CN104862379A - Detection kit for human leucocyte antigen genes - Google Patents

Abstract

The invention belongs to the technical field of biological medicines and relates to a detection kit for human leucocyte antigen (HLA) genes--HLA-B*59: 01 genes, and the HLA-B*59: 01 genes can serve as marker genes for forecasting methazolamide caused SJS (Stevens-Johnson syndrome) and TEN (Toxic epidermal necrolysis). According to the detection kit, after DNA extracted from the peripheral blood of a patient, the PCR-SSO method is adopted to detect the HLA-B*59 :01 genes. The detection kit for HLA-B*59: 01 genes can be used for examining before application of methazolamide and screening targeted medicines for treating methazolamide caused SJS and TEN; and examination can guide clinical medication to reduce incidence of SJS and TEN caused by methazolamide, and the screening mainly acts on HLA-B*59: 01 molecules in a targeted manner, so that interaction between the HLA-B*59: 01 molecules and methazolamide or other metabolic products during attack.

Description

A detection kit for human leukocyte antigen gene

technical field

The invention belongs to the field of biomedicine and reagent detection, and relates to a test kit for detecting human leukocyte antigen gene; Two types of cutaneous adverse drug reactions (Stevens-Johnson syndrome and toxic epidermal necrolysis) were associated.

Background technique

Drug eruption refers to the inflammatory reaction of skin and mucous membranes caused by drugs entering the human body through various channels. Stevens-Johnson syndrome (Stevens-Johnson syndrome, SJS) and toxic epidermal necrolysis (Toxic epidermal necrolysis, TEN) belong to the same spectrum of severe skin adverse drug reactions. exfoliation and necrosis of severe hepatic and renal dysfunction (Bastuji-Garin S, Rzany B, Stern RS, Shear NH, Naldi L and Roujeau JC. Clinical classification of cases of toxicepidermal necrolysis, Stevens-Johnson syndrome, and erythema multiforme. Arch Dermatol 1993;129:92-96.). According to statistics, the annual incidence rates of TEN and SJS are 0.4-1.2/1000000 and 1.2-6/1000000 respectively (Roujeau JC and Stern RS.Severe adverse reactions to drugs.N Engl J Med1994;331:1272-1285.), but The mortality rate is as high as 1-5% and 20-30%, respectively (Roujeau JC and Stern RS. Severe adverse reactions to drugs. N Engl J Med1994; 331:1272-1285. Rzany B, Mockenhaupt M, Baur S, Schroder W, Stocker U, Mueller J, Hollander N, Bruppacher R and Schopf E. Epidemiology of erythema exsudativum multiformemajus, Stevens-Johnson syndrome, and toxic epidermal necrolysis in Germany(1990-1992): structure and results of ple lin-aColregulation 9; 49:769-773.).

Methazolamide is a sulfonamide carbonic anhydrase inhibitor, which is mainly used clinically to reduce elevated intraocular pressure caused by glaucoma and other diseases (Fritsch PO and Sidoroff A.Drug-induced Stevens-Johnson syndrome/toxic epidermal necrolysis.Am J Clin Dermatol2000;1:349-360. Mockenhaupt M, Viboud C, Dunant A, Naldi L, Halevy S, BouwesBavinck JN, Sidoroff A, Schneck J, Roujeau JC and Flahault A. with emphasis on recently marketed drugs. The EuroSCAR-study. J Invest Dermatol 2008;128:35-44.). Studies have shown that methazolamide is a common drug causing SJS and TEN (Sud RN and Grewal SS. Stevens Johnson syndrome due to Diamox. Indian J Ophthalmol1981; 29:101-103. Flach AJ, Smith RE and Fraunfelder FT. Stevens-Johnson syndrome associated with methazolamide treatment reported in two Japanese-American women. Ophthalmology 1995;102:1677-1680.).

Human leukocyte antigen (Human leukocyte antigen, HLA) gene expression product is human leukocyte antigen, which plays a vital role in the immune response. Studies have found that drug eruptions caused by several drugs, including carbamazepine, abacavir, and allopurinol, are associated with HLA alleles (Bharadwaj, M., et al., Drug hypersensitivity and human leukocyteantigens of the major histocompatibility complex.Annu Rev PharmacolToxicol,2012.52:p.401-31.Cao,Z.H.,et al.,HLA-B*58:01allele isassociated with augmented risk for both mild and severe cutaneous adversereactions induced by allopurinol in Han Chinese.Pharmacogenomics ,2012.13(10):p.1193-201.Roujeau,J.C.,et al.,Genetic susceptibility to toxic pidermal necrolysis.Arch Dermatol,1987.123(9):p.1171-3.). In the study of four Japanese patients with SJS caused by methazolamide, Shirato et al. found that three of them carried HLA-B*59 (Shirato S, Kagaya F, Suzuki Y and Joukou S.Stevens-Johnson syndrome induced by methazolamide treatment. Arch Ophthalmol 1997;115:550-553.). In a Korean study, Kim et al. performed HLA-A,-Band-C typing on five cases of SJS caused by methazolamide, and found that HLA-B*59:01 was highly correlated with SJS caused by methazolamide (p<0.001; odds ratio: 249.8; 95% CI: 13.4–4813.5). (Kim SH, Kim M, Lee KW, Kim SH, KangHR, Park HW and Jee YK. HLA-B*5901 is strongly associated withmethazolamide-induced Stevens-Johnson syndrome/toxic epidermalnecrolysis. Pharmacogenomics2010;11:879-884.)

After searching the existing technical literature, it is found that there is no correlation between the HLA-B*59:01 gene and the pathogenesis of SJS and TEN caused by methazolamide in the Han population in mainland China, and it can be used as a genetic marker to screen for this type. Reports on drug eruptions.

