DE4411594C1 - Test kit for detecting HLA gene alleles by PCR amplification - Google Patents

Abstract

Test kit for determn. of HLA histocompatibility antigens by specific PCR amplification comprise a thermostable DNA polymerase, a DNA matrix, four deoxynucleotide 5'-triphosphates (dATP, dGTP, dTTP and dCTP), a betaine-contg. PCR buffer, and two allele-specific oligodeoxynucleotide primers. The last three nucleotides at the 3' end of the primers must correspond to the target allelic sequence, the rest having at least 80% homology with the allelic sequence.

Description

The invention relates to a test kit for determining Histocompatibility antigens (HLA) in DNA samples specific PCR amplification.

HLA alleles play an important role in rejection of grafts, in immune and autoimmune reactions. HLA-B27 in particular is strong with certain forms of Spondyloarthropathies are associated and can help this early on from similar clinical pictures divorce.  

Areas of application of the invention are medical Diagnostics, the pharmaceutical industry and the mole biological-biological research.

Genetic testing in a wide range of research and clinical routine are increasingly based on the Polymerase chain reaction (PCR) performed. With that the tests significantly cheaper and more reliable made. The PCR was developed in 1987 (Mullis, K.B. et al., Methods in Enzymology 155, 335-342, 1987) with her can treat small amounts of DNA by treating individual complementary strands of this DNA with a molecular excess of two oligonucleotide primers and their extension to form a DNA template quickly and safely increased (EP-A-200 362, EP-A-201 184, EP- A-258 017).

Also in US 5,023,171 is a process for the production of recombinant DNA using the polymerase chain reaction (PCR), wherein the recombinant double-stranded DNA amplified by PCR in the presence of oligo-a and oligo-d (SOE; gene splicing by overlap extension).

With the help of PCR, HLA class II Alleles routinely examined (Olerup et al., Tissue Antigens 39, 225-235, 1992).

Adrian V. S Hill et al (The Lancet, Vol. 337, March 16, 640-642, 1991) used HLA-B specific PCR and Hybridization with a B * 2703 specific oligo nucleotide and found evidence that this is for West Africa (Gambia) characteristic subtype as the only one probably not associated with Bechterev's syndrome. HLA-B specific PCR and subsequent hybridization with Oligonucleotides Specific for subtypes B * 2701 bis B * 2706 was also described by O. Dominguez et al., Immunogenetics 36, 277-282, 1992).

The similarity between organ HLA antigens donors and recipients is crucial for the success of organ transplants. Furthermore are some alleles of these genes with resistance or Susceptibility to certain diseases associated.  

B27 has one of the strongest positive genes among the HLA genes Associations with two overlapping clinical Diseases on, acute anterior uveitis (Baarsma, G. S., 1992, Current Eye Research, 11 supl., 1-9) and Spondyloarthropathies (MacLean, L. 1992, Ann. Rheum. Dis. 51, 929-931).

The latter are also, but weaker, with the Antigens B7, Bw22, B60 (Stein, M. et al., 1990, J. Rheum. 17, 1337-1339) and possibly also B44 (Thomson, G. T. D. et al., 1992, Clin. Immunology, 64, 227-232) and B62 associated. The B27-associated risk for Bechterevs Syndrome increases approximately threefold when B60 (B * 4001) is on the other HLA haplotype is present (Robinson, W. P., 1989, Arthritis and Rheumatism, 31, 1135-1140).

Of the other B27-associated clinical pictures Subforms are preferably associated with other HLA antigen. B13, B16 and B17 (B57 and B58) can help "rheumatic" psoriatic arthritis from "spondyli table "(B27) (Salvarani, C. et al., 1989, J. Exp. Rheum., 7, 391-396; Torre-Alonso, J.C. et al., 1991, Brit. J. Rheumatol., 30, 245-250). A similar Differentiation with the help of B62 is probably with Crohn's disease possible.

