Kylt Coryza Real-Time PCR Detection - www.kylt.eu
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For in vitro Veterinary
Diagnostics only.
Kylt® Coryza
Real-Time PCR Detection
www.kylt.euDIRECTION FOR USE
Rev003, Mar 2020
Kylt® Coryza
PCR Detection
Revision No. Amendments
003 New Layout
A. General
Kylt® Coryza PCR Detection kits are intended for the specific detection of the etiological agent of Infectious Coryza
(Avibacterium paragallinarum). The kits are suitable for the analysis of samples from birds, such as swab samples and
exsudates of sinus infraorbitalis, trachea, lung and air sacs as well as material derived from cultural processes of the
aforementioned samples.
Kylt® Coryza comprises all reagents and controls of Coryza. The qualitative testing with Kylt® Coryza kits is based
on Polymerase Chain Reaction (PCR). By using specific oligonucleotides the target gene of interest in a samples is
specifically amplified. Following amplification, the PCR reaction is analyzed by agarose gel electrophoresis for qualitative
test results. By analyzing the detection of the target gene in the samples and the Negative Control and Positive Control
per run the Coryza-specific status of a sample can be evaluated in the end. This way, results can be achieved within a
few hours after sample receipt.
These kits were developed for use by trained laboratory personnel following standardized procedures. This Direction
For Use must be followed strictly.
Kylt® Coryza | PCR Detection 2/7B. Reagents and Materials
The following Kylt® Coryza kits are available and comprise the following reagents:
100 Reactions 25 Reactions
Reagent Colour of Lid Article No 31048 Article No 31049 Store at
2x PCR-Mix white 4 x 280 µl 1 x 280 µl ≤ -18 °C
10x Loading Dye white 4 x 60 µl 1 x 60 µl ≤ -18 °C
Primer-Mix white 4 x lyophilizate 1 x lyophilizate ≤ -18 °C
(final 160 µl each) (final 160 µl each)
Positive Control red 4 x lyophilizate 2 x lyophilizate ≤ -18 °C
(final 20 µl each) (final 20 µl each)
Negative Control blue 1 x 1 ml 1 x 1 ml ≤ -18 °C
After receipt, the components are immediately stored at the corresponding temperatures listed above. Avoid repeated
freezing and thawing of all the reagents and keep them thawed as short as possible. If occasional processing of few
samples only is expected you may prepare appropriate aliquots of reagents before storage at ≤ -18 °C. Prepare aliquots
in such a way that freeze-thaw-cycles are reduced to a maximum of three. The Negative Control can alternatively be
stored at +2°C to +8°C.
The components are to be used within the indicated shelf life (see box label). The components of different batches may
not be mixed.
Before its first use, rehydrate the Positive Control: add 20 μl of Negative Control per vial, briefly incubate at room
temperature and mix thoroughly by repeated vortexing. It is recommended to generate aliquots of suitable volumes and
store them at ≤ -18 °C.
Before its first use, rehydrate the Primer Mix: add 160 μl of Negative Control per vial, briefly incubate at room temperature
and mix thoroughly by repeated vortexing. It is recommended to generate aliquots of suitable volumes and store them
at ≤ -18 °C.
C. Equipment and Reagents not included
This detection method can be used on all commercially available PCR thermal cyclers.
Apart from the disposables, the following further devices are needed and are not included in the Kylt® Coryza kits:
DNA preparation kit / protocol (e.g. Kylt® DNA Extraction-Mix II or Kylt® RNA / DNA Purification products)
Table top microcentrifuge
Micropipettes covering volumes of 1 µl to 1000 µl
Centrifuge for PCR tubes or plates
PCR thermal cycler
Equipment, media and disposables for agarose gel electrophoresis
We recommend the exclusive use of certified Nuclease-free disposables as well as powder-free protective gloves.
Please wear gloves during the entire experimental procedure. Gloves need to be changed frequently, especially after
spillage or suspected contaminations.
Kylt® Coryza | PCR Detection 3/7D. Control Reactions
The Positive Control allows for control of the specificity and efficiency of the reagents and the reaction itself, including
the performance of the PCR and of the PCR thermal cycler.
The Negative Control allows for exclusion of contaminations. The sample testing is only valid if both, Positive and
Negative Controls, are used and verified for validity in every PCR run.
E. Protocol (see also „Protocol At A Glance“ at the end of this Direction For Use)
The overall protocol of the analysis consists of the following main workflow:
1. Sample Preparation
2. DNA Preparation
3. Reaction Setup and Amplification (PCR)
4. Agarose Gel Electrophoresis
5. Data Analysis – Validity and Qualitative Result
We recommend proceeding through the protocol without interruption to avoid potential degradation of the processed
samples and reagents. If necessary, you may store the final DNA preparation at ≤ -18 °C until further processing. Avoid
repeated freezing and thawing of the DNA preparations.
1. Sample Preparation
We recommend pooling of at most five samples or samples from five individuals, respectively, per DNA preparation.
Pool swabs in a sufficient volume of sterile buffer (e.g. 1 ml of Normal Saline or 0.1 x TE), let the swabs soak for an
adequate period of time and finally wash out the swabs by thorough pulse-vortexing.
The supernatant is used for DNA preparation.
For Kylt® DNA Extraction the supernatant is (fully) transferred to a conical screw cap tube (please refer to 2 "DNA
Preparation").
Small swabs may directly be immersed in lysis buffer, if applicable.
Material derived from cultural processes, i.e. colony material, is directly transferred into respective tubes, such as conical
screw cap tube; therefore a little amount of a single colony is picked with a sterile loop wire or sterile pipette tip and
transferred to the tube.
