CN1952170A - Liquid-phase gene chip system for human leukocyte antigen gene typing - Google Patents

Abstract

The invention involves biomedical field, particularly involves the detecting method and agent of the human leukocyte antigen genotyping. The method comprises the steps of: designing and synthesizing a set of probe by selecting the main variability region of three sites of HLA-A, -B, -DRB1 with most abundant polymorphism in human leukocyte antigen gene complexes, covalent-linking the probe to the fluorescent microspheres vector surface, each different fluorescent microspheres coupling a probe, balanced-mixing probes with different fluorescent microspheres to compose the liquid gene chip. The invention carries out balanced amplification of a number of gene fragments, the probe hybrids with target gene fragments in liquid environment, which has more hybridization efficiency compared with liquid-solid phase hybridization of traditional gene chip. In addition, the hybridization can be carried out in the ordinary 96-well plates, facilitates the pipetting operation with the use of multi-channel pipetting gun, the whole hybridization is convenient, rapid, which can effectively carry out high-throughput gene-typing.

Description

A kind of liquid phase Gene Chip system that is used for HLA gene's somatotype

Technical field

The present invention relates to biomedical sector, relate in particular to human leucocyte antigen (HLA) (Human Leukocyte Antigen, HLA) detection method of gene type and reagent.

Background technology

The HLA gene is positioned at human chromosome 6p21.3, and the about 4000kb of total length is the most complicated system of genetic polymorphism in the Human genome.HLA is the highest gene complex of polymorphism in the known so far, and existing known have more than 2000 allelotrope approximately.According to structure and characteristic distributions, the HLA mixture can be divided three classes, and is referred to as I, II, III genoid respectively.The I genoid comprise HLA-A ,-B ,-C ,-E ,-F ,-G ,-H ,-J ,-K ,-totally ten kinds of L.The II genoid has 27 kinds of DR, DP, DQ series etc., and its molecule mainly is distributed in the antigen presenting cell surface.In addition, also have some to constitute the III genoid of complement component.The various combination of these genes constitute vary individual HLA genotype and corresponding specificity.The polymorphism of HLA gene and the genetic predisposition of disease have obvious relation.The HLA gene product controlling immune response to cause of disease, to tolerance or repulsion and the oncological surveillance of graft.So clinically especially on the transplantation medicine, must be to carrying out the HLA somatotype for acceptor.Traditional HLA typing method mainly contains three classes: serology, cytology and molecular Biological Detection method.Serology, cytology HLA classifying method have many weak points on somatotype accuracy, resolving power and experimental implementation.Serology and cytological method are eliminated gradually both at home and abroad in recent years, the main biological method of developer molecule is come somatotype.Present molecular biological method has multiple, mainly comprise pvuii restriction fragment (RFLP) detection technique, PCR/SSO (sequence specific oligonucleotide probetyping) technology, PCR/SSP technology, wherein anti-phase SSO is most widely used genotyping technique.

Since the nineties, gene chip (genechip) technical development is very fast.So-called gene chip is meant the dna fragmentation of various particular sequences (or is claimed probe, probe), is integrated on solid phase carrier such as glass-chip or the silicon by means such as little point sample or molecule operating and microelectronic chip technology.During application, this chip that is integrated with numerous probes is hybridized with the testing sample DNA after handling by DNA basepairing rule (A and T, G and C pairing).If the sequence of sample DNA and probe match fully, this accurate pairing is in conjunction with detecting by marking signal.If unpaired, meaning has sudden change, and this sudden change can be the identification of somatotype software.Gene chip can be applicable to key areas such as the research of gene discriminating, rapid DNA sequencing, transgenation polymorphism and activity of gene expression mensuration in fundamental research.It can be used as the comprehensive diagnos of molecular genetic disease in medical science, be applied to medical jurisprudence and cancer research etc.

The principal feature of gene chip is an integrated probe in a large number in very little zone.Traditional gene chip is to encode to probe by the point sample position on the solid phase carrier, and software is according to the detection signal of this adress analysis sample.The advantage of this gene chip is can highly integrated probe, promptly utilizes a chip to detect simultaneously and reaches hundreds thousand of signals.Because the high complexity of DNA sample, the detected result repeatability of traditional gene chip generally is not fine.In addition, traditional gene chip is owing to be that solid-liquid is hybridized mutually, and very high to the control requirement of hybridization, probe also can influence the result of hybridization each other, and then the Interference Detection signal.When realizing high throughput testing, traditional gene chip also needs expensive detecting instrument system.

Chinese patent CN1185353C has explained a kind of method of design of gene chip, will be at the allelic probe stationary of HLA gene's complex body on various membrane matrixs, with enzyme connection substrate development process detection signal.The stability of this method and repeatability are undesirable, realize that high throughput testing is also difficult.

Therefore, this area presses for high-throughput, good stability, repeatability, preferable HLA genotype tests method and the reagent of specificity.

Summary of the invention

One of purpose of the present invention provides the specific probe that a cover is used for HLA gene's somatotype;

Another object of the present invention provides a kind of gene chip of being made up of above-mentioned probe and carrier, can not realize high-throughput and stability, repeatability, the insufficient problem of specificity to overcome prior art;

Another object of the present invention provides the Auele Specific Primer that a cover is used for HLA gene's somatotype;

Another object of the present invention provides the test kit that a kind of HLA gene's of being used for somatotype detects;

A further object of the present invention provides a kind of method of the HLA gene's of detection somatotype.

Design of the present invention is such:

Select in HLA gene's complex body the rich H LA-A of polymorphism ,-B ,-three gene locuss of DRB1 carry out gene type.According to the allelotrope sequence that three gene locuss have been reported, select each allelic main region of variability design, a synthetic cover probe, again probe is connected to carrier, form the gene chip that a cover is used for HLA gene's somatotype.

HLA-A ,-B ,-three gene probes of DRB1 design site 5 ' end and 3 ' is held, design, synthesize the upstream and downstream primer that is used for pcr amplification respectively, form a cover be used to increase sample HLA-A to be checked ,-B ,-the allelic PCR primer of DRB1, primer 5 ' end all can carry out biotin labeling.Use this primer, to HLA-A ,-B ,-the allelic main region of variability of DRB1 carries out pcr amplification.

The gene fragment to be detected that amplification is obtained is added in the said gene chip, hybridizes under suitable condition, analyzes results of hybridization, can determine the genotype of sample to be checked.

A first aspect of the present invention discloses one group of probe that is used for HLA gene's somatotype, and sequence sees the following form:

Table 1a, the allelic probe of differentiation HLA-A

The probe numbering	Sequence (5 '------3 ')	Length (b)	Probe location
				PA01	TGAGGTATTTCTTCACATCCGCATCGCCGTGGGC	34	E2
PA02	GAGGTATTTCTACACCTCTTCATCGCCGTGGGC	33	E2
				PA03	CGGTTCGACAGCTGGAGGGGCCGGAGTTACCGAGTGGA CCT	41	E2
PA04	GCAGGAGAGGCCTGAGTATTG	21	E2
				PA05	AGGAGGGGCCGGAGTAAGGCCCACTCACAG	30	E2
PA06	TATTGGGACCTGCAGACACGG	21	E2
				PA07	GACACGGAATATGAAGGCCCA	21	E2
PA08	GGAATGTGAAGGCCCAGTCAC	21	E2
				PA09	GGCCCACTCACAGACTGACCG	21	E2
PA10	TCACAGATTGACCGAGTGGAC	21	E2
				PA11	ACAGACTGACCGAGTGGACCT	21	E2
PA12	GAGAGCCTGCGGATCGCGCT	20	E2
				PA13	ACCGAGCGAACCTGGGGACC	20	E2
PA14	CGAGCCAGAGGATGGGGCCCAGTCACAGAC	30	E2
				PA15	TCACAGACTCACCGAGTGGAC	21	E2
PA16	GACGAGGAGACAGGGAAAGTG	21	E2
				PA17	AGGTATTTCTACACCTCCGTGATCGCAGTGGGCTAC	36	E2
PA18	CGGAACACACGGAATCGGATCGCGCTCCGC	30	E2
				PA19	TGGGACCAGGAGACATTTTTGGCCCAGTCACAGAC	35	E2
PA20	TTGGGACCGGAACACACGGA	20	E2
				PA21	AAGGCCCAGTCACAGCGAGTGGACCTGGG	29	E2
PA22	CCTGCGGATCGCGCTCCGCTA	21	E2
				PA23	ACACGGAAAGTGAAGGCTTTCGAGTGGACCTGGG	34	E2
PA24	TGGGACCAGGAGACATTTTTCCTGGGGACCCTGCG	35	E2
				PA25	GCGGTTCGACAGCCGCGCTCCGCTACTA	28	E2
PA26	TCATCGCAGTGGGCTGTTCGACAGCGACGC	30	E2
				PA27	CTACACCTCCGTGTCGGCCCACTCACAGAC	30	E2
PA28	GGACCGGAACACACGCGAGCGAACCTGG	28	E2
				PA29	CTGACCGAGAGAACCTGGGGA	21	E2
PA30	AGCAGGAGGGTCCGGAGTATT	21	E2
				PA31	TTTCTACACCTCCGTGTCTTTAGGCCCAGTCACAGA	36	E2
PA32	GCCCACTCACAGACTCACCG	20	E2
				PA33	GGAGACACGGAAAGTGAAGGC	21	E2
PA34	CAGCTCAGACCACCAAGC	18	E3
				PA35	GAGGCGGCCCGTGTGGCGGA	20	E3
PA36	CCTACCTGGATGGCACGTGCG	21	E3