Contents of the invention

The object of the present invention is to provide a kit for detecting human leukocyte antigen gene--HLA-B*59:01 gene and its application method.

A further object of the present invention is to provide the above kit for screening the application of methazolamide-induced skin adverse reactions, specifically, by detecting the presence of the HLA-B*59:01 gene to evaluate patients taking methazolamide. Whether there is a potential for SJS and TEN after methazolamide. At the same time, it can be used to guide clinical medication, thereby reducing the occurrence of SJS and TEN caused by methazolamide.

In one aspect, the present invention provides a detection kit for human leukocyte antigen gene.

The human leukocyte antigen gene is HLA-B*59:01 gene;

The kit contains reagents for detecting the nucleic acid or protein of the human leukocyte antigen gene.

The kit contains amplification primers for human leukocyte antigen genes; or labeled probes.

Alternatively, the kit contains specific antibodies to human leukocyte antigen genes.

The nucleotide sequence of the related HLA allele-HLA-B*59:01 gene of SJS and TEN caused by methazolamide of the present invention is as shown in SEQ NO.1:

(a) Sequence features:

* Length: 3333 base pairs

*Type: nucleic acid

* Chain type: double chain

*Topology: linear

(b) Molecular type: Genomic DNA (gDNA library)

(c) Assumption: No

(d) Antisense: no

(e) Primary source: human

(f) SEQ NO.1 nucleic acid sequence is described as follows:

gatcaggacgaagtcccatgtcccggacggggctctcagggtctcaggct50ccgagggccgcgtctgc

attggggaggcgcagcgttggggattccccact

100cccacgagtttcacttcttctcccaacctatgtcgggtccttcttccagg

150atactcgtgacgcgtccccatttcccactccccatgggtgtcgggtgtct

200agagaagccaatcagtcgccggggtcccagttctaaagtccccacgca

250cccacccggactcagagtctcctcagacaccgagatgcgggtcacggcac

300cccgaaccctcctcctgctgctctggggggccctggccctgaccgagacc350

tgggccggtgagtgcgggtcgggagggaaatggcctctgtgggggaggagc

400gagggggaccgcaggcggggggcgcaggacccggggagccgcgccggggagga

450gggtctggcgggtctcagcccctcctcgcccccaggctcccactccatga

500ggtatttctacaccgccatgtcccggcccggccgcggggagccccgcttc

550atcgcagtgggctacgtggacgacacgcagttcgtgaggttcgacagcga

600cgccgcgagtccgagagaggagccgcgggcgccgtggatagagcaggagg

650ggccggagtattgggaccggaacacacagatcttcaagaccaacacacag

700acttaccgagagaacctgcggatcgcgctccgctactacaaccagagcga

750ggccggtgagtgaccccggcccggggcgcaggtcacgactccccatcccc

800cacgtacggcccgggtcgccccgagtctccgggtccgagatccgcctccc

850tgaggccgcgggacccgcccagaccctcgaccggcgagagccccaggcgc

900gtttacccggtttcattttcagttgaggccaaaatccccgcgggttggtc950

ggggcggggcggggctcgggggacggggctgaccgcggggccggggccag

1000ggtctcacacttggcagacgatgtatggctgcgacctggggccggacggg

1050cgcctcctccgcgggcataaccagttagcctacgacggcaaggattacat

1100cgccctgaacgaggacctgagctcctggaccgcggcggacaccgcggctc1150

agatcacccagcgcaagtgggaggcggcccgtgtggcggagcagctgaga1200

gcctacctggagggcacgtgcgtggagtggctccgcagatacctggagaa1250

cgggaaggagacgctgcagcgcgcgggtaccaggggcagtggggagcctt1300

ccccatctcctataggtcgccggggatggcctcccacgagaagaggagga1350

aaatgggatcagcgctagaatgtcgccctcccttgaatggagaatggcat1400

gagttttcctgagtttcctctgagggccccctcttctctctaggacaatt1450

aagggatgacgtctctgaggaaatggaggggaagacagtccctagaatac1500

tgatcaggggtcctctttgacccctgcagcagccttgggaaccgtgactt1550

ttcctctcaggccttgttctctgcctcacactcagtgtgtttggggctct1600

gattccagcacttctgagtcactttacctccactcagatcaggagcagaa1650

gtccctgttccccgctcagagactcgaactttccaatgaataggagatta1700

tcccaggtgcctgcgtccaggctggtgtctgggttctgtgccccttcccc1750

accccaggtgtcctgtccattctcaggctggtcacatgggtggtcctagg1800

gtgtcctatgagagatgcaaagcgcctgaattttctgactcttcccatca1850

gaccccccaaagacacacgtgacccaccacccccatctctgaccatgaggc1900

caccctgaggtgctgggccctgggcttctaccctgcggagatcacactga

1950cctggcagcgggatggcgaggaccaaactcaggacactgagcttgtggag

2000accagaccagcaggagatagaaccttccagaagtgggcagctgtggtggt

2050gccttctggagaagagcagagatacacatgccatgtacagcatgaggggc2100

tgccgaagcccctcaccctgagatggggtaaggaggggggatgaggggtca2150

tatctcttctcagggaaagcaggagcccttctggagcccttcagcagggt2200

cagggcccctcgtcttcccctcctttcccagagccatcttcccagtccac2250

catccccatcgtgggcattgttgctggcctggctgtcctagcagttgtgg2300

tcatcggagctgtggtcgctactgtgatgtgtagggaggaagagctcaggt2350

agggaaggggtgaggggtggggtctgggttttcttgtcccactgggggtt2400

tcaagccccaggtagaagtgttccctgcctcattactgggaagