Instructions for Use. HLA-Ready Gene. Intended Use: For molecular biological (SSP) typing of HLA class I and II antigens.

Transcription

1 INNO-TRAIN Diagnostik GmbH Niederhöchstädter Str. 62 D Kronberg / Taunus Tel Instructions for Use HLA-READY GENE QMS HLA-Ready Gene Intended Use: For molecular biological (SSP) typing of HLA class I and II antigens.. REACTIONS/ TESTS/ PRODUCT ARTICLE NO. COLOUR TEST PACK HLA-Ready Gene A Low green HLA-Ready Gene B Low yellow HLA-Ready Gene C Low orange HLA-Ready Gene ABC Low blue HLA-Ready Gene ABDR Low colourless HLA-Ready Gene DR Low red HLA-Ready Gene DQ Low blue 8 48 HLA-Ready Gene DRDQ Low violet HLA-Ready Gene B27 Low yellow 1 32 HLA-Ready Gene B27 Low yellow 1 96 HLA-Ready Gene B5/57 cross yellow 8 12 HLA-Ready Gene Coeliac Disease blue/orange 2 32 HLA-Ready Gene B57 Low green 1 32 HLA-Ready Gene Narcolepsy red 1 32 TABLE OF CONTENTS 1. GENERAL PRODUCT INFORMATION PRINCIPLE OF THE METHOD HLA-READY GENE CLASS I HLA-READY GENE CLASS II MATERIAL COMPONENTS OF THE HLA-READY GENE KITS STORAGE AND SHELF LIFE ADDITIONALLY REQUIRED MATERIALS METHOD PROCEDURE OBTAINING SAMPLES PRECAUTIONS PCR / AMPLIFICATION GEL DETECTION EVALUATION PERFORMANCE DATA/PERFORMANCE ASSESSMENT GENERAL WARNINGS AND PRECAUTIONS TROUBLE SHOOTING EXPLANATION OF SYMBOLS LITERATURE... 7

2 Page 2 1. GENERAL PRODUCT INFORMATION 1.1 Principle Of The Method INNO-TRAIN s molecular biological SSP detection systems are based on the Polymerase Chain Reaction (PCR), which enables amplification of defined DNA sequences. After successful amplification, the genomic DNA target sequence is present in a detectable concentration. The term SSP, Sequence Specific Priming, describes a variant of PCR, in which only the sequence of the primer at the 3 end is responsible for specification of the allele to be identified. For complete PCR SSP analysis, several amplifications are performed in parallel. PCR samples in which the primer binds to its specific target have a specific amplificate following PCR, while samples without this primer-specific binding do not. Primers are also present for amplification of the sample s internal. If no specific product is present after PCR, the amplificate of this positive must be clearly detectable. Evaluation of the result is performed by agarose gel electrophoresis. In the electric field the amplificates separate according to their size. Under UV light, the bands are visualized by intercalating ethidium bromide and can be photographed for documentation HLA-Ready Gene Class I INNO-TRAIN s HLA-Ready Gene Class I test system enables low-resolution typing of HLA-A*, -B* and - C*, comparable with serological HLA typing methods. The prealiquoted and dried primer cocktails are amplified. The positive amplificate (HGH) in each reaction tube is present in two different sizes (800 bp & 1070 bp). The large amplificate (HGH, 1070 bp) codes the tube row of the PCR tube block to prevent incorrect gel loading. Therefore the 1070 bp HGH amplificate is in the second tube of the second row and the third tube of the third row, whereas the other reaction tubes contain the small HGH fragment (800 bp). The first tube of the first row (black mark) contains the negative (430 bp HGH amplificate), with the exception of HLA-Ready Gene B27 low, B57 low and B5/57cross (additional negative tube = contamination ) HLA-Ready Gene Class II INNO-TRAIN s HLA-Ready Gene Class II test system enables low-resolution typing of HLA-DRB1*, DRB3*, DRB4*, DRB5* and DQB1*, comparable with serological HLA typing methods. The prealiquoted and dried primer cocktails are amplified. The positive amplificate (HGH) in each reaction tube is present in two different sizes (430 bp & 800 bp). The large amplificate (HGH, 800 bp) codes the tube row of the PCR tube block to prevent incorrect gel loading. Therefore the 800 bp HGH amplificate is in the second tube of the second row and the third tube of the third row, whereas the other reaction tubes contain the small HGH fragment (430 bp). The first tube of the first row (black mark) contains the negative (430 bp HGH amplificate), with the exception of HLA-Ready Gene DQ low, HLA-Ready Gene Coelic Disease and Narcolepsy (additional negative tube = contamination ). 2. MATERIAL 2.1. Components of the HLA-Ready Gene Kits. 96-tube PCR trays with labelled cover foil (HLA-Ready Gene A, B, C, DR, DQ, ABC, ABDR, DRDQ, B5/57 cross) Contain the pre-aliquoted and dried primer cocktails, consist of sequence specific and internal primers. Primer set no.1 is in the first reaction tube (black mark) of each PCR block. Each tray consists of reaction tube blocks (RT blocks) which are connected by the covering foil. One RT block is required for one typing. 8 PCR Strips (HLA-Ready Gene B27, B57, Coeliac Disease, Narcolepsy) Contain pre-aliquoted and dried primer cocktails that consist of sequence-specific and internal primers. Depending on the kit, one or two tubes of an 8-tube strip, which can be cut as necessary, are required for one typing (see the following table). The primer cocktails consist of HLA and HGH primers (internal ). reaction tubes with 0.5 ml of PCR buffer (ReadyPCR) Contain dntps, PCR buffer, cresol red, glycerine 8-well lid strips, colourless or 1 silicone mat, for 96 well trays Instructions for use