Kylt® Coryza | PCR Detection 4/72. DNA Preparation
a) Kylt® DNA Extraction (requires Kylt® DNA Extraction-Mix II)
For detailed information, please refer to the Direction For Use of Kylt® DNA Extraction-Mix II.
b) Kylt® RNA/DNA Purification products
All kinds of sample matrices, including pure isolates and swabs, may be processed with Kylt® RNA/DNA Purification
products (please refer to chapter F “Related Products”).
For detailed information on the DNA preparation process, please refer to the respective Direction For Use.
c) Alternative methods
All kinds of sample matrices, including pure isolates and swabs, may be processed with appropriate DNA preparation
kits or appropriate in-house methods.
For detailed information on the DNA preparation process, please refer to the Direction For Use or Standard Operating
Procedure of the specific kit or in-house method, respectively.
3. Reaction Setup and Amplification (PCR)
Before each use, briefly vortex and spin down the 2x PCR-Mix, rehydrated Primer-Mix and Negative Control.
To determine the total number of reactions needed, count the number of samples and add two more for the Negative
Control and the Positive Control.
Prepare a Master-Mix contraining the 2x PCR-Mix, 10x Loading Dye and the Primer-Mix for the appropriate number of
reactions. Then add 18 μl of the Master-Mix to reach of the PCR tubes or wells of plate ("cavity"). The PCR is set up in
the given order:
Volume (µl)
Reagent per Reaction e.g. n=7
2x PCR-Mix 10 µl 70 µl
10x Loading Dye 2 µl 14 µ
Primer-Mix 6 µl 42 µl
Total Master-Mix 18 µl 126 µl
dispense 18 µl per reaction
Template (Negative Control / DNA preparation / Positive Control) 2.0 µl
Total Reaction 20.0 µl
Kylt® Coryza | PCR Detection 5/7Add 2 μl of the Negative Control to the corresponding cavity and seal it individually, if possible.
Add 2 μl of each sample DNA to the corresponding cavities and seal them individually, if possible.
To minimize risk of potential cross-contaminations, 2 μl of the Positive Control are added to the corresponding cavity
after all previous samples and control reactions are set up. Before each use, briefly vortex and spin down the rehydrated
Positive Control (see also chapter B “Reagents and Materials”).
If not already done, finally seal the cavities. It is recommended to briefly spin them down before the start of the PCR run.
Place the cavities in the PCR thermal cycler and run the test with the parameters as given below.
Step No Description Temperature Duration
1 Activation of Polymerase 94 °C 3 min
2 Denaturation 94 °C 30 sec
3 Annealing 57 °C 30 sec 35 cycles
4 Extension 72 °C 60 sec
5 Post-PCR cooling (optional) 7 °C hold
Please follow the specified instructions of your PCR thermal cycler as recommended by the manufacturer.
4. Agarose Gel Electrophoresis
General
The expected Coryza-product size is 510 bp, you may run any standard agarose gel electrophoresis method appropriate
for this product size. An appropriate method is described in the following:
Prepare a 2% standard agarose gel for separation of the DNA sample after PCR amplification.
The PCR reactions already contain loading buffer including electrophoresis tracking dyes that migrate at approximately
4 kbp and 50 bp and are ready for loading on agarose gel following PCR.
Load the wells of agarose gel electrophoresis with 5 µl of the PCR reactions from the DNA sample(s), Positive Control
and Negative Control, respectively. Load at least an additional well of agarose gel with an appropriate volume of e.g. an
100 bp reference DNA ladder. Make notes of the position of sample(s), controls and ladder.
Run the electrophoresis at a voltage of approximately 15 V/cm (the distance in cm refers to the distance between
electrodes) for 45 min to 60 min.
Following electrophoresis, stain the gel with appropriate amount of nucleotide / intercalating dye (e.g. ethidium bromide,
GelRed Nucleic Acid or SYBR green) and visualize by using the corresponding technique. For more details, please refer
to the Direction For Use of the dye.
Kylt® Coryza | PCR Detection 6/75. Data Analysis – Validity and Qualitative Result
The readily stained agarose gel must give discrete bands of expected sizes for control reactions and the reference DNA
ladder. The actual PCR test analysis starts with the validity check of the entire PCR run. Therefore, check the results
of Positive Control and Negative Control for presence / absence of expected product size for Coryza. Afterwards, the
specific status of Coryza of each sample is analyzed by looking for presence / absence of the expected product size.
Test Evaluation
The PCR test run is only valid, if the Negative Control is negative and the Positive Control is positive with regard to the
expected Coryza-specific product size of 510 bp.
A sample is negative for Coryza, if absence of Coryza-specific product with size of 510 bp is observed.
A sample is positive for Coryza, if presence of Coryza-specific product with size of 510 bp is observed.
G. Ordering information
For a fast and efficient service please send your order to orders@kylt.eu and please provide the following information:
Delivery address
Invoice address
Purchaser contact telephone number
End user name and telephone number (if different)
Purchase order number
Product name and cataloge number
Quantity and size of products
Indicate if your account is VAT exempt
Production:
AniCon Labor GmbH | Muehlenstr. 13 | D-49685 Hoeltinghausen | Germany | www.kylt.eu | info@kylt.eu
Development, manufacturing and distribution of Kylt® In-Vitro Diagnostica
is certified according to ISO 9001:2015.
c.PCR.Coryza.02, Rev003, March 2020
Kylt® is a registered trademark.
For veterinary use only. For in vitro use only. Regulatory requirements vary by country, not all of the products described
herein may be available in your geographic area.
© 2020 AniCon Labor GmbH. All rights reserved. The trademark mentioned herein is the property of AniCon Labor GmbH
or their respective owners.
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