
and Internal
Control Mix
PCR grade
Water
Tube N° 1
(Positive Control)
Tube N° 2
Tube N° 3
Tube N° 4
Tube N° 5
Tube N° 6
Tube N° 7

40 Cycles
Ct value Result
Marginal Positive
Negative

2.1.-Assess the Ct value when amplification curve of Standard
tube 1, 2, 3, 4, 5, 6 passes the threshold line. However, four
tubes are sufficient for standard curve. (tube1-tube4).
2.2.- Calculate quantitative value to compare with Ct value of
unknown samples and curve of Standard tube 1, 2, 3, 4, 5, 6.
2.3.- When you visualized result in the Real Time PCR platform
you must see just one amplification curve for Positive Control.
You must not see an Internal Control amplification curve.
3.1.- Each Ct value standard should be as follows.
Standard 1 < Standard 2 < Standard 3 < Standard 4 <
Standard 5 < Standard 6.
3.2.- R-value of standard curve should be 0.900 – 0.999.
R-value represent how well the experimental data fit the
regression line. A significant difference in observed Ct values
between replicates will lower the R-value.
3.3.- The standard curve slope result should be all negative.
3.4.- The desired amplifcation efficiencies vary from 90% to
110%. The theoretical maximum of 100% indicates that the
polymerase enzyme is functioning at its maximum capacity.
Low reaction efficiencies may be caused by poor primer
design or by suboptimal reaction conditions. Reaction
efficiencies >110 may indicate pipetting error in your serial
dilutions or coamplication of nonspecific products, such as
primer-dimers.

1.-Positive control:
Green
Yellow
Orange
Red
Crimson
Channel Source Detector Dyes
470 (Nm)
530 (Nm)
585 (Nm)
625 (Nm)
680 (Nm)
520 (Nm)
550 (Nm)
610 (Nm)
660 (Nm)
710 (Nm) Quasar 705, Lightcycler Red
705, Alexa Fluor 680
FAM, Sybr green1, Fluorescein,
Eva green, Alerxa fluour 488,
Joe, Vic, Hex, Tet, Cal Fluorgold
540, YaKima Yellow, Chods ZX™
Rox, Cal Fluor Red 610, Cy3.5,
Texas Red, Alexa Fluor 568
Cy5, Quasar 670, Lightcycler, Red
640, Alexa Fluor 633, Aeon Zw™.
kellú ZZ™

3) Test validation:

Visual explanation FAQ:

* Remember: Run a positive control and negative control for
each 12 samples. For reaction mix you must use Universal
qPCR Master Mix.
*You must use quencher and reporter dye to setup your
software (see table 8 and 9) and run the following channel:
The Positive control assay uses a kellú ZZ™ dye and should
be detected through the Green channel of your real time
PCR instrument (see table 8 and 9).
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 Acetobacter
sp copy number / Ct value.

Alternatively, the positive control can be used at a single
dilution Acetobacter sp on where full quantitative analysis of
the sample is not required. Each time the kit is used, at least
one positive control reaction must be included in the run.
Particularly, due to amount of this reagent, you
should run a positive control for each 12 samples. A
positive result indicates that the primer and probes for
detecting the target Acetobacter sp gene worked properly
in that particular experimental scenario. If a negative result is
obtained the test results should be invalid and must be
repeated (see Table 11). Sealing all other samples and
negative controls before pipetting the positive control into
the positive control well tube.

2.-Internal Control:

The internal control is included in Primer, Probes and Internal
Control Mix along to the target pathogen detection. In order
to interpreted results, read the yellow channel. The internal
control assay uses a Chods ZX™ dye and should be detected
through the Yellow channel of your real time PCR instrument
and gives a Ct value of 28 (+/-5) depending on the level of
sample dilution and concentration. A positive result through
the Yellow channel therefore indicates that PCR conditions
are suitable for detection of the target pathogen gene. If a
negative result is obtained through the Yellow channel the
results should be analyzed by combination of result

3.-Negative control:

To conform absence of contamination a negative control
reaction should be included every time the kit is used. Particularly, due to amount of this reagent, you should run a
negative control for each 12 samples. In this instance the
PCR grade water should be used in place of template. A
negative result indicates that the reagents have not become
contaminated. If a positive result and Ct value less than 36 is
obtained, the results should be analyzed and check if a
correct amplification curve was obtained. When you obtain a
clear amplification curve you should consider repeat your
assay due to probably the sample was contaminated

-520 nm
●FAM
●SYBR Green
●kellú ZZ™
Green
-550 nm -580 nm
●TAMRA
●NED
●CY3
-610 nm
●ROX
●TEXAS RED
-670 nm
●CY5
●Aeon Zw™
Red
Pure Dyes

●HEX
●JOE
●VIC
●Chods ZX™