3 Protocol Sheets and Interpretation Help Worksheet (PCR protocol) Evaluation table (Worksheet) Specificity table Interpretation table 1 Interpretation table 2 Page 3 (Allele groups) (List of frequent alleles with details of frequency and serological equivalents) CD containing Instructions for use Protocol Sheets and Interpretation Help Kit- and Lotspecific INNO-TRAIN evalutation software (exept KIR, B27 low, B57 low, B5/57 cross, Coeliac Disease and Narcolepsy) Typing kit -file for the Score TM software (exept KIR, B27 low, B57 low, B5/57 cross and Coeliac Disease) Article Typings per PCR tubes per ReadyPCR Coverstrips test kit typing buffer 0,5ml A, DR, C ABDR, ABC DRDQ B DQ Neg. B 27, B 57, Narcolepsy Neg. B5/57 cross Neg. Coeliac Disease 2.2. Storage And Shelf Life Neg. Misc PCR tubes negative 1 integrated 32 PCR tubes with negative (=contamination ) 1x GenLadder 1x positve DNA 1x negative DNA PCR tubes with negative (=contamination ) 1 integrated 32 PCR tubes with negative (=contamination ) 1x GenLadder 1x positve DNA 1x negative DNA The systems are despatched chilled. After receipt the kit should be stored at 20 to 30 C. The shelf life of the individual components and the entire system is stated on the respective label Additionally Required Materials For amplification For gel detection Distilled water Agarose gel apparatus, e.g. INNO-TRAIN Sample DNA (50 ng/µl, ratio A 260/280 > 1.7) Agarose, ethidium bromide, e.g. INNO-TRAIN Taq polymerase (5 U/µl), e.g. Power source INNO-TRAIN or Ampli-Taq DNA Polymerase * Pipettes ( µl) TBE buffer (running buffer), e.g. INNO-TRAIN Pipette tips 100 bp ladder, e.g. INNO-TRAIN Thermocycler (see below) UV transilluminator, photo documentation * Use of other DNA polymerase enzymes must be validated by the user.