B60 and B15-DR4 haplotypes may also help Differentiate between subforms and rheumatic diseases (Sanders, P.A. et al., 1988, Tiss. Ant., 33, 21-29; Charles, P.J. et al., 1991, Disease Markers, 9, 97-101; Brand, C.A. et al., 1992, Ann. Rheumatism. Dis., 51, 173-176). The haplotype B44-C4A * 3C4BQ * O-DR4 is becoming increasingly common Patients with Feltys syndrome in rheumatoid arthritis before (Hammond, A. et al., 1992, Clin. Exp. Imm., 88, 163-168).  

B60 and haplotype B14-DR1 are also milder Forms of 21-hydroxylase deficiency in Northern Europe Population associated (Speiser, P.W. et al., 1988, N. Engl. J. Med., 319, 19-23; Sinnott, P.J. et al., 1991, Hum. Genet., 87, 361-366; Azziz, R. et al., 1991, J. Clin. Endocrinol. Metab., 73, 1327).

Although different variants of the PCR reaction can be developed safely This reaction is generally not predicted to succeed become. The selection of the for is essential Structure of the primer suitable for the matrix. But even if that desired sequence known and that for hybridization suitable primer is selected is a successful one Amplification not yet guaranteed. Numerous examples show that after knowing the desired sequence selected primer not for the expected amplification has led.

The aim of the invention is a test kit for determination of histocompatibility antigens based on the PCR Develop reaction. It is based on the task to build suitable primers and the reaction to design conditions so that a safe Amplification is guaranteed.

The invention is implemented according to claim 1, which Subclaims are preferred variants. Essential feature are the primers used in the test kit, which at the 3'-end in the last three nucleotides with the one you are looking for Allele sequence match and in the rest of the sequence too are at least 80% homologous with the sequence sought, and the betaine buffer according to the invention. It was found that Base mismatches at the 3'-end of the primer after Extension by Taq polymerase are bothersome especially the last base pair is crucial.  

Mismatches on the fourth and the further base pairs from 3'-end removed hinder the DNA polymerase reaction practically no more.

The test kits according to the invention each contain the specific primers for individual HLA antigens according to Claims 3-19.

Below is an overview of those with the test kits according to the invention, determinable HLA-B alleles and the 3'- and 5'- according to the invention contained in the test kit Given primer. The test kits according to the invention can also each one of the 3'- and 5'-primers mentioned below other than the combination described. In addition, simultaneous amplifications are different Alleles and allele groups possible if several of the 3'- and 5'-primers described below in the same Reaction can be used ("Multiplex-PCR"). Corresponding can the test kits according to the invention in a special Embodiment also include several 3'- and 5'-primers.  

The primers for the internal controls are in the Test kits according to the invention contain:

The test kits according to the invention can be used in PCR known and usual buffers. In a preferred embodiment, however, they contain one Betaine (N, N, N-trimethylglycine) buffer for PCR Amplification.

The buffer can contain 200-1000 mM betaine. It exists in a preferred variant from 500-800 mM betaine and 1-6 mM magnesium chloride. In another preferred Variant consists of 500-800 mM betaine, 1-6 mM magnesium chloride, 6-14 mM N-tris (hydroxy methyl) methylglycine with pH 8.3 and 80-120 µg / ml bovine Serum albumin V.

The selection of the buffer according to the invention is based on one on the finding that already small Salt concentrations in the PCR medium amplification certain HLA-B alleles. Of the In contrast to the usual, buffers therefore contain Do not buffer potassium or ammonium chloride.  

Betain also combines a number of advantages more conventional, previously in PCR and reverse Transcription (RT) cosolvents used:

It is chemically inert and non-ionic, i.e. H. it can with a number of other cosolvencies and salts combined to find optimal reaction conditions for the to create the respective primer and template.

It also makes transcription easier for GC-rich people Domains. Since only GC base pairs are destabilized, primers can be chosen so that their hybridization is affected as little as possible with the target sequence.