PA37	CTGGAGGGCCGGTGCGTGGA	20	E3
				PA38	CTGGAGGGCACGTGCGTGGA	20	E3
PA39	CCTGCGCTCTTGGTAGCAGCAGAGAGCTGGAGTGGCTCC	39	E3
				PA40	GCGGAGCAGCAGAGAGCCTAC	21	E3
PA41	CAAGTGGGAGACGGCCCATG	20	E3
				PA42	GGGTCGGACTGGCGCTTCCT	20	E3
PA43	TCGCCCTGAAAGAGGACCTGC	21	E3
				PA44	GAGCAGTTGAGAGCCCGTGGAGTGGCTC	28	E3
PA45	GAGCAGTTGAGAGCCTACCT	20	E3
				PA46	ACATGGCGGCTCAGAAAGTGGACGGGCTCCGC	32	E3
PA47	GAGCAGTGGAGAGCCTACCT	20	E3
				PA48	CGTGGGGTCGGACTTTGGTACCAGCAGGACGC	32	E3
PA49	TACCAGCAGGACGCTTACGAC	21	E3
				PA50	GTGCGTGGACGGGCTCCGCAG	21	E3
PA51	TACCACCAGTACGCCAGTGGGAGGCGGCCC	30	E3
				PA52	GGAGCAGTTGAGAGCGCGTGGACGGGCTCC	30	E3
PA53	TCAGATCACCCAGCGCAAGTG	21	E3
				PA54	GGTACCGGCAGGACGAGCACAAGTGGGAGG	30	E3
PA55	TCTCACACCATCCAGAGAGCAGTGGAGAGCC	31	E3
				PA56	GGGTACCGGCAGGACGCCTA	20	E3
PA57	CATCGCCCTGAAAGAGGGCGGCTCAGATCAC	31	E3
				PA58	CGGCCCATGAGGCGTGGAGGGCACGTGC	28	E3
PA59	ACCTGCGCTCTTGGAGACATGGCAGCTGTGGAGTGGCTC	39	E3
				PA60	TCTCACACCATCCAGATGATGTAT	24	E3
PA61	ACCACCAGTACGCCTACTTTGATCACCAAGCGCAA	35	E3
				PA62	CCCATGTGGCGGAGTTTTTCGTGGAGTGGCTCCG	34	E3
APC2	GCTACTACAACCAGAGCGAGG	21	E2
				APC3	GACGGCAAGGATTACATCGCC	21	E3
ANC	GACGGCAAGCATTACATCG	19	E3

Table 1b, the allelic probe of differentiation HLA-B

The probe numbering	Sequence (5 '------3 ')	Length (b)	Probe location
				PB01	TGCAAGGCCAAGGCACAGACT	21	E2
PB02	CTGCGCACCGCGCTCCGCTACTAC	24	E2
				PB03	ACTGACCGAGAGAGCCTGCGG	21	E2
PB04	CTGCGGATCGCGCTCCGCTAC	21	E2
				PB05	CAGATCTCCAAGACCAACACA	21	E2
PB06	ACACAGATCTACAAGGCCCAGGCA	24	E2
				PB07	ACACAGATCTTCAAGACCAAC	21	E2
PB08	ACACAGATCTGCAAGACCAAC	21	E2
				PB09	GCCACGAGTCCGAGGAAGGAG	21	E2
PB10	AGACTTACCGAGAGAGCCTGCGG	23	E2

PB11	CGGACCCTGCTCCGCTACTAC	21	E2
				PB12	GCCGCGAGTCCGAGAGAGGAG	21	E2
PB13	CGGAACACACAGATCTCCAAGACC	24	E2
				PB14	TGGGACCGGGAGACACAGATC	21	E2
PB15	ATGAAGGCCTCCGCGCAGACT	21	E2
				PB16	CGGGAGACACAGATCTCCAAGACC	24	E2
PB17	CCGAGGACGGAGCCCCGGGCG	21	E2
				PB18	GAGGTATTTCCACACCGCCAT	21	E2
PB19	ACACCGCCATGTCCCTCCTGCGGAACCTGCG	31	E2
				PB20	CGTGGATAGAGCAGGTTTGGCCCAGGCACAGAC	33	E2
PB21	AGACCAACACACAGACTGACC	21	E2
				PB22	ACGACACCCAGTTCGATTCGAGAGAGGAGCCGC	33	E2
PB23	CTTCATCGCAGTGGGTTACCGAGAGAGGAGCCG	33	E2
				PB24	GACGACACCCAGTTCGATTCCGGGAGACACAGAT	34	E2
PB25	ACGACACCCAGTTCGATACCGAGGATGGCGCCC	33	E2
				PB26	TGCGGAACCTGCGCGGCTAC	20	E2
PB27	ACGACACGCAGTTCGAATGCGGATCGCGCTCC	32	E2
				PB28	ACACAGATCTGCAAGCGGATCGCGCTCCGC	30	E2
PB29	GCTTCATTGCAGTGGTTTTTCGGATCGCGCTCCGC	35	E2
				PB30	CGGAACACACAGATCCGGATCGCGCTCCGC	30	E2
PB31	CGGGAGACACAGATCCGGATCGCGCTCCGC	30	E2
				PB32	GTATTTCCACACCGCCAAGCCTGCGGAACCTG	32	E2
PB33	ACGACACCCAGTTCGCAAGGCCCAGGCACA	30	E2
				PB34	GCCGTGGGTGGAGCAGGAG	19	E2
PB35	AGAAGTACAAGCGCCAGGCAC	21	E2
				PB36	CAGATCTCCAAGACCAACATCGCGCTCCGC	30	E2
PB37	GACGCCACGAGTCCGATGGCGCCCCG	26	E2
				PB38	CCGAGGATGGCGCCCCCGGAACACACAGAT	30	E2
PB39	AGTCCGAGGATGGCGCCCCTGCGGATCGCGCTC	33	E2
				PB40	GACTTACCGAGAGAACCTGC	20	E2
PB41	ATTTCTACACCTCCGTGTTGCGGAACCTGCGCG	33	E2
				PB42	GTCCGAGAGAGGAGCTTTGACCGGGAGACACAG	33	E2
PB43	GCTTCATCTCAGTGGTTTCAAGGCCCAGGCACA	33	E2
				PB44	ACACCTCCGTGTCCTCATCACCGTGGGACTACAACCAG	38	E2
PB45	ACCGGGAGACACAGAATTCCTGCGGAACCTGCG	33	E2
				PB46	GTCCGAGGAAGGAGCTTATGCGGAACCTGCGCG	33	E2
PB47	CCGAGAGAGGAGCTTCCGGAACACACTGGAACCTGCGC G	39	E2
				PB48	CCGAGAGAGGAGCTTCGGAACACACTGGAACCTGCGCG	38	E2
PB49	TTCATCGCAGTGGCCGGAACACACATCTACAACCAG	36	E2
				PB50	CAGATCTACAAGTTTCCAACACACAGAGCTACAACCAG AGC	41	E2
PB51	GGCGAGTGCGTGGAGTGGCTC	21	E3

PB52	GTGCGTGGACGGGCTCCGCA	20	E3
				PB53	CAGAGGATGTCTGGCTGCGAC	21	E3
PB54	GAGCAGCTGAGAACCTACCTG	21	E3
				PB55	GGGCATGACCAGTACGCCTAC	21	E3
PB56	GTGGCGGAGCAGGACAGAGCCTAC	24	E3
				PB57	CGGAGCAGCGGAGAGCCTACC	21	E3
PB58	ATCTCCCAGCGCAAGTTGGAG	21	E3
				PB59	CGCGGGTATGACCAGGACGCC	21	E3
PB60	CGCGGGTACCACCAGGACGCC	21	E3
				PB61	CGCGGGCATAACCAGTTCGCCTAC	24	E3
PB62	AAGGACACGCTGGAGCGCGCG	21	E3
				PB63	CGTGAGGCGGAGCAGTGGAGA	21	E3
PB64	CTGGAGGGCACGTGCGTGGAG	21	E3
				PB65	CGGCTGCGACCTGGGGCCGGA	21	E3
PB66	GGGCATGACCAGTCCGCCTACGAC	24	E3
				PB67	GGGTCTCACACCCTCCAGAGGATG	24	E3
PB68	CGTGAGGCGGAGCAGCTGAGA	21	E3
				PB69	TGCGTGGAGTCGCTCCGCAGA	21	E3
PB70	CGCGGCGGACACGGCGGCTC	20	E3
				PB71	CAGAGGATGTACGGCTGCGAC	21	E3
PB72	ACCAGTTAGCCTACGGGACCTGAGCTCCTG	30	E3
				PB73	GGACCTGAGCTCCTGTTTGTGGAGTGGCTCCGC	33	E3
PB74	CGGACACGGCGGCTCTGGGAAGGAGACGCTG	31	E3
				PB75	CTCCAGAGCATGTACGGGCATAACCAGTACGCC	33	E3
PB76	GCGGGCATGACCAGTGACGGACACCGCGGCTC	32	E3
				PB77	CATGACCAGTCCGCCTTTTCGCAGATACCTGGAG	33	E3
PB78	GGGCATAACCAGTACGCCTAC	21	E3
				PB79	CGGAGCAGGACAGAGATAGGAGTCGCTCCGCAG	33	E3
PB80	GATGTATGGCTGCGACTGACCAGTCCGCCTA	31	E3
				PB81	CACACTTGGCAGACGTTAGGAGGGCCTGTGCGT	33	E3
PB82	CATCGCCCTGAACGTTTGACCTGCGCTCCTG	33	E3
				PB83	CACCCTCCAGAGGATGTGAGGGCACGTGCGTG	32	E3
PB84	AGCAGCTGAGAGCCTTTTCGCAGACACCTGGAG	33	E3
				PB85	GAGCAGCTGAGAGCCTACCTG	21	E3
PB86	GAGGGCCTGTGCGTGTTTCGCAGACACCTGGAG	33	E3
				PB87	CCTCCAGAGCATGTACGTTTGCTCAGATCTCCCAGCG	37	E3
PB88	GGTCTCACACTTGGCAGAATTCTGCGACCTGGGGCC	36	E3
				PB89	TCCAGGTGATGTATGGCTGCG	21	E3
PB90	CATGACCAGTTCGCCTACGAC	21	E3
				PB91	GCGGACACGGCGGCTGAGCAGCGGAGAGCC	32	E3
PB92	ACATCATCCAGAGGATGACCCTGAACGAGGACC	33	E3
				PB93	GTGAGGCGGAGCAGCGGAGA	20	E3