4 Page 4 3. METHOD PROCEDURE 3.1 Obtaining Samples EDTA or citrate blood should be used for the procedure as heparin residues following DNA extraction can inhibit the PCR. In DNA extraction from blood, the leukocytes act as the DNA source but in general genomic DNA of any origin (hairs, buccal swab, etc.) can be used for the method. The sample DNA should be dissolved in distilled water or Tris buffer (10mM Tris, ph 7-8, max. 1mM EDTA) in a concentration of ng/µl. If the DNA concentration is less than 25ng/µl the volume for the mastermix can be adjusted. The ratio OD 260 /OD 280 should be 1.8 ±10%. Optimal typing results are obtained if 50 ng of DNA are used per reaction mix (in reference to frequent alleles (frequency 1:1000) comparable with serological HLA typing methods). Occasionally DNA samples that are extracted by automated systems or magnetic beads (e.g. BEXS 12 by INNO-TRAIN) that have a concentration of less than 25 ng/µl can be used successfully with a lower concentration. 3.2 Precautions To avoid contamination within the PCR method. Spacial separation of the pre-pcr area (DNA isolation, storage, PCR sample) from the post- PCR area (thermocycler, gel electrophoresis, analysis) Components of the post-pcr area must not get into the pre-pcr area. Use pipette tips with aerosol protection in the pre-pcr area. To perform molecular biological methods in HLA typing. Experience in HLA and DNA diagnostics is necessary. 3.3 PCR / Amplification The procedure and pipetting volumes per PCR test are the same in all HLA-Ready Gene kits Thaw the PCR blocks, Ready PCR and DNA sample (conc. approx. 50 ng/µl). Before removing the cover foil from the PCR blocks, check the lot number and expiry date of the primers against the protocol forms. Primer mix no.1 is in the black marked well. Produce the master mix (see table), mix well and pipette before adding DNA 10 µl in the negative tube. Add DNA to the remaining mastermix, mix well and dispense 10µl aliquots in the remaining wells of the PCR block. Article PCR tubes per typing PCR water (6 µl/well) ReadyPCR (3 µl/well) Taq Polymerase (0.08 µl/well) sample DNA (1 µl/well) Volume µl/typing A, DR, C ABDR, ABC DRDQ B DQ, B5/57 cross B27, B57, Narcolepsy 8 + 1Neg Neg Coeliac Disease 2 + 1Neg The stated volumes contain a pipetting excess and are for a single test. The protocols contain details for calculating parallel samples. Close the wells with cover strips / PCR mat and transfer to thermocycler. Check that the samples are sealed and start PCR.

5 Page 5 PCR-Programme (for all HLA-Ready Gene kits) 1x Initial 10x cycles 20x cycles hold 96 C 2 min. 96 C 15 sec. 96 C 15 sec. 4 C endless 65 C 60 sec. 61 C 50 sec. 72 C 30 sec. The programme has been validated with thermocyclers PE 9600, PE 9700 (Applied Biosystems/Perkin Elmer),PTC 100 (MJ Research/Biorad) and T-Professional 96 (Biometra). If the PE 9700 is used, the emulation should correspond to that of the PE 9600 (modified ramp setting: 1 C/sec). Use of other thermocyclers must be validated by the user. 3.4 Gel Detection Band evaluation is performed by agarose gel electrophoresis. The INNO-TRAIN gel system enables sample loading with an 8-channel pipette using only 100 ml agarose to separate 96+6 samples. Make up 2 % agarose gel with 1x TBE buffer or use INNO-TRAIN ready to use agarose gel. Heat the gel and then cool to C. Add ethidium bromide to obtain a final concentration of 0.7 µg/ml or add 3 drops of the ready to use INNO-TRAIN EtBr solution per 100 ml of gel and pour the gel. Place the sample combs in the liquid gel and leave it to set for minutes. Put the gel in the electrophoresis chamber and add 1x TBE as running buffer. Load the gel with complete amount of amplificate per gel slot and add 3 µl of molecular weight marker (100 bp ladder, INNO-TRAIN) also per row of samples. Run the electrophoresis at 8-10 V/cm (INNO-TRAIN gel system: 200 volt for 20 minutes). Evaluation under UV light with the protocol forms supplied with the kit. 4. EVALUATION PCR products specific for HLA and belonging to the internal become visible in the gel under UV light. The amplificate of the internal sequence should be detectable as a band in all samples that are negative in their specific reaction. In positive specific reactions, the internal band may be suppressed because of competitive primer effects. The molecular weight marker added to the gel is used to determine fragment size. Evaluation starts with documentation of the HLA-specific PCR products. To do this, the pattern of the specific bands is entered in the enclosed result protocol. In a few HLA-Ready Gene kits there are PCR tests with several specific primer pairs (Multiplex PCR samples). You will find further information on evaluating all the band patterns that occur in the interpretation tables of the protocol sheets. For documentation the gel is photographed with a digital or polaroid camera. 5. PERFORMANCE DATA/PERFORMANCE ASSESSMENT As part of the performance assessment studies, all HLA-Ready Gene SSP systems were tested with pretyped (serological/molecular biological) samples. In all systems the results agreed 100%. As part of the production final, each primer mix is tested for correct positivity and negativity. A safe typing result is obtained if 50 ng of DNA are used per reaction mix. The composition of the primer mixes allows reliable identification of the antigens listed in the interpretation tables. The accuracy and reproducibility of the reactivity of the individual primer mixes were checked using samples with known antigens. 6. GENERAL WARNINGS AND PRECAUTIONS Ethidium bromide is a mutagenic and carcinogenic fluorescent dye, which must be used only when wearing gloves. UV light can cause burns of the skin and retina. Suitable UV face protection should therefore be worn. In samples of human origin, there is still a potential risk of infection even after DNA extraction. Gloves and labcoats should therefore be worn when performing the PCR SSP method and all recommendations on handling infectious material should be followed. Reagents should not be used after their expiry date.