General destabilization of dsDNA, e.g. B. by glycerol, dimethyl sulfoxide or formamide, also lowers the hybridization temperature of the primer and thus has both positive and negative effects on the yield of DNA polymerase reactions. Betain is unique among non-ionic cosolvents in its ability to bind and thereby stabilize AT base pairs. Since non-ionic cosolvents generally destabilize dsDNA, a specific destabilization of GC-rich domains in DNA and RNA takes place overall in the presence of betaine. However, betaine is considerably cheaper than the deoxyguanidine analogues that have been successfully used for this purpose in sequencing and PCR and can also be used in RT. AT-rich primers such as B. (dT) _12-18 in RT, however, are less affected than GC-rich domains in the template.

Magnesium chloride is an essential cofactor for DNA Polymerases, however, the optimal concentration depends on the PCR strongly from the respective primers or templates from. In the presence of betaine, this optimal one expands Area, whereby the optimization, in particular of  Co-amplifications ("multiplex-PCR"), simplified.

Furthermore, it is known that both NaCl and heparin can occur as contaminants in DNA samples. In concentrations between 2.5 · 10 ^-4 -10 ^-3 U / µl heparin has similar effects to NaCl. In the prior art, heparinate digestion or pretreatment of heparin-containing DNA with Chelex 100 has therefore been proposed (Francesca Poli, Rosa Catteano, Loretta Crespiatico, Angelea Nocco and Girolamo Sirchia, PCR Methods and Applications, 1993, 2, 356-358).

It has now been found that the PCR yields, in particular of HLA-B in DNA samples containing heparin in Presence of betaine can be significantly improved. These findings are preferably shown in Betaine concentrations of 500-800 mM. In present of 0.8 M betaine is up to 10 times higher heparin concentrations tolerated.

So betaine does the purification of salt or heparin DNA samples containing largely superfluous what before especially in the routine examination of clinical and forensic samples is beneficial.

In addition, in contrast to the cosolvencies, betaine is DMSO, Formamide and methyl mercury hydroxide are non-toxic and a natural protection, the cells in the course of evolution against thermal and ionic denaturation of their proteins have developed.

The activity of Taq polymerase and the reverse Transcriptase is not noticeable through betaine impaired.  

As a temperature-stable DNA polymerase according to the invention preferably Taq polymerase used.

The advantages of the test kit according to the invention over the State of the art are in the following points

• a) The betaine buffer makes the reaction more robust different methods of DNA preparation and NaCl Impurities, as already stated.
• b) The primers used according to the invention are selected for maximum specificity and robustness, the following points being important
  • Detection of as few HLA alleles as possible,
  • as far as possible avoidance of 3′-deoxy-thymidine in primers or in the complementary position on all known alleles of HLA class I,
  • - Check the last 10 nucleotides for common sequence motifs in the human genome (comparison with a database of human sequences: Genbank Release 81.0, 2/94) such as B. Preserved structures of the immunoglobulin family to reduce the risk of PCR artifacts.
• c) The number of PCRs required for B27 amplification Cycles is less than in the prior art.
• d) The kit according to the invention can be designed flexibly to some or more of those with Spondylo arthropathy-associated B7 cross-reactive antigens B60 (B40), B27, Bw22 and B7 as well as the possibly associated alleles B44 and B62 (B15) or that with Psoriatic arthritis associated allele B13 too determine.  
• e) The internal controls (Gene XA / XB) contain one well documented markers for an HLA class III Locus, through which in some cases the one with the HLA-B Allelic associated MHC haplotype can be assessed can.
• f) The internal controls have been carefully selected (Gene XA / XB or TNFβ) and tested to ensure that the HLA-B product is among all Conditions are amplified equally well or better.
• g) The amplified HLA-B products vary in their Length of about 400-800 nucleotides, so that the Possibility to have multiple HLA determinations in the same Reaction volume in order to reduce costs and Save labor.

In summary, it can be stated that the test kit according to the invention to a significant advance in the diagnosis of spondyloarthritis and the like Diseases.

The invention also relates to the use of HLA allele-specific oligodeoxynucleotide primers that at the 3′-end in the last three nucleotides with the searched allele sequence match and in the rest Sequence at least 80% homologous to the allele sequence and a betaine-containing buffer for determination of histocompatibility antigens in DNA samples specific PCR amplification and in particular for Amplification of a fragment of the HLA-B gene containing Sequences from exon 2 to exon 3.

The invention is intended to be described in the following by means of embodiments are explained in more detail.

Embodiments a) Reaction approach

The amplifications are carried out in DNA thermal cyclers (e.g. the DNA thermal cycler 480 from Perkin-Elmer-Cetus) in 500 µl "Eppendorf" reaction vessels carried out.

DNA is made by digesting samples to be examined Proteinase K and subsequent phenol / chloroform extraction won. Amounts of 50-1000 ng in a 20 ul reaction volume used.

The approach also includes:

10 mM N-tris (hydroxymethyl) methylglycine, pH 8.3 at 20 ° C (tricin)
100 µg / ml bovine serum albumin (BSA V)
0.2 mM per nucleotide triphosphate (dATP, dCTP, dGTP, dTTP)
3 ng / µl per primer
0.025 U / µl Taq polymerase

with TNFβ as control:
600 mM N, N, N-trimethylglycine (betaine)
3 mM magnesium chloride
with genes XA / XB as control:
800 mM N, N, N-trimethylglycine (betaine)
4 mM magnesium chloride

b) temperature steps

The PCR programs are in the following temperature steps carried out:

According to the invention, they are found in the presence of betaine Hybridization temperatures that are about 10 ° C lower lie than the theoretical denaturation temperature of the Primer, and MgCl₂ concentrations between 3 and 4 mM as advantageous.

c) detection

Successful amplification of internal control and optionally an allele-specific HLA-B fragment is by electrophoresis in a 1.5% agarose gel, 0.5 × TBE buffer checked. DNA bands are stained with ethidium bromide and fluorescence under a UV lamp made visible. The length of the PCR products obtained is a control for the specificity of PCR for HLA-B.  

d) results

HLA-B27, B7, B8, B41, B42, B60, B61 and B73 were successfully amplified under the above conditions. The specificity of the HLA-B PCR was tested using the following standard cell lines:
There were positive controls

• a) for B27 and its subtypes: HOM2, JESTHOM, WT24, LS40 as well as the non-standardized lines LH, NW, R69 and Wewak,
• b) for B60 (B * 40012): SLE, MADURA, MT14B, PE117, BRU, RLO, LS40 (B27 / B60)
• c) for B7: HHKB, SAVC, LD2B, R69 (B7 / B27)
• d) for B8: VAVY, LH (B8 / B27)
• e) for B42: RSH
• f) for B41: RLO (B38 / B41)
• g) for B61 (B * 4002): SWEIG.

Numerous other representative, standard for the most common HLA-B alleles lines or lines whose allele sequence to one of the two primers used was completely complementary (e.g. B14 and B16 with B7CREG1 in the B27-PCR).

From 51 both serologically as well as with the help of PCR HLA-B27 donors examined became positive in 43 cases Agreement between the two tests and in 6 cases negative match found. A sample of one Patients with Bechterev were serologically negative and B27 positive based on the PCR result. Once no PCR was Product from a serologically positive healthy donor  receive. The specificity of the PCR products has continued by gel electrophoresis (size) and in the Amplification products of 22 different completely HLA-B typed B27 heterozygous samples by digestion confirmed with restriction enzymes.

Nonspecific (co) amplifications were found under the above Conditions not observed.

To the influence of tetramethylammonium chloride, betaine and NaCl towards the amplification of B7, B8 and B27 four buffers containing 50 mM KCl and 1.5 mM MgCl₂ tested: two commercially available (Boehringer Mannheim, Promega) with Tris and two of their own with Tris or tricine.

It was found that 50 mM KCl as well as low Impurities with 5-10 mM NaCl (final) the PCR of the HLA B alleles of the internal controls only at 70 mM NaCl Hinder concentrations. Improved amplification will however achieved in the presence of 50 mM TMAC1 and optimal PCR of HLA-B7, B8 and B27 at 0.6-1.0 M betaine and 2.5-4 mM MgCl₂ concentrations. Above these limits Betaine inhibits PCR with the above called primers. During the amplification of the internal Controls in the absence of isostabilizing substances more robust than the HLA-B PCR, this reverses Ratio in the presence of betaine and TMAC1 um.

References for information on the genomic position of the primers

Accession numbers refer to Genbank Release 81.0 (2/94). The Positions of the HLA-B primers were given as they are on the sequence X03945 of the allele would be HLA-B * 2705, regardless of possible 3′-mismatches. The Length of the PCR products for the different primer combinations were determined also stated as if a fragment of the sequence X03945 were amplified.

• a) HLA-B primer: Accession X03945
  Authors: Weiss, EH, Kuon, W., Doerner, C., Lang, M. and Riethmüller, G.
  Title: Organization, sequence and expression of the HLA-B27 gene:
  A molecular approach to analyze HLA and disease associations
  Journal: Immunobiology 170, 367-380 (1985)
• b) XA / XB Primer: Accession X71937
  Authors: Bristow, J., Tee, MK, Gintelman, SE, Mellon, SH and Miller, WL
  Title: Tenascin-X: a novel extracellular matrix protein encoded by the human XB gene overlapping P450c21B
  Journal: J. Cell Biol. 122, 265-278 (1993)
• c) TNFβ primer: Accession M16441
  Authors: Nedospasov SA, Shakhov AN, Turetskaya RL, Mett VA, Azizov MM, Georgiev GP, Korobko VG, Dobrynin VN, Filippov SA, Bystrov NS, Boldyreva EF, Chuvpilo SA, Chumakov AM, Shingarova LN, Ovchinnikov YA;
  Title: "Tandem arrangement of gene coding for tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) in the human genome";
  Journal: Cold Spring Harb. Symp. Quant. Biol. 51: 611-624 (1986).
• d) B * 40011: Accession M27540
  Authors: Arnot D., Lillie JW, Auffray C., Kappes D., Strominger JL;
  Title: "Inter-locus and intra-allelic polymorphisms of HLA class I antigen gene mRNA"; (Figure 3. The nucleotide sequences of the three clones JY103, (LB4.2, and LB45.)
  Journal: Immunogenetics 20: 237-252 (1984).
• e) B * 40012: Accession M95530
  Authors: Kawaguchi, G., Kato, N., Kashiwase, K., Karaki, S., Kohsaka, T., Akaza, T., Kano, K. and Takiguchi, M.
  Title: Structural analysis of HLA-B40 epitopes
  Journal: Hum. Immunol. 36, 193-198 (1993)

Claims (26)

1. Test kit for the determination of histocompatibility anti genes (HLA) in DNA samples by specific PCR amplification, including temperature-stable DNA polymerase, a DNA template, the deoxynucleotide 5′-triphosphates dATP, dGTP, dTTP and dCTP, a buffer for the polymerase chain reaction and two allele-specific oligo deoxynucleotide primers, characterized in that the buffer contains betaine and the primers used at the 3 'end in the last three nucleotides match the allele sequence sought and in the rest of the sequence are at least 80% homologous to the allele sequence. 2. Test kit according to claim 1 for the determination of HLA-B Alleles. 3. Test kit according to claim 1 or 2 for determining a HLA-B27 allele, characterized in that it has a 3′-primer with the sequence 5 ′ CCA CGT CGC AGC CAT ACA TA 3 ′, specific for the alleles B * 2701, B * 2702, B * 2703, B * 27051, B * 27052, B * 2706 and either a 5′-primer with the sequence 5 ′ GCC GCG AGT CCG AGA GA 3 ′, specific for the alleles and serological Specificities B7, B8, B14, B * 1509, B * 1510, B16, B27, B42, BSS, B56, B73  or a 5 'primer with the sequence 5' CGC CGC GAG TCC GAG AG 3 ', specific for the alleles and serological speci B7, B8, B14, B * 1509, B * 1510, B16, B27, B42, B48, B54, B55, B56, B73, B79 or a 5′-primer with the sequence 5 ′ CTC CCA CTC CAT GAG GTA TTT CC 3 ′, specific for the alleles and serological speci B18, B27, B37, B40, B41, B45, B49, B50, B73 includes. 4. Test kit according to claim 1 or 2 for determining the B27 Subtypes B * 2701, B * 2703, B * 2704, B * 2705, B * 2706 and for Differentiation of these subtypes from B * 2702, thereby characterized that he is a 5'-primer with the sequence 5 ′ GAC CGA GAG AAC CTG CGC AC 3 ′, specific for the alleles and serological Specificities B13, B27 (with the exception of the subtype B * 2702), B37, B44, B47 and a 3′-primer with the Sequence 5 ′ CCA CGT CGC AGC CAT ACA TA 3 ′, specific for the alleles B * 2701, B * 2702, B * 2703, B * 2704, B * 27051, B * 27052, B * 2706 includes.   5. Test kit according to claim 1 or 2 for determining a HLA-B44 allele, characterized in that it has a 5′-primer with the sequence 5 ′ GAC CGA GAG AAC CTG CGC AC 3 ′, specific for the alleles and serological speci B13, B27 (with the exception of subtype B * 2702), B37, B44, B47 and the 3'-primer has the sequence 5 'CAC CAG GTA TCT GCG GAG CG 3'. 6. Test kit according to claim 1 or 2 for determining a HLA-B45 allele, characterized in that it has a 5′-primer with the sequence 5 ′ CTC CCA CTC CAT GAG GTA TTT CC 3 ′, specific for the alleles and serological speci B18, B27, B37, B40, B41, B45, B49, B50, B73 and a 3′-primer with the sequence 5 ′ CAC CAG GTA TCT GCG GAG CG 3 ′. 7. Test kit according to claim 1 or 2 for determining a HLA-B7 allele, characterized in that it has a 5′-primer with the sequence 5 ′ GTA TTG GGA CCG TAA CAC ACA GAT CTA 3 ′, specific for the alleles and serological speci B7, B * 40011, B42, B54, B55, B56 and one 3′-primer with the sequence 5 ′ CGT AGC CAC TCC ACG CAC TC 3 ′, specific for the alleles and serological speci B7, B13, B27, B40, B47, B * 4801, B73. 8. Test kit according to claim 1 or 2 for determining the B7 subtypes B * 0701, B * 0702 and for distinction of these subtypes of B * 0703, characterized in that he a 5'-primer with the sequence 5 ′ GTA TTG GGA CCG TAA CAC ACA GAT CTA 3 ′, specific for the alleles and serological speci B * 0701, B * 0702, B * 40011, B42, B54, B55, B56 and a 3'-primer with the sequence 5 'CGT AGC CAC TCC ACG CAC TC 3', specific for the alleles and serological speci B7, B13, B27, B40, B47, B * 4801, B73. 