PB94	CTGCGACCTGGGGCTACATCGCCCTGAATCCCGTGTGGC GGAG	43	E3
				PB95	CAGCTGAGAGCCTACAAAAGGGCCTGTGCGTGG	33	E3
PB96	TCCAGAGCATGTACGGCTGCG	21	E3
				BPC2	ACTACAACCAGAGCGAGGCCG	21	E2
BPC3	ATTACATCGCCCTGAACGAGGAC	23	E3
				BNC	CATCGCCGTCAACGAGGAC	19	E3

Table 1c distinguishes the allelic probe of HLA-DRB1

The probe numbering	Sequence (5 '------3 ')	Length (b)
			PD01	AGCTTAAGTTTGAATGTCATTTCT	24
PD02	GAGCAGGTTAAACATGAGTGTCAT	24
			PD03	GTGGCAGGGTAAGTATAAGTG	21
PD04	GAAGCAGGATAAGTTTGAGTG	21
			PD05	GGAGGAGGTTAAGTTTGAGTG	21
PD06	ATAACCAGGAGGAGTCCGTGC	21
			PD07	CAGAAGGACATCCTGGAAGAC	21
PD08	GCGGTGACGGAGCTTTTCTGCTGCGGAGCACT	32
			PD09	GAGTACTCTACGTCTGTTTTGGAGCAGAAGCGGG	35
PD10	GGGCCCTGGTGGACTTTGTTGTGGAGAGCTTC	32
			PD11	GGACATCCTGGAAGACGAGC	20
PD12	AGTACTCTACGTCTGATTTCGGCCTGATGAGGAG	34
			PD13	GGAGTACGTGCGCTTTTTGGCCCTGGTGGACA	32
PD14	GTGCGGTTACTGGAGAGACAC	21
			PD15	GCCTGATGCTGAGTACTGGA	20
PD16	CCTGATGAGGAGTACTTTTCTGGAAGACGAGCGG	34
			PD17	GACATCCTGGAAGACAAGCGG	21
PD18	GGGAGTTCCGGGCTTACCTGGAAGACGAGCG	31
			PD19	GAGGAGTTCGTGCGCATTCCTGATGAGGAGTAC	33
PD20	TGATGAGGAGCACTGGAACAG	21
			PD21	AGATACTTCTATCACCAAGAGGAG	24
PD22	GAGGAGAACGTGCGCATTGTGGACAACTACTGC	33
			PD23	ACTCTACGGGTGAGTGTATTGGGAGTACCGGGC	33
PD24	GTTCCTGGACAGATACTATTCTGATGCCGAGTACT	35
			PD25	CAGAAGGACCTCCTGGAAGAC	21
PD26	GGAGTACTCTACGTCTGTTTGTGGACAATTACTGCA	36
			PD27	TGGAGTACTCTACGTCTTTTGCCTGACGCTGAGTA	35
PD28	GGGTGAGTGTTATTTCTTCAATGG	24
			PD29	CCTGGACAGATACCTTCGACAGCGACCCTGATGCGGAG	38
PD30	CTACGTCTGAGTGCCTTCGACAGCGACTGGGTTGTGGAG AG	41
			PD31	CTACGTCTGAGTGGGAGTTCCGGGCAAGGACCTCCTGG	38
PD32	GTACTCTACGTCTGAGTGCGGTACCTGGAC	30

PD33	GACATCCTGGAGGACAGGCG	20
			PD34	CTTGGAGTACTCTACGCTTCCATAACCAGGAG	32
PD35	CCTGGAGAGATACTTCGAGGAGAACGTGCGC	31
			PD36	AGGAGAACGTGCGCTCCTGATGCCGAGTACTG	32
PD37	GAGGAGTTCGTGCGCTGCTGCGGAGCACT	29
			PD38	GAAGACGAGCGGGCGGTTGGTGAGAGCTTC	30
PD39	GGTTCCTGGACAGATACGCCTAGCGCCGAGT	31
			PD40	GGAGTACTCTACGGGTGAGTG	21
PD41	TCTACGTCTGAGTGTCCCTGGAAGACAGGCG	31
			PD42	CCTGGAAGACAGGCGACGGGGTTGTGGAGA	30
PD43	CTGATGCCGAGTACTGCCTGGAAGACAGGC	30
			PD44	TGGCAGCCTAAGAGGTCCTGGAAGACAGGCG	31
PD45	GAGGAGTCCGTGCGCGGGAGTTCCGGGC	28
			PD46	GGCCTAGCGCCGATGGAGCAGAGGCGGG	28
PD47	GGTTCCTGGACAGATACGAAGGACTTCCTGGA	32
			PD48	GGAGTACGTGCGCTTCCTGATGAGGAGTACTG	32
PD49	GGAGAGATACTTCCATAGTGGACACCTACTGC	32
			PD50	CAGGAGGAGAACGTGCGCCTGGAAGACGAGCGG	33
PD51	TCCTGGAAGACGAGCGGGGGTTGGTGAGAGC	31
			PD52	CGGCCTAGCGCCGAGCTGGAGCAGAGGCGG	30
PD53	CTGGAGCAGGCGCGGGGGGTTGGTGAGAGC	30
			PD54	TTCTTCAATGGGACGGTTTTTGGACTTCCTGGAAGAC	37
PD55	GGTGCGGTTCCTGTGAGTACGTGCGCCGACGTGGGGGA	38
			PD56	GCGGTTCCTGGACAGTTTTTGGCCTGATGCCGAGT	35
PD57	GGAGTACGTGCGCATGATGAGGAGTACGAAGGACTTCCT GGA	42
			PD58	CTTCGACAGCGAGCCGAGTACTGGACTGGAAGACGAGC	38
PD59	AGGAGTTCGTGCGCCCTGATGCCGAGAGGGTTGTGGAGA	39
			PD60	TGGAGTACTCTACGATCGACAGCGACTGGGGCTGTGGAG AG	41
PD61	GTACTCTACGTCTGATTTGTGCGGTACCTGGAC	33
			PD62	AGGAGAACGTGCGCTATTCCTGATGCCGAGTACTG	35
PD63	TCCTGGACAGATACTATTCTGGAGCAGATTTGTGGACAC CTACTG	45
			PD64	GAGTACTCTACGTCTGTTTTTCTGGAGCAGAGGCGG	36
PD65	GTACTCTACGTCTGAGTTGATGAGGAGTACTGG	33
			PD66	GGCCTGATGCCGAGTTTTTTGGGCCCTGGTGGACA	35
PD67	GTACTCTACGGGTGAGTCGCTTCGACAGCGAC	32
			PD68	CTCTACGTCTGAGTGTCTTACCTGGAAGACAGGC	34
PD69	CTGATGCCGAGTACTGTTACCTGGAAGACAGGC	33
			PD70	CTGGACAGATACTTACTTCGACAGCGATAGGACATCCTG GA	41
PD71	AGAGGAGTACGTGCGTTCCTGGAAGACAGGC	31
			PD72	GGTTCCTGGACAGATACATTGAAGGACTTCCTGGA	35

PD73	GAGTACTCTACGTCTGATTTTTCTGGAGCAGAAGCGG	37
			PD74	TTCTTCAATGGGACGGGGACTTCCTGGAAGAC	32
PD75	CCTGGAGAGATACTTCGGAAGACGAGCGGGC	31
			PD76	CCTGGACAGATAAGAGCAGAAGCGGGTCACCTACTGCAG	39
PD77	GCTTCGACAGCGACTGGCCTGTCGCCGATGGAAGACAGG CG	41
			PD78	TCTACGTCTGAGTGTCTTTTTCCGCGGTGGACACCT	36
PD79	CTGATGCCGAGTACTCTGGAAGACAGGCGG	30
			PD80	CTAAGAGGGAGTGTGGCAGTACCGGGCAAGACAGGCG	37
PD81	GCTTCGACAGCGACTCTGATGCCGAGTGAGCAGAGGCGG G	40
			PD82	AGCCTAAGAGGGACTTCGACAGCGACTGAGCAGGCGCG GGC	41
PD83	CCTGGAGAGATACGAGCAGAGGCGGGGACACCTACTGC	38
			PD84	CCTGGACAGATACGATGCCGAGTACTAGGTTGGTGAGAG C	40
PD85	TGCGGTTCCTGTGAGGAGTACGTGCGTACGTGGGGGA	37
			PD86	GCAGCCTAAGAGGGATTTGGGGAGTACCGGGCG	33
PD87	CTGGACAGATACATTCGACAGCGATCTGGAGCGGAGGC	38
			PD88	TCGACAGCGACGTAGGGCCCTGGTGGAGGTTGTGGAGAG C	40
PD89	CTTCGACAGCGTGGGAGTTCCGGGCAAGGACTTCCTGG	38
			PD90	TCTACGTCTGAGTTTCGACAGCGACTGAGCAGAAGCGGG	39
DPC	GCGCTTCGACAGCGACGTGGG	21
			DNC	CGCTTCGACTCCGACGTGG	19

In preferred scheme of the present invention, probe carries out following modification:

X-(5 '-probe-3 ')

X=NH wherein ₂-(CH ₂-CH ₂) _n-O-; N is 3～6 integer, preferred n=3;

A second aspect of the present invention provides a cover by above-mentioned probe and carrier-bound gene chip, and said carrier comprises polystyrene microsphere, glass-chip or the various membrane matrix of surface through carboxyl modified.

In a preferred scheme, carrier is a fluorescent microsphere, and promptly a kind of surface is through the polystyrene microsphere of carboxyl modified, the inner embedding fluorescent substance of microballoon, and the fluorescence that different fluorescent substances sends is used to discern the kind of microballoon.The probe that 5 ' end is amido modified and the carboxyl on fluorescent microsphere surface make probe be covalently bonded in microsphere surface by condensation reaction.Every kind of fluorescent microsphere coupling joins a kind of probe, and the kind of fluorescent microsphere is corresponding one by one with the probe kind.To just form a cover liquid phase gene chip in conjunction with the fluorescent microsphere balanced mix of different probe together.Such fluorescent microsphere can be bought by commercial sources, for example, can buy the product of U.S. Luminex Corporation.

A third aspect of the present invention discloses one group of PCR primer that is used for HLA gene's somatotype, and sequence sees the following form:

Table 2a, second exon of HLA-A gene and the PCR primer sequence of the 3rd exon

Primer #	Sequence (5 ' 3 ')	Length (b)	GC％	Tm
					AE2F1	CGCCTCTGYGGGGAGAAGCAA	21	67	71.6
AE2R1	GCCCGTCCGTGGGGGATGA	19	74	70.5
					AE2R2	GGGGATGAGGGGTCSTGACCTGC	23	70	72.7
AE3F1	GCCCAGGCGCCTTTACCCGGTT	22	68	74
					AE3F2	GGGAGAGGCCCAGGCGCCTT	20	75	71.6
AE3R1	GGGAGGCCAGCCCGGGAGAYCTA	23	70	73.5
					AE3R2	GTCCCAAWTGTCTCCCCTCCTT	22	54.5	72

Among the table 2a, AE2F1, AE2R2 and AE2R1 are the primer of amplification HLA-A gene second exon; AE3F1, AE3F2, AE3R1 and AE3R2 are the primer of amplification the 3rd exon, and for persons skilled in the art, the appropriate combination of above-mentioned PCR primer all can make second and third exon amplification.

Table 2b, second exon of HLA-B gene and the PCR primer sequence of the 3rd exon

Primer #	Sequence (5 ' 3 ')	Length (b)	GC％	Tm
					BE2F1	GGGAGGAGCGAGGGGACCSCAG	22	72.7	73.6
BE2R1	CCCGGGCCGGGGTCACTCA	19	79	72.8
					BE2R2	GGGGGATGGGGAGTCGTGACCT	22	68	70.2
BE3F1	CCCGGTTTCATTTTCAGTTGAGGCCA A	27	48	73.9
					BE3R1	GGCCATCCCCGSCGACCTATAGGAGA T	27	63	76.4

Among the table 2b, BE2F1, BE2R1 and BE2R2 are the primer of amplification HLA-B gene second exon: BE3F1, BE3R1 are the primer of amplification the 3rd exon, for persons skilled in the art, the appropriate combination of above-mentioned PCR primer all can make second and third exon amplification.

The PCR primer sequence of second exon of table 2c, HLA-DRB1 gene

Primer #	Sequence (5 ' 3 ')	Length (b)	GC％	Tm
					DE2F1	CCGGATCCTTCGTGTCCCCACAGCACG	27	66.7	72.6
DE2F2	CGTTCGTGTCCCCACAGCACGTT	23	61	71.1
					DE2F3	CGTTCTTGTCCCCCCAGCACGTT	23	61	71.3
DE2R1	TCGCCGCTGCACTGTGAAGCTC	22	64	70.7
					DE2R2	TgCTYACCTCgCCKCTgCAC	20	74	68

Among the table 2c, DE2F1, DE2F2, DE2F3, DE2R1 and DE2R2 are the primer of amplification HLA-DRB1 gene second exon; For persons skilled in the art, the appropriate combination of above-mentioned PCR primer all can make second to show the son amplification.

A fourth aspect of the present invention, the test kit that provides a kind of HLA gene's of being used for somatotype to detect comprises:

HLA-A ,-B ,-probe of three gene locuss of DRB1 and the gene chip that coupling connection carrier is formed;

Amplification HLA-A ,-B ,-the allelic PCR primer of DRB1.

Wherein HLA-A ,-B ,-probe sequence of three gene locuss of DRB1 sees Table 1, preferred 5 ' holds amido modified probe, more preferably has the polymerization degree and be the probe of 3～6 polyoxyethylene glycol arm, most preferably have the polymerization degree and be the probe of 3 polyoxyethylene glycol arm.

Wherein the surface that produces with the preferred U.S. of the carrier Luminex Corporation company of probe coupling connection is through the fluorescence polystyrene microsphere of carboxyl modified, the about 5.5um of microsphere diameter.

Wherein increase HLA-A ,-B ,-the allelic PCR primer sequence of DRB1 sees Table 2.

A fifth aspect of the present invention discloses a kind of method of the HLA gene's of detection somatotype, comprises the steps:

(1) sample disposal

Sample to be checked extracts genomic dna with ordinary method, as the pcr amplification template;

(2) multiplex PCR amplification

The primer system of second exon of HLA-A gene and the pcr amplification of the 3rd exon: PCR is AE2F1: AE2R2: AE2R1: AE3F1: AE3R1: AE3F2: AE3R2=1: 2: 0.1: 1: 2: 0.1: 0.1, wherein 5 of AE2R2 and two primers of AE3R1 ' end carries out biotin labeling, the concentration of primer AE2F1 is 0.1～0.4uM, and the suitableeest is 0.2uM;

Second exon of HLA-B gene and the PCR of the 3rd exon: the present invention has realized balanced simultaneously second and third two exons of amplification, the primer system of PCR is BE2F1: BE2R1: BE2R2: BE3F1: BE3R1=1: 2: 0.1: 1.5: 3, wherein 5 of BE2R1 and two primers of BE3R1 ' end carries out biotin labeling, and the concentration of primer BE2F1 is 0.1～0.4uM, and the suitableeest is 0.2uM;

The primer system of the PCR:PCR of second exon of HLA-DRB1 gene is DE2F1: DE2F2: DE2F3: DE2R1: DE2R2=1: 0.5: 0.5: 2: 0.1, wherein 5 of primer DE2R1 ' end carries out biotin labeling, and the concentration of primer DE2F1 is 0.1～0.2uM, and the suitableeest is 0.15uM;

Primer sequence sees Table 2;

(3) hybridization

With HLA-A ,-B ,-probe of three gene locuss of DRB1 is suspended in the hybridization solution with the gene chip that Luminex Corporation fluorescent microsphere coupling joint group becomes;

Pcr amplification product is added the hybridization system, and 60 ℃ of incubation 15min. are hatched the phycoerythrin (SAPE) of reacted microballoon and streptavidin modification again;

Probe kind and sequence see Table 1;

(4) interpretation as a result

With Luminex instrument read data information, with HLA somatotype software analysis result.

Beneficial effect of the present invention is embodied in:

(1) PCR system of the present invention is a multiplex PCR, and a plurality of dna fragmentations simultaneously can increase.The proportioning of various primers has realized the equilibrium amplification of a plurality of gene fragments in the Design of length of the dna fragmentation by primer sequence design, amplification, the primer system.

(2) select crucial downstream primer to carry out end mark in the primer system of the present invention, each dna fragmentation that makes amplification obtain all has biotin labeling, the convenient detection.

(3) probe system of the present invention comprises a plurality of allelotrope of three gene locuss.HLA-A, B, the allelic quantity of DRB1 three classes proved conclusively at present are respectively 324,587 and 389.Because human each individuality has two allelotrope, so HLA-A, B, the possible allelotrope combination of DRB1 have 324 * (324-1)/2=52326 kind, 589 * (589-1)/2=173166 kind, the combination of 389 * (389-1)/2=75466 kind respectively.In addition, because the allelic dna sequence dna of HLA is not at random, be not a simple mathematical problem so how to improve the working efficiency of probe.The present invention adjusts kind, site, the length of probe by a large amount of experimental studies, designs the probe system of a cover hybridization efficiency height, relative equilibrium.

(4) the present invention adopts the Luminex technology, is coupled to the probe of fluorescent microsphere and allelotrope fragment that pcr amplification obtains and hybridizes in liquid phase environment, and with respect to the liquid-solid phase hybridization of traditional gene chip, hybridization efficiency is higher, better effects if.In addition, crossover process can be carried out on common 96 orifice plates, can use hyperchannel to carry out pipetting as 8 passages or the 12 passage volley of rifle fires, and therefore whole crossover process is convenient fast, can effectively realize high throughput testing.

Embodiment

The liquid phase gene chip

Traditional gene chip is meant and (or claims probe with the dna fragmentation of various particular sequences, probe), be integrated on solid phase carrier such as glass-chip or the silicon by means such as little point sample or molecule operating and microelectronic chip technology, encode to probe by the point sample position on the solid phase carrier, and software is according to the monitor signal of this adress analysis sample.