6 Page 6 7. TROUBLE SHOOTING Problem Possible cause Solution DNA smear in the gel tracks; Background bands; Non-specific PCR products High DNA concentration Impure or degraded DNA Contamination Incorrect amplification conditions Primer dimer bands evaluated as positive specific reactions. Re-measure DNA conc.; Reduce the employed DNA concentration gradually. Measure OD 260 /OD 280 (a quotient of 1.8 is optimal) and evaluate on agarose gel. Reextract DNA if necessary. Check Negative Control. Check sample DNA and re-extract if necessary. Use filter tips. Check the thermocycler. The thermocyclers PE9600/9700, PTC100 (ramp 1 C/sec.) have been validated for performing the method. Check band sizes. Weak bands; Suppression of reactions Low DNA concentration Impure or degraded DNA Taq or Sample DNA not added to PCR Mix Insufficient mixing of the DNA or reactions Tubes not correctly sealed Incorrect amplification conditions No Ethidium Bromid in gel PCR product leaked out of gel pocket Re-measure DNA conc.; Increase the employed DNA concentration gradually. Measure OD 260 /OD 280 (a quotient of 1.8 is optimal) and evaluate on agarose gel. Reextract DNA if necessary. Repeat PCR. Dissolve DNA at C. Vortex well after preparing the master mix. Check the seal of the cover strips or PCR mat (use press-on mat). Check the thermocycler. The thermocyclers PE9600/9700, PTC100 (ramp 1 C/sec.) have been validated for performing the method. Staining of gel with EtBr. Correct gel preparation with straight pipette tips Further information and telephone assistance at: or mail: support@inno-train.de 8. EXPLANATION OF SYMBOLS Note accompanying documents Lot number Expiry date Article number Follow instructions for use Manufacturer Observe upper temperature limit In vitro diagnosticum Content sufficient for <n> tests

7 9. LITERATURE Page 7 1. Mullis KB, Faloona F: Specific synthesis of DNA in vitro via polymerase catalysed chain reaction. Meth. Enzym. 1987;155: Newton CR, Graham A, Heptinstall E, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF: Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Research 1989;17: Olerup O, Zetterquist H: HLA DR typing by PCR amplification with sequence specific primers (PCR SSP) in two hours: An alternative to serological DR typing in clinical practice including donor recipient matching in cadaveric transplantation. Tissue Antigens 1992;39: Bunce M, O Neill CM, Barnardo MCNM, Krausa P, Browning MJ, Morris PJ, Welsh KI: Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP). Tissue Antigens 1995;46: Bunce M, Young NT, Welsh KI: Molecular HLA Typing The brave new world. Transplantation 1997;64: Marsh SGE, Albert ED, Bodmer WF, Bontrop RE, Dupont B, Erlich HA, Geraghty DE, Hansen JA, Hurley CK, Mach B, Mayr WR, Parham P, Petersdorf EW, Sasazuki T, Schreuder GMTh, Strominger JL, Svejgaard A, Terasaki PJ, Trowsdale J: Nomenclature for factors of the HLA system, Tissue Antigens 2005;65: