English - Product Documentation

DOT129v1 Instructions for Use Biofortuna SSPGo
TM
HLA No Template Control Kit BF-40-02
Page 1 of 7
Instructions for Use Biofortuna SSPGoTM HLA No Template Control Kit BF-40-02
CE revision 2, July 2014
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DOT129v1 Instructions for Use Biofortuna SSPGo
TM
HLA No Template Control Kit BF-40-02
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1. Intended Use
The Biofortuna SSPGo No Template Control (NTC) Kit contains supplementary tests for PCR amplicon contamination
to be used in conjunction with Biofortuna SSPGo kits that do not have an integral NTC.
2. Introduction
PCR is a sensitive technique that is susceptible to contamination with DNA amplicon from a previous PCR.
Contamination can lead to false positive amplification in subsequent PCRs, which can lead to incorrect genotyping.
PCR amplicon can contaminate reagents and samples, as well as laboratory equipment such as pipettes. Reagents
and equipment should be monitored regularly for signs of contamination. The NTC test is used to detect potential
PCR or DNA contamination present in the DNA diluent used for a test sample.
3. Test Description
Each SSPGo No Template Control kit consists of 12 strips of 8 PCR reactions containing freeze-dried PCR buffer,
polymerase and primers specific for the HLA-DRA gene and will produce an 187bp amplicon from human genomic
DNA or amplified DNA. All Biofortuna kits utilise a DRA amplicon as an internal control, therefore any contamination
from the use of Biofortuna SSPGo kits will have co-amplification of the DRA gene, and will be detected by the NTC
test primers.
4. Kit Contents
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12 strips of 8 PCR reactions, each containing pre-dispensed freeze dried primers, polymerase, dNTPs* and
buffer. The eight reaction strip format is shown below.
Reaction
Dye
1
Red
Use
No Template Control: Sample 1
2
Purple No Template Control: Sample 2
3
Purple No Template Control: Sample 3
4
Purple No Template Control: Sample 4
5
Purple No Template Control: Sample 5
6
Purple No Template Control: Sample 6
7
Purple No Template Control: Sample 7
8
Purple No Template Control: Sample 8
12x8 PCR caps
1x instructions for use
1x Certificate of Analysis
The MSDS can be downloaded from the Biofortuna website www.biofortuna.com. If you are unable to
download from the website please contact your local distributor.
*CleanAmp™ dNTPs are licensed from Trilink Biotechnologies Inc for use in Biofortuna SSPGo products.
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DOT129v1 Instructions for Use Biofortuna SSPGo
TM
HLA No Template Control Kit BF-40-02
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5. Reagents and Equipment Not Supplied
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Appropriate calibrated pipettors and sterile tips e.g. P10 pipettor with 10µl filter tips.
DNA isolation kit/equipment.
UV spectrophotometer.
Polypropylene tubes.
Sterile molecular grade water.
A thermal cycler with the following specifications should be used:
 96 well thermal cycler with heated lid with a temperature of 104°C for oil-free operation
 Ramp rate of 1.0oC/sec.
 Temperature range of 4.0°C to 99.9°C
 Temperature accuracy of ±0.25°C for the range of 35°C to 99.9°C
 Temperature calibration traceable to a reference standard
 Program the thermal cycler using the PCR Cycling Parameters in Section 8 below.
Note: For specific thermal cycler information refer to the manufacturer’s user manual. Thermal cycler should be
calibrated according to ASHI (American Society of Histocompatibility and Immunogenetics) or EFI (European
Federation of Immunogenetics) accreditation rules.
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Gel electrophoresis reagents (agarose, 0.5x TBE, 1000bp DNA molecular weight marker, 10mg/ml Ethidium
Bromide).
Gel electrophoresis equipment (gel tanks, power supply, gel documentation system with UV transilluminator).
Note: any change in the specified conditions, such as thermal cycler ramp rates, may affect interpretation of
the test results.
6. Safety and Warnings
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Tests should only be carried out by appropriately trained personnel.
Handle all reagents in accordance with Good Laboratory Practice.
Keep pre- and post-PCR areas separate. Do not bring any post-PCR materials back to the pre-PCR area.
Biohazard Warning: Treat all blood products as potentially infectious.
Biohazard Warning: Ethidium Bromide is a potential carcinogen. If used, always wear gloves, a laboratory coat
and protective eye glasses.
Biohazard Warning: Take care when using UV sources - always wear gloves, a laboratory coat and protective
eye glasses. Never view the UV light source directly.
Material Safety Data Sheets are available from www.biofortuna.com.
7. Storage and Stability
Biofortuna SSPGo kits should be stored at 2-28°C. Once PCR vessels are removed from the foil pouches the reagents
should be re-hydrated with DNA within 3 hours. Refer to packaging for expiration date. Do not use products after
the printed date.
Do not use kits if the foil pouch is ripped or perforated or if there is no desiccant bag present.
Using the sealing sheets or caps provided only, ensure PCR vessels are sealed tightly after adding DNA as omitting
this may lead to evaporation during PCR amplification. Pay particular attention to edges and corners.
Note (1): If necessary, the out-of-pouch non-hydrated PCR plates and strips may be held for up to 3 hours prior to
addition of DNA at a temperature of up to 21oC and humidity of no more than 60%.
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DOT129v1 Instructions for Use Biofortuna SSPGo
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HLA No Template Control Kit BF-40-02
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Note (2): Once hydrated with DNA, PCR strips and plates from freshly opened pouches can be stored for up to 24
hours at 2–8°C before the PCR step provided that the wells are well sealed to avoid evaporation.
8. Directions for Use
Note:
A frequent source of contamination is PCR pipettes and it is advisable to use a known contamination free pipette to
load sample into PCR reactions, including this NTC test.
1. Open the foil pouch, and label appropriately; one strip can be used to test between one and eight samples. Once
opened the PCR reactions should be used within 3 hours.
2. For each DNA sample genotyped add 10µl of the water or diluent used to one of the reactions of the NTC strip.
3. If a positive control is required use 10µl of 10ng/µl human genomic DNA. This will result in a positive test
amplicon of 187bp.
4. Cap the reactions with the provided caps and proceed with the standard SSPGo PCR parameters as shown below.
RE-SUSPENSION NOTE: Ensure the PCR mixes are re-suspended with the samples within 3 hours of removing the
tray from the foil pouch.
PCR PLATE/STRIP HEIGHT PROFILE NOTE: It is recommended that the height profile of plates and strips are
equivalent when placed in the same PCR machine. Different height profiles can cause poor contact with the PCR
machines heated lid. This may result in poor or failed PCR amplification.
PCR Parameters
The following PCR parameters should be used. Ensure ramp speeds of at least 1°C per second and enable the heated
lid. Please refer to the thermacycler manufacturer’s user manual for full instructions for use. Thermalcyclers should
be calibrated according to the American Society of Histocompatibility and Immunogenetic (ASHI) or European
Federation of Immunogenetics (EFI) accreditation rules.
Denature
94°C
5 minutes
Denature
Anneal
Extend
96°C
66°C
72°C
15 seconds
50 seconds
30 seconds
10 cycles
Denature
Anneal
Extend
96°C
64°C
72°C
15 seconds
50 seconds
30 seconds
20 cycles
HOLD 15°C
Gel Electrophoresis
These instructions apply to horizontal agarose gel electrophoresis: Prepare a 2% agarose gel in 0.5x TBE buffer.
When the gel is cooled to about 60°C add ethidium bromide to a final concentration of 0.5µg/ml. Cast gel and insert
microtitre format combs (e.g. 12x8 wells with 9mm spacing). Once set, remove the combs and cover gel in 0.5x TBE
buffer. Transfer a minimum of 5µl and a maximum of 10µl from each tray or strip reaction to the corresponding well
on the gel, noting the position of each reaction. A 100bp ladder can be useful to aid size determination. Run gel for
20 minutes at 10V/cm.
Refer to your electrophoresis system manufacturer’s instructions for use for specific equipment details. Gels should
be imaged using a UV gel documentation system with UV transilluminator.
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DOT129v1 Instructions for Use Biofortuna SSPGo
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HLA No Template Control Kit BF-40-02
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9. Interpretation
Record the results using the sample record table on page 6 of this IFU. A 187bp amplicon will be observed if there is
SSPGo PCR contamination or DNA contamination present. Any smears or bands of different sizes may also indicate
PCR contamination, but primer-dimer and other primer extension artefacts of less than 100bp should be ignored. A
positive result for any diluent sample indicates that the genotyping of that sample is invalid and should be repeated
with another DNA sample using different reagents.
10. Quality Assurance and Control
Assay testing: Using PCR amplicon from a Biofortuna Kit, the NTC Test was performed on the amplicon undiluted and
then in dilutions from 1x101 to 1x1015. The amplicon was detected in dilutions up to and including 1x1015.
The NTC Test was performed on gDNA at 100ng/µl and then in dilutions from 1x101 to 1x1015. The DNA was detected
in dilutions up to 1x103.
11. References
1) Bunce M et al Tissue Antigens. 1995 Nov;46(5):355-67.
2) Saiki RK et al. Nature. 1986 Nov 13-19;324(6093):163-6.
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DOT129v1 Instructions for Use Biofortuna SSPGo
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HLA No Template Control Kit BF-40-02
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12. Biofortuna NTC Test Sample Record Sheet
It is recommended that this sample record sheet is photocopied prior to use as the NTC Kit has sufficient tests for 96
samples (twelve strips of eight tests).
Sample 1 Description.
Sample 2 Description.
Sample 3 Description.
Sample 4 Description.
Sample 5 Description.
Sample 6 Description.
Sample 7 Description.
Sample 8 Description.
NTC Sample record
Reaction
Dye
1
Red
2
Purple
3
Purple
4
Purple
5
Purple
6
Purple
7
Purple
8
Purple
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Sample ID
Date of test
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Result
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DOT129v1 Instructions for Use Biofortuna SSPGo
TM
HLA No Template Control Kit BF-40-02
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13. Guide to Symbols Used
X
P
i
M
V
H
28:
2: s
g
Number of Tests
EC Representative
Consult Instructions for Use
Site of Manufacture
In Vitro Diagnostic
Expiry Date YYYY-MM-DD
Storage Temperature
Lot Number
Distributed by
Global Trade Item Number
14. Manufacturer Contact Details
Biofortuna Ltd
1 Hawkshead Road
Croft Business Park
Bromborough, CH62 3RJ, UK
T: +44 (0) 151 334 0182
E: info@biofortuna.com
W: www.biofortuna.com
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