The liquid phase gene chip is that probe has been coupled to covalent manner on the microballoon of support effect, and diameter of micro ball is generally about 5.5um, and material comprises inert materials such as polystyrene; Microsphere surface is modified with carboxyl; Microballoon inside is embedded with fluorescent substance, and can distinguish the kind of microballoon according to the ratio of fluorescence.Its technological core is that microballoon comprises fluorescent substance, and microballoon is dyed different fluorescence color respectively, can encode to probe by the fluorescence of microballoon inside.The fluorescent microsphere balanced mix of mark different probe, can form a cover liquid phase gene chip.

Probe, design of primers

The gene order data of designing probe of the present invention take from EMBL ( Http:// www.ebi.ac.uk/), adopt the PrimerPrimer5.0 software design.

The gene order that the present invention designs primer take from GENBANK ( Http:// www.ncbi.nlm.nih.gov/), adopt Primer Primer5.0 software design, specific as follows:

The reference sequences of second exon of HLA-A gene and the PCR design of primers of the 3rd exon is taken from GENBANK:AJ278305; The reference sequences of second exon of HLA-B gene and the PCR design of primers of the 3rd exon is taken from GENBANK:AJ309047; The reference sequences of the PCR design of primers of second exon of HLA-DRB1 gene is taken from GENBANK:AF142465, AJ556173 AF489519.

The coupling connection of probe and carrier

The coupling of probe and fluorescent microsphere is associated in the MES damping fluid (pH4.5) of 0.1mM and carries out, as condensing agent, make the amino of probe 5 ' end and the carboxyl covalent attachment of microsphere surface with EDC (1-enthyl-[3dimethylaminopropyl] carbodiimide hydrchloride).

Gene PCR amplification to be detected

The present invention is the same to the pcr amplification system of three class HLA genes.Reaction volume is 10～100ul, generally uses 20～25ul reaction system.(template DNA concentration is 20～100ng/ul) and the usefulness of template DNA is measured 2ul.Taq enzyme dosage is 1U.The concentration of every kind of dNTP is 200uM.Mg ²⁺Concentration is 1.5mM.Cycling condition is 94 ℃, 5 minutes.95 ℃, 20 seconds; 60 ℃, 30 seconds; 72 ℃, 15 seconds; 10 circulations.95 ℃, 15 seconds; 60 ℃, 15 seconds; 72 ℃, 15 seconds; 30 circulations.72 ℃, 10 minutes.

By adopting biotin labeled PCR primer, sample to be checked (genomic dna) has biotin labeling through the amplified production of pcr amplification.

Hybridization

Biotin labeled PCR product is hybridized with the covalently bound probe of microsphere surface that is suspended in the hybridization solution, and post-hybridization washing is hatched the phycoerythrin (SAPE) of reacted microballoon and streptavidin modification again; Hybridization male microballoon combines with the specificity of biotin labeled gene fragment to be checked by probe, and vitamin H combines with avidin streptavidin specificity, makes hybridization male microballoon have phycoerythrin (SAPE) fluorescent reporter molecule.

Detect and result's judgement

After the phycoerythrin that microballoon and fluorescent reporter molecule streptavidin modify is hatched, microballoon is resuspended in the washings, transfers on the reading plate, be directly used in Luminex instrument read data information.During detection, shine fluorescent microsphere simultaneously with two bundle laser, beam of laser is determined the fluorescent microsphere kind, thereby the intensity for hybridization of the label probe on another bundle laser determination fluorescent microsphere gives analyte quantitative.The processing of the raw data that detection is read and genotype deterministic process, also can finish by computer software, for example, (number of accepting is the V1.0 version " gene type expert software (HLA-GT) " that registers by Shanghai Fudan Zhangjiang biomedical Co., Ltd: 2005111554) finish.

Sample source

A4008 A4009 A4010 A4011 A4012 A4013 totally 6 duplicate samples derives from the Shanghai Blood Center.

Main agents and equipment

General primer and biotin labeled primer have 5 of polyoxyethylene glycol arm and ' hold amido modified probe synthetic by the living worker's biotechnology in Shanghai service company; (Luminex xMAP CarboxylatedMicrospheres, L100-C101～C200) purchase the company in Luminex Corporation to fluorescent microsphere; Test set is the Luminex detector, purchases the company in Luminex Corporation; Taq enzyme and other PCR reagent are purchased the company in Promega, and MES purchases the company in SIGMA, and EDC purchases the company in Pierce, and the phycoerythrin (SAPE) that streptavidin modifies is purchased the company in Molecular Probe, and other reagent is homemade analytical reagent.

Below in conjunction with embodiment, the present invention is described further, but the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, work such as Sambrook for example, " molecular cloning laboratory manual " (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

The extracting of embodiment one, genomic dna

A4008 A4009 A4010 A4011 A4012 A4013 blood sample, with the DNA extraction test kit extracting genomic dna of the living worker's biotechnology in Shanghai service company, concentration is 20～100ng/ul, OD _260/280=1.5～1.8.

The pcr amplification of embodiment two, second and third exon of HLA-A gene

Preparation 10X damping fluid: 100mM Tris-HCl, pH9.0,100mM KCl, 80mM (NH ₄) ₂SO ₄, 0.5%NP-40,20mM MgSO ₄, the 0.22um membrane filtration.Glycerine adds distilled water, and 121 ℃, 15 minutes, autoclaving was standby.

Preparation P Mix solution: primer AE2F1, AE2R2, AE2R1, AE3F1, AE3R1, AE3F2 and AE3R2 add aseptic double-distilled water respectively and are dissolved into the solution that concentration is 10uM.With the primer solution that has prepared, in following ratio AE2R2: AE3R1: AE2F1: AE3F1: AE2R1: AE3F2: AE3R2=2: 2: 1: 1: 0.1: 0.1: 0.1 (V/V) mixes, mixing.

Preparation D Mix solution: with aseptic double-distilled water preparation 5mM dNTP solution (containing each 5mM of dATP, dTTP, dGTP and dCTP).

The DNA that embodiment one described sample extracts respectively gets 2ul in the PCR reaction tubes, puts ice bath.Add 10X damping fluid 2ul; 50% glycerine 5ul; Add D Mix solution 1ul; Add P Mix solution 2ul and add aseptic double-distilled water 9.7ul; Add Taq enzyme 0.3ul.Centrifugal 13000rpm, 10 seconds.

The PCR response procedures is as follows: 94 ℃, and 5 minutes.95 ℃, 20 seconds; 60 ℃, 30 seconds; 72 ℃, 15 seconds; 10 circulations.95 ℃, 15 seconds; 60 ℃, 15 seconds; 72 ℃, 15 seconds; 30 circulations.72 ℃, 10 minutes.

The pcr amplification of embodiment three, second and third exon of HLA-B gene

Preparation P Mix solution: primer BE2F1: BE2R1: BE2R2: BE3F1: BE3R1 adds aseptic double-distilled water respectively and is dissolved into the solution that concentration is 10uM.With the primer solution that has prepared, in following ratio BE2F1: BE2R1: BE2R2: BE3F1: BE3R1=1: 2: 0.1: 1.5: 3 (V/V) mix, mixing.

The DNA that embodiment one described sample extracts carries out application of sample and pcr amplification by the method for embodiment two.

The pcr amplification of embodiment four, HLA-DRB1 gene second exon

Preparation P Mix. solution: primer DE2F1, DE2F2, DE2F3, DE2R1 and DE2R2 add aseptic double-distilled water respectively and are dissolved into the solution that concentration is 10uM.With the primer solution that has prepared, in following ratio DE2F1: DE2F2: DE2F3: DE2R1: DE2R2=1: 0.5: 0.5: 2: 0.1 (V/V) mixes, mixing.

The DNA that embodiment one described sample extracts carries out application of sample and pcr amplification by the method for embodiment two.

Embodiment five, the design of HLA-A gene type gene chip and detection

Oligonucleotide probe: 5 ' end is amido modified, and totally 65, its concrete sequence nucleotide sequence sees Table 1a.Second exon has 34 probes, is respectively probe numbering PA01～PA33 and APC2; The 3rd exon has 31 probes, is respectively probe numbering PA34～PA362 and APC3 and ANC.The positive contrast of APC2 and APC3 can be indicated the amount of second and third exon of pcr amplification respectively, also can be applicable to the calculating of second and third exon probe threshold value in addition.Whether the negative contrast of ANC can indicate pcr amplification and follow-up hybridization contaminated, also can be applicable to the calculating of second and third exon probe threshold value in addition.

Method by embodiment five is carried out probe mark and counting.

Embodiment two pcr amplification products are got 5ul and are put in 96 orifice plates, one hole, add 5000 every kind of the fluorescent microspheres of above-mentioned 65 kinds of marks, add 35ul 1.5 X TMAC hybridization solutions (pH8.0), cumulative volume 50ul.95 ℃, 3 minutes.60 ℃, 15 minutes.Add 80ul washings (50Mm Tris-HCl, pH8.0,0.1%TritonX-100,0.1M NaCl), mixing, centrifugal 5 minutes of 1300g abandons supernatant.Add 50ul SAPE solution (with the preparation of 1 x TMAC solution, element-phycoerythrin conjugate concentration is 4ug/ml to the chain parent), resuspended precipitation, 60 ℃, 6 minutes.Add the 80ul washings, mixing, centrifugal 5 minutes of 1300g abandons supernatant.Add the 80ul washings, resuspended precipitation is put Luminex detector reading.

With gene type software (HLA-GT) the V1.0 version interpretation that Shanghai Fudan Zhangjiang biomedical Co., Ltd registers, the genotype of this sample is:

HLA-A

Mark this shop genotype 1 specificity 1 genotype 2 specificitys 2

A4008 A ^*11 A11 A ^*31 A31

A4009 A ^*01 A1 A ^*02 A2

A4010 A ^*11 A11 A ^*11 A11

A4011 A ^*11 A11 A ^*30 A30

A4012 A ^*02 A2 A ^*30 A30

A4013 A ^*11 A11 A ^*24 A24

SSP method validation: the Micro SSP that uses U.S. ONE LAMBDA company product ^TMThe HLA test kit is pressed the operation of test kit specification sheets, analyzes above-mentioned sample, comes to the same thing.