9. Test kit according to claim 1 or 2 for determining a HLA-Bw22 allele, characterized in that it has a 5′-primer with the sequence 5 ′ GAA CAC ACA GAT CTA CAA GGC CC 3 ′, specific for the alleles and serological speci B * 0701, B * 0702, B42, B54, B55, B56 and one 3′-primer with the sequence 5 ′ TGT AAT CCT TTC CGT CGT AGG CTA 3 ′, specific for the alleles and serological speci B13, B45, B49, B50, B54, B55, B56. 10. Test kit according to claim 1 or 2 for determining a HLA-B13 allele, characterized in that it has a 3′-primer with the sequence 5 ′ TGT AAT CCT TTC CGT CGT AGG CTA 3 ′, specific for the alleles and serological speci B13, B45, B49, B50, B54, B55, B56 and either a 5′-primer with the sequence 5 ′ GAC CGA GAG AAC CTG CGC AC 3 ′, specific for the alleles and serological speci B13, B27 (with the exception of subtype B * 2702), B37, B44, B47 or a 5′-primer with the sequence 5 ′ CGC GAG TCC GAG GAT GGC 3 ′, specific for the alleles and serological speci b * 1501, B * 1502, B * 1504, B * 1505, B * 1506, B * 1507, B * 1516, B * 1517, B13, B46 and B57 included. 11. Test kit according to claim 1 or 2 for determining the HLA alleles B14, B18 or B73, characterized thereby records that he is a 3'-primer with the sequence 5 ′ CTC CTT CCC GTA CTC CAG GTG 3 ′, specific for the alleles and serological speci B14, B18, B * 5101, B * 5103, B * 5104, B52 and B73 such as a 5′-primer for the amplification of B14 and B73 with the sequence 5 ′ GCC GCG AGT CCG AGA GA 3 ′, specific for the alleles and serological speci B7, B8, B14, B * 1509, B * 1510, B16, B27, B42, B48, B54, B55, B56, B73 or a 5′-primer with the  Sequence 5 ′ CGC CGC GAG TCC GAG AG 3 ′, specific for the alleles and serological speci B7, B8, B14, B * 1509, B * 1510, B16, B27, B42, B48, B54, B55, B56, B73, B79 such as a 5′-primer for the amplification of B18 and B73 with the sequence 5 ′ CTC CCA CTC CAT GAG GTA TTT CC 3 ′, specific for the alleles and serological speci B18, B27, B37, B40, B41, B45, B49, B50, B73 includes. 12. Test kit according to claim 1 or 2 for specific Amplification of alleles and serological specificities Bw21 or B45, characterized in that it has a 3′-primer with the sequence 5 ′ GAG GAG GCG CCC GTC G 3 ′, specific for the alleles and serological speci B35, B45, B * 4802, B49, B50, B53 and B58 and a 5′-primer with the sequence 5 ′ CTC CCA CTC CAT GAG GTA TTT CC 3 ′, specific for the alleles and serological speci B18, B27, B37, B40, B41, B45, B49, B50, B73 includes.   13. Test kit according to claim 1 or 2 for determining the Allels B * 4001 using B41 / B * 4001-specific Amplification, characterized in that it has a 5′-primer with the sequence 5 ′ CTC CCA CTC CAT GAG GTA TTT CC 3 ′, specific for the alleles and serological speci B18, B27, B37, B40, B41, B45, B49, B50, B73 and a 3'-primer with the sequence 5 'CC GCG CGC TCC AGC TTG 3', specific for B7, B8, B * 40011, B * 40012, B41, B42 and B * 4801 included. 14. Test kit according to claim 13, characterized in that to distinguish between B * 40011 and B41 Reamplification of the primers from claim 13 PCR product obtained a 3'-primer with the sequence 5 ′ CGT AGC CAC TCC ACG CAC TC 3 ′, specific for the alleles and serological speci B7, B13, B27, B40, B47, B * 4801, B73 is. 15. Test kit according to claim 1 or 2 for determining the Allele group B * 1501, B * 1505, B * 1507 and B * 1517, thereby characterized that he is a 5'-primer with the sequence 5 ′ CGC GAG TCC GAG GAT GGC 3 ′, specific for the alleles and serological speci b * 1501, B * 1502, B * 1504, B * 1505, B * 1506, B * 1507, B * 1516, B * 1517, B13, B46 and B57 and a 3′-primer with the sequence 5 ′ CCC CAC GTC GCA GCC G 3 ′, specific for the alleles and serological speci b * 1501, B * 1505, B * 1507, B * 1509, B * 1510, B * 1517, B7, B8, B16 B18, B * 2707, B * 3505, B * 4001, B * 4002, B * 4003, B * 4005, B42, B * 4401, B * 4402, B * 4403, B * 4405, B * 4801, B * 5602, B79 included. 