Embodiment six, the design of HLA-B gene type gene chip and detection

Oligonucleotide probe is amido modified with 5 ' end, and totally 99, its concrete sequence nucleotide sequence sees Table six.Second exon has 51 probes, is respectively probe numbering PB01～PA50 and BPC2; Second exon has 48 probes, is respectively probe numbering PB51～PB96 and PC3 and BNC.The quantity of second and third exon of pcr amplification is indicated in the positive contrast of BPC2 and BPC3 respectively, also can be applicable to the calculating of second and third exon probe threshold value in addition.Whether the negative contrast of BNC can indicate pcr amplification and follow-up hybridization contaminated, also can be applicable to the calculating of second and third exon probe threshold value in addition.

Method by embodiment five is carried out probe mark and counting.

Embodiment three pcr amplification products are got 5ul and are put in 96 orifice plates, one hole, add 5000 every kind of the fluorescent microspheres of above-mentioned 99 kinds of marks, add 35ul 1.5 X TMAC hybridization solutions (pH8.0), cumulative volume 50ul.95 ℃, 3 minutes.60 ℃, 15 minutes.Adding 80ul washings (50Mm Tris-HCl, pH8.0,0.1%TritonX-100,0.1MNaCl), and mixing, centrifugal 5 minutes of 1300g abandons supernatant.Add 50ul SAPE solution, resuspended precipitation, 60 ℃, 6 minutes.Add the 80ul washings, mixing, centrifugal 5 minutes of 1300g abandons supernatant.Add the 80ul washings, resuspended precipitation is put Luminex detector reading.

With gene type software (HLA-GT) the V1.0 version interpretation that Shanghai Fudan Zhangjiang biomedical Co., Ltd registers, the genotype of this sample is:

HLA-B

Mark this shop genotype 1 specificity 1 genotype 2 specificitys 2

A4008 B ^*40 B60 B ^*58 B58

A4009 B ^*40 B60 B ^*57 B57

A4010 B ^*15 B75 B ^*56 B56

A4011 B ^*13 B13 B ^*13 B13

A4012 B ^*13 B13 B ^*46 B46

A4013 B ^*15 B62 B ^*35 B35

The SSP method validation:

Use the Micro SSPTM HLA test kit of U.S. ONE LAMBDA company product, press the operation of test kit specification sheets, analyze above-mentioned sample, come to the same thing.

Embodiment seven, the design of HLA-DRB1 gene typing chips and detection

Oligonucleotide probe is amido modified with 5 ' end, and totally 92, its concrete sequence nucleotide sequence sees Table seven.Second exon has 92 probes, is respectively probe numbering PD01～PA90 and DPC and DNC.The positive contrast of DPC, one can be indicated the amount of pcr amplification second exon, also can be applicable to the calculating of the second exon probe threshold value in addition.Whether the negative contrast of DNC, one can indicate pcr amplification second exon and follow-up hybridization contaminated, also can be applicable to the calculating of the second exon probe threshold value in addition.

Method by embodiment five is carried out probe mark and counting.

Embodiment four pcr amplification products are got 5ul and are put in 96 orifice plates, one hole, add 5000 every kind of the fluorescent microspheres of above-mentioned 92 kinds of marks, add 35ul 1.5 X TMAC hybridization solutions (pH8.0), cumulative volume 50ul.95 ℃, 3 minutes.60 ℃, 15 minutes.Adding 80ul washings (50Mm Tris-HCl, pH8.0,0.1% TritonX-100,0.1MNaCl), and mixing, centrifugal 5 minutes of 1300g abandons supernatant.Add 50ul SAPE solution, resuspended precipitation, 60 ℃, 6 minutes.Add the 80ul washings, mixing, centrifugal 5 minutes of 1300g abandons supernatant.Add the 80ul washings, resuspended precipitation is put Luminex detector reading.

The detection data are as follows:

With gene type software (HLA-GT) the V1.0 version interpretation that Shanghai Fudan Zhangjiang biomedical Co., Ltd registers, the genotype of this sample is:

HLA-DRB1

Mark this shop genotype 1 specificity 1 genotype 2 specificitys 2

A4008 DRB1 ^*11 DR11 DRB1 ^*13 DR13

A4009 DRB1 ^*07 DR7 DRB1 ^*14 DR14

A4010 DRB1 ^*13 DR13 DRB1 ^*13 DR6

A4011 DRB1 ^*07 DR7 DRB1 ^*15 DR15

A4012 DRB1 ^*04 DR4 DRB1 ^*07 DR7

A4013 DRB1 ^*04 DR4 DRB1 ^*08 DR8

The SSP method validation:

Use the Micro SSPTM HLA test kit of U.S. ONE LAMBDA company product, press the operation of test kit specification sheets, analyze above-mentioned sample, come to the same thing.

Claims (11)

1, one group of probe that is used for HLA gene's somatotype is characterized in that probe sequence is as follows: first group of HLA-A

PA01 TGAGGTATTTCTTCACATCCGCATCGCCGTGGGC

PA02 GAGGTATTTCTACACCTCTTCATCGCCGTGGGC

PA03 CGGTTCGACAGCTGGAGGGGCCGGAGTTACCGAGTGGACCT

PA04 GCAGGAGAGGCCTGAGTATTG

PA05 AGGAGGGGCCGGAGTAAGGCCCACTCACAG

PA06 TATTGGGACCTGCAGACACGG

PA07 GACACGGAATATGAAGGCCCA

PA08 GGAATGTGAAGGCCCAGTCAC

PA09 GGCCCACTCACAGACTGACCG

PA10 TCACAGATTGACCGAGTGGAC

PA11 ACAGACTGACCGAGTGGACCT

PA12 GAGAGCCTGCGGATCGCGCT

PA13 ACCGAGCGAACCTGGGGACC

PA14 CGAGCCAGAGGATGGGGCCCAGTCACAGAC

PA15 TCACAGACTCACCGAGTGGAC

PA16 GACGAGGAGACAGGGAAAGTG

PA17 AGGTATTTCTACACCTCCGTGATCGCAGTGGGCTAC

PA18 CGGAACACACGGAATCGGATCGCGCTCCGC

PA19 TGGGACCAGGAGACATTTTTGGCCCAGTCACAGAC

PA20 TTGGGACCGGAACACACGGA

PA21 AAGGCCCAGTCACAGCGAGTGGACCTGGG

PA22 CCTGCGGATCGCGCTCCGCTA

PA23 ACACGGAAAGTGAAGGCTTTCGAGTGGACCTGGG

PA24 TGGGACCAGGAGACATTTTTCCTGGGGACCCTGCG

PA25 GCGGTTCGACAGCCGCGCTCCGCTACTA

PA26 TCATCGCAGTGGGCTGTTCGACAGCGACGC

PA27 CTACACCTCCGTGTCGGCCCACTCACAGAC

PA28 GGACCGGAACACACGCGAGCGAACCTGG

PA29 CTGACCGAGAGAACCTGGGGA

PA30 AGCAGGAGGGTCCGGAGTATT

PA31 TTTCTACACCTCCGTGTCTTTAGGCCCAGTCACAGA

PA32 GCCCACTCACAGACTCACCG

PA33 GGAGACACGGAAAGTGAAGGC

PA34 CAGCTCAGACCACCAAGC

PA35 GAGGCGGCCCGTGTGGCGGA

PA36 CCTACCTGGATGGCACGTGCG

PA37 CTGGAGGGCCGGTGCGTGGA

PA38 CTGGAGGGCACGTGCGTGGA

PA39 CCTGCGCTCTTGGTAGCAGCAGAGAGCTGGAGTGGCTCC

PA40 GCGGAGCAGCAGAGAGCCTAC

PA41 CAAGTGGGAGACGGCCCATG

PA42 GGGTCGGACTGGCGCTTCCT

PA43 TCGCCCTGAAAGAGGACCTGC

PA44 GAGCAGTTGAGAGCCCGTGGAGTGGCTC

PA45 GAGCAGTTGAGAGCCTACCT

PA46 ACATGGCGGCTCAGAAAGTGGACGGGCTCCGC

PA47 GAGCAGTGGAGAGCCTACCT

PA48 CGTGGGGTCGGACTTTGGTACCAGCAGGACGC

PA49 TACCAGCAGGACGCTTACGAC

PA50 GTGCGTGGACGGGCTCCGCAG

PA51 TACCACCAGTACGCCAGTGGGAGGCGGCCC

PA52 GGAGCAGTTGAGAGCGCGTGGACGGGCTCC

PA53 TCAGATCACCCAGCGCAAGTG

PA54 GGTACCGGCAGGACGAGCACAAGTGGGAGG

PA55 TCTCACACCATCCAGAGAGCAGTGGAGAGCC

PA56 GGGTACCGGCAGGACGCCTA

PA57 CATCGCCCTGAAAGAGGGCGGCTCAGATCAC

PA58 CGGCCCATGAGGCGTGGAGGGCACGTGC

PA59 ACCTGCGCTCTTGGAGACATGGCAGCTGTGGAGTGGCTC

PA60 TCTCACACCATCCAGATGATGTAT

PA61 ACCACCAGTACGCCTACTTTGATCACCAAGCGCAA

PA62 CCCATGTGGCGGAGTTTTTCGTGGAGTGGCTCCG

APC2 GCTACTACAACCAGAGCGAGG

APC3 GACGGCAAGGATTACATCGCC

ANC GACGGCAAGCATTACATCG

Second group of HLA-B

PB01 TGCAAGGCCAAGGCACAGACT

PB02 CTGCGCACCGCGCTCCGCTACTAC

PB03 ACTGACCGAGAGAGCCTGCGG

PB04 CTGCGGATCGCGCTCCGCTAC

PB05 CAGATCTCCAAGACCAACACA

PB06 ACACAGATCTACAAGGCCCAGGCA

PB07 ACACAGATCTTCAAGACCAAC

PB08 ACACAGATCTGCAAGACCAAC

PB09 GCCACGAGTCCGAGGAAGGAG

PB10 AGACTTACCGAGAGAGCCTGCGG

PB11 CGGACCCTGCTCCGCTACTAC

PB12 GCCGCGAGTCCGAGAGAGGAG

PB13 CGGAACACACAGATCTCCAAGACC

PB14 TGGGACCGGGAGACACAGATC

PB15 ATGAAGGCCTCCGCGCAGACT

PB16 CGGGAGACACAGATCTCCAAGACC

PB17 CCGAGGACGGAGCCCCGGGCG

PB18 GAGGTATTTCCACACCGCCAT

PB19 ACACCGCCATGTCCCTCCTGCGGAACCTGCG

PB20 CGTGGATAGAGCAGGTTTGGCCCAGGCACAGAC

PB21 AGACCAACACACAGACTGACC

PB22 ACGACACCCAGTTCGATTCGAGAGAGGAGCCGC

PB23 CTTCATCGCAGTGGGTTACCGAGAGAGGAGCCG

PB24 GACGACACCCAGTTCGATTCCGGGAGACACAGAT

PB25 ACGACACCCAGTTCGATACCGAGGATGGCGCCC

PB26 TGCGGAACCTGCGCGGCTAC

PB27 ACGACACGCAGTTCGAATGCGGATCGCGCTCC

PB28 ACACAGATCTGCAAGCGGATCGCGCTCCGC

PB29 GCTTCATTGCAGTGGTTTTTCGGATCGCGCTCCGC

PB30 CGGAACACACAGATCCGGATCGCGCTCCGC

PB31 CGGGAGACACAGATCCGGATCGCGCTCCGC

PB32 GTATTTCCACACCGCCAAGCCTGCGGAACCTG

PB33 ACGACACCCAGTTCGCAAGGCCCAGGCACA

PB34 GCCGTGGGTGGAGCAGGAG

PB35 AGAAGTACAAGCGCCAGGCAC

PB36 CAGATCTCCAAGACCAACATCGCGCTCCGC

PB37 GACGCCACGAGTCCGATGGCGCCCCG

PB38 CCGAGGATGGCGCCCCCGGAACACACAGAT

PB39 AGTCCGAGGATGGCGCCCCTGCGGATCGCGCTC

PB40 GACTTACCGAGAGAACCTGC

PB41 ATTTCTACACCTCCGTGTTGCGGAACCTGCGCG

PB42 GTCCGAGAGAGGAGCTTTGACCGGGAGACACAG

PB43 GCTTCATCTCAGTGGTTTCAAGGCCCAGGCACA

PB44 ACACCTCCGTGTCCTCATCACCGTGGGACTACAACCAG

PB45 ACCGGGAGACACAGAATTCCTGCGGAACCTGCG

PB46 GTCCGAGGAAGGAGCTTATGCGGAACCTGCGCG

PB47 CCGAGAGAGGAGCTTCCGGAACACACTGGAACCTGCGCG

PB48 CCGAGAGAGGAGCTTCGGAACACACTGGAACCTGCGCG

PB49 TTCATCGCAGTGGCCGGAACACACATCTACAACCAG

PB50 CAGATCTACAAGTTTCCAACACACAGAGCTACAACCAGAGC

PB51 GGCGAGTGCGTGGAGTGGCTC

PB52 GTGCGTGGACGGGCTCCGCA

PB53 CAGAGGATGTCTGGCTGCGAC

PB54 GAGCAGCTGAGAACCTACCTG

PB55 GGGCATGACCAGTACGCCTAC

PB56 GTGGCGGAGCAGGACAGAGCCTAC

PB57 CGGAGCAGCGGAGAGCCTACC

PB58 ATCTCCCAGCGCAAGTTGGAG

PB59 CGCGGGTATGACCAGGACGCC

PB60 CGCGGGTACCACCAGGACGCC

PB61 CGCGGGCATAACCAGTTCGCCTAC

PB62 AAGGACACGCTGGAGCGCGCG

PB63 CGTGAGGCGGAGCAGTGGAGA

PB64 CTGGAGGGCACGTGCGTGGAG

PB65 CGGCTGCGACCTGGGGCCGGA

PB66 GGGCATGACCAGTCCGCCTACGAC

PB67 GGGTCTCACACCCTCCAGAGGATG

PB68 CGTGAGGCGGAGCAGCTGAGA

PB69 TGCGTGGAGTCGCTCCGCAGA

PB70 CGCGGCGGACACGGCGGCTC

PB71 CAGAGGATGTACGGCTGCGAC

PB72 ACCAGTTAGCCTACGGGACCTGAGCTCCTG

PB73 GGACCTGAGCTCCTGTTTGTGGAGTGGCTCCGC

PB74 CGGACACGGCGGCTCTGGGAAGGAGACGCTG

PB75 CTCCAGAGCATGTACGGGCATAACCAGTACGCC

PB76 GCGGGCATGACCAGTGACGGACACCGCGGCTC

PB77 CATGACCAGTCCGCCTTTTCGCAGATACCTGGAG

PB78 GGGCATAACCAGTACGCCTAC

PB79 CGGAGCAGGACAGAGATAGGAGTCGCTCCGCAG

PB80 GATGTATGGCTGCGACTGACCAGTCCGCCTA

PB81 CACACTTGGCAGACGTTAGGAGGGCCTGTGCGT

PB82 CATCGCCCTGAACGTTTGACCTGCGCTCCTG

PB83 CACCCTCCAGAGGATGTGAGGGCACGTGCGTG

PB84 AGCAGCTGAGAGCCTTTTCGCAGACACCTGGAG

PB85 GAGCAGCTGAGAGCCTACCTG

PB86 GAGGGCCTGTGCGTGTTTCGCAGACACCTGGAG

PB87 CCTCCAGAGCATGTACGTTTGCTCAGATCTCCCAGCG

PB88 GGTCTCACACTTGGCAGAATTCTGCGACCTGGGGCC

PB89 TCCAGGTGATGTATGGCTGCG

PB90 CATGACCAGTTCGCCTACGAC

PB91 GCGGACACGGCGGCTGAGCAGCGGAGAGCC

PB92 ACATCATCCAGAGGATGACCCTGAACGAGGACC

PB93 GTGAGGCGGAGCAGCGGAGA

PB94 CTGCGACCTGGGGCTACATCGCCCTGAATCCCGTGTGGCGGAG

PB95 CAGCTGAGAGCCTACAAAAGGGCCTGTGCGTGG

PB96 TCCAGAGCATGTACGGCTGCG

BPC2 ACTACAACCAGAGCGAGGCCG

BPC3 ATTACATCGCCCTGAACGAGGAC

BNC CATCGCCGTCAACGAGGAC

The 3rd group of HLA-DRB1

PD01 AGCTTAAGTTTGAATGTCATTTCT

PD02 GAGCAGGTTAAACATGAGTGTCAT

PD03 GTGGCAGGGTAAGTATAAGTG

PD04 GAAGCAGGATAAGTTTGAGTG

PD05 GGAGGAGGTTAAGTTTGAGTG

PD06 ATAACCAGGAGGAGTCCGTGC

PD07 CAGAAGGACATCCTGGAAGAC

PD08 GCGGTGACGGAGCTTTTCTGCTGCGGAGCACT

PD09 GAGTACTCTACGTCTGTTTTGGAGCAGAAGCGGG

PD10 GGGCCCTGGTGGACTTTGTTGTG GAGAGCTTC

PD11 GGACATCCTGGAAGACGAGC

PD12 AGTACTCTACGTCTGATTTCGGCCTGATGAGGAG

PD13 GGAGTACGTGCGCTTTTTGGCCCTGGTGGACA

PD14 GTGCGGTTACTGGAGAGACAC

PD15 GCCTGATGCTGAGTACTGGA

PD16 CCTGATGAGGAGTACTTTTCTGGAAGACGAGCGG

PD17 GACATCCTGGAAGACAAGCGG

PD18 GGGAGTTCCGGGCTTACCTGGAAGACGAGCG

PD19 GAGGAGTTCGTGCGCATTCCTGATGAGGAGTAC

PD20 TGATGAGGAGCACTGGAACAG

PD21 AGATACTTCTATCACCAAGAGGAG

PD22 GAGGAGAACGTGCGCATTGTGGACAACTACTGC

PD23 ACTCTACGGGTGAGTGTATTGGGAGTACCGGGC

PD24 GTTCCTGGACAGATACTATTCTGATGCCGAGTACT