16. Test kit according to claim 1 or 2 for determining the Allele B42 using B7 / B42-specific Amplification, characterized in that it is either a 5'-primer with the sequence 5 ′ GAA CAC ACA GAT CTA CAA GGC CC 3 ′, specific for the alleles and serological speci B * 0701, B * 0702, B42, B54, B55, B56 or a 5'-primer with the sequence 5 'GTA TTG GGA CCG TAA CAC ACA GAT CTA 3', specific for the alleles and serological speci B7, B42, B54, B55, B56 and a 3'-primer with the sequence 5 'CC GCG CGC TCC AGC TTG 3', specific for the alleles and serological species B7, B8, B * 40011, B * 40012, B41, B42 and B * 4801, includes and the absence of B * 0701 / B * 0702 through the primer pair is excluded according to claim 8. 17. Test kit according to claim 1 or 2 for determining a HLA-B57 allele, characterized in that it a 5'-primer with the sequence 5 ′ CGC GAG TCC GAG GAT GGC 3 ′, specific for the alleles and serological speci b * 1501, B * 1502, B * 1504, B * 1505, B * 1506, B * 1507, B * 1516, B * 1517, B13, B46 and B57 and a 3′-primer with the sequence 5 ′ CCA CGT CGC ATC CAT ACA TCA C 3 ′, specific for the allele of the serological species b57. 18. Test kit for the determination of HLA-B alleles and Allele groups according to claim 1 or 2, characterized characterized in that it each one of the claims 3 to 17 called 3'-primers and one of each in the Claims 3 to 17 mentioned 5'-primer in any Combination contains. 19. Test kit according to claim 18, characterized in that he several of those mentioned in claims 3 to 17 3'- and 5'-primers for simultaneous amplification ver different alleles and allele groups in the same Contains reaction ("Multiplex-PCR"). 20. Test kit according to one of claims 1 to 19, characterized characterized in that it serves as internal control for Amplification of a fragment of the XB gene and, in the corresponding MHC haplotypes, in addition to the XA gene a 5'-primer with the sequence 5 ′ AAC TGC AGA GCG ACT TCC ATT C 3 ′, and a 3′-primer with the sequence 5 ′ AGG TCA TGC AGG GG TAG TCC A 3 ′.   21. Test kit according to one of claims 1 to 5, 7 to 10, 15 to 19, characterized in that it is used for Amplification of a fragment of the TNFβ gene as internal Control a 5′-primer with the sequence 5 ′ CGT GCT TCG TGC TTT GGA CTA 3 ′, and a 3 ′ primer with the sequence 5 ′ AGC TGG TGG GGA CAT GTC TG 3 ′. 22. Test kit according to one of claims 1 to 21, characterized characterized as a thermostable DNA polymerase Contains Taq polymerase. 23. Test kit according to one of claims 1 to 22, characterized characterized that the buffer 200-1000 mM betaine contains. 24. Test kit according to one of claims 1 to 23, characterized characterized in that the buffer 500-800 mM betaine and Contains 1-6 mM magnesium chloride. 25. Use of HLA allele-specific oligodeoxy nucleotide primers at the 3′-end in the last three Nucleotides with the allele sequence sought match and at least in the rest of the sequence 80% are homologous to the allele sequence, and one Betain-containing buffer for the determination of Histocompatibility antigens in DNA samples specific PCR amplification. 26. Use according to claim 25 for the amplification of a Fragment of the HLA-B gene containing sequences from Exon 2 to Exon 3.