PD25 CAGAAGGACCTCCTGGAAGAC

PD26 GGAGTACTCTACGTCTGTTTGTGGACAATTACTGCA

PD27 TGGAGTACTCTACGTCTTTTGCCTG ACGCTGAGTA

PD28 GGGTGAGTGTTATTTCTTCAATGG

PD29 CCTGGACAGATACCTTCGACAGCGACCCTGATGCGGAG

PD30 CTACGTCTGAGTGCCTTCGACAGCGACTGGGTTGTGGAGAG

PD31 CTACGTCTGAGTGGGAGTTCCGGGCAAGGACCTCCTGG

PD32 GTACTCTACGTCTGAGTGCGGTACCTGGAC

PD33 GACATCCTGGAGGACAGGCG

PD34 CTTGGAGTACTCTACGCTTCCATAACCAGGAG

PD35 CCTGGAGAGATACTTCGAGGAGAACGTGCGC

PD36 AGGAGAACGTGCGCTCCTGATGCCGAGTACTG

PD37 GAGGAGTTCGTGCGCTGCTGCGGAGCACT

PD38 GAAGACGAGCGGGCGGTTGGTGAGAGCTTC

PD39 GGTTCCTGGACAGATACGCCTAGCGCCGAGT

PD40 GGAGTACTCTACGGGTGAGTG

PD41 TCTACGTCTGAGTGTCCCTGGAAGACAGGCG

PD42 CCTGGAAGACAGGCGACGGGGTTGTGGAGA

PD43 CTGATGCCGAGTACTGCCTGGAAGACAGGC

PD44 TGGCAGCCTAAGAGGTCCTGGAAGACAGGCG

PD45 GAGGAGTCCGTGCGCGGGAGTTCCGGGC

PD46 GGCCTAGCGCCGATGGAGCAGAGGCGGG

PD47 GGTTCCTGGACAGATACGAAGGACTTCCTGGA

PD48 GGAGTACGTGCGCTTCCTGATGAGGAGTACTG

PD49 GGAGAGATACTTCCATAGTGGACACCTACTGC

PD50 CAGGAGGAGAACGTGCGCCTGGAAGACGAGCGG

PD51 TCCTGGAAGACGAGCGGGGGTTGGTGAGAGC

PD52 CGGCCTAGCGCCGAGCTGGAGCAGAGGCGG

PD53 CTGGAGCAGGCGCGGGGGGTTGGTGAGAGC

PD54 TTCTTCAATGGGACGGTTTTTGGACTTCCTGGAAGAC

PD55 GGTGCGGTTCCTGTGAGTACGTGCGCCGACGTGGGGGA

PD56 GCGGTTCCTGGACAGTTTTTGGCCTGATGCCGAGT

PD57 GGAGTACGTGCGCATGATGAGGAGTACGAAGGACTTCCTGGA

PD58 CTTCGACAGCGAGCCGAGTACTGGACTGGAAGACGAGC

PD59 AGGAGTTCGTGCGCCCTGATGCCGAGAGGGTTGTGGAGA

PD60 TGGAGTACTCTACGATCGACAGCGACTGGGGCTGTGGAGAG

PD61 GTACTCTACGTCTGATTTGTGCGGTACCTGGAC

PD62 AGGAGAACGTGCGCTATTCCTGATGCCGAGTACTG

PD63 TCCTGGACAGATACTATTCTGGAGCAGATTTGTGGACACCTACTG

PD64 GAGTACTCTACGTCTGTTTTTCTGGAGCAGAGGCGG

PD65 GTACTCTACGTCTGAGTTGATGAGGAGTACTGG

PD66 GGCCTGATGCCGAGTTTTTTGGGCCCTGGTGGACA

PD67 GTACTCTACGGGTGAGTCGCTTCGACAGCGAC

PD68 CTCTACGTCTGAGTGTCTTACCTGGAAGACAGGC

PD69 CTGATGCCGAGTACTGTTACCTGGAAGACAGGC

PD70 CTGGACAGATACTTACTTCGACAGCGATAGGACATCCTGGA

PD71 AGAGGAGTACGTGCGTTCCTGGAAGACAGGC

PD72 GGTTCCTGGACAGATACATTGAAGGACTTCCTGGA

PD73 GAGTACTCTACGTCTGATTTTTCTGGAGCAGAAGCGG

PD74 TTCTTCAATGGGACGGGGACTTCCTGGAAGAC

PD75 CCTGGAGAGATACTTCGGAAGACGAGCGGGC

PD76 CCTGGACAGATAAGAGCAGAAGCGGGTCACCTACTGCAG

PD77 GCTTCGACAGCGACTGGCCTGTCGCCGATGGAAGACAGGCG

PD78 TCTACGTCTGAGTGTCTTTTTCCGCGGTGGACACCT

PD79 CTGATGCCGAGTACTCTGGAAGACAGGCGG

PD80 CTAAGAGGGAGTGTGGGAGTACCGGGCAAGACAGGCG

PD81 GCTTCGACAGCGACTCTGATGCCGAGTGAGCAGAGGCGGG

PD82 AGCCTAAGAGGGACTTCGACAGCGACTGAGCAGGCGCGGGC

PD83 CCTGGAGAGATACGAGCAGAGGCGGGGACACCTACTGC

PD84 CCTGGACAGATACGATGCCGAGTACTAGGTTGGTGAGAGC

PD85 TGCGGTTCCTGTGAGGAGTACGTGCGTACGTGGGGGA

PD86 GCAGCCTAAGAGGGATTTGGGGAGTACCGGGCG

PD87 CTGGACAGATACATTCGACAGCGATCTGGAGCGGAGGC

PD88 TCGACAGCGACGTAGGGCCCTGGTGGAGGTTGTGGAGAGC

PD89 CTTCGACAGCGTGGGAGTTCCGGGCAAGGACTTCCTGG

PD90 TCTACGTCTGAGTTTCGACAGCGACTGAGCAGAAGCGGG

DPC GCGCTTCGACAGCGACGTGGG

DNC CGCTTCGACTCCGACGTGG

2, a kind of gene chip that is used for HLA gene's somatotype is formed by carrier with the crosslinked probe of carrier, it is characterized in that, comprises one group or its combination in the said three groups of probes of claim 1.

3, gene chip as claimed in claim 2 is characterized in that, said carrier is sheet glass, silicon chip and membrane matrix.

4, gene chip as claimed in claim 2 is characterized in that, said carrier is to contain the microballoon that inertia macromolecular material fluorescent substance, diameter 4～7um is made.

5, gene chip as claimed in claim 4 is characterized in that, said carrier is the polystyrene microsphere through carboxyl modified.

6, gene chip as claimed in claim 2 is characterized in that, it is 5 ' terminal modified that said probe carries out, and represents with following formula:

X-(5 '-probe-3 ')

X=NH wherein ₂-(CH ₂-CH ₂) _n-O-; N is 3～6 integer.

7, each said gene chip of claim 4～6 is characterized in that, the carrier microballoons covalent cross-linking of a kind of probe and a kind of specific fluorescent with one group of fluorescent microsphere balanced mix that probe is crosslinked, is promptly formed a kind of gene chip.

8, one group of Auele Specific Primer that is used for HLA gene's somatotype is characterized in that, primer is selected from a group or its combination in following three groups:

First group of HLA-A

AE2F1 CGCCTCTGYGGGGAGAAGCAA

AE2R1 GCCCGTCCGTGGGGGATGA

AE2R2 GGGGATGAGGGGTCSTGACCTGC

AE3F1 GCCCAGGCGCCTTTACCCGGTT

AE3F2 GGGAGAGGCCCAGGCGCCTT

AE3R1 GGGAGGCCAGCCCGGGAGAYCTA

AE3R2 GTCCCAAWTGTCTCCCCTCCTT

Second group of HLA-B

BE2F1 GGGAGGAGCGAGGGGACCSCAG

BE2R1 CCCGGGCCGGGGTCACTCA

BE2R2 GGGGGATGGGGAGTCGTGACCT

BE3F1 CCCGGTTTCATTTTCAGTTGAGGCCAA

BE3R1 GGCCATCCCCGSCGACCTATAGGAGAT

The 3rd group of HLA-DRB1

DE2F1 CCGGATCCTTCGTGTCCCCACAGCACG

DE2F2 CGTTCGTGTCCCCACAGCACGTT

DE2F3 CGTTCTTGTCCCCCCAGCACGTT

DE2R1 TCGCCGCTGCACTGTGAAGCTC

DE2R2 TgCTYACCTCgCCKCTgCAC

9, primer as claimed in claim 8 is characterized in that, probe has biotin labeling.

10, a kind of test kit that is used for the detection of HLA gene's somatotype is characterized in that, comprises said gene chip of claim 2 and/or the said primer of claim 8.

11, a kind of method of HLA gene's somatotype is characterized in that, comprises the steps:

(1) sample disposal

Sample to be checked extracts genomic dna with ordinary method, as the pcr amplification template;

(2) multiplex PCR amplification

The primer system of the pcr amplification of HLA-A gene: PCR is AE2F1: AE2R2: AE2R1: AE3F1: AE3R1: AE3F2: AE3R2=1: 2: 0.1: 1: 2: 0.1: 0.1, the concentration of primer AE2F1 was 0.1～0.4uM;

The primer system of the pcr amplification of HLA-B gene: PCR is BE2F1: BE2R1: BE2R2: BE3F1: BE3R1=1: 2: 0.1: 1.5: 3, and the concentration of primer BE2F1 is 0.1～0.4uM;

The primer system of the pcr amplification of HLA-DRB1 gene: PCR is DE2F1: DE2F2: DE2F3: DE2R1: DE2R2=1: 0.5: 0.5: 2: 0.1, and the concentration of primer DE2F1 is 0.1～0.2uM;

(3) hybridization

Respectively with HLA-A ,-B ,-gene chip of DRB1 probe and the crosslinked composition of fluorescent microsphere is suspended in the hybridization solution;

Pcr amplification product is added the hybridization system that contains corresponding chip, and 60 ℃ of incubation 15min. are hatched the phycoerythrin (SAPE) of reacted microballoon and streptavidin modification again;

(4) interpretation as a result

With Luminex detector read data information, with HLA somatotype software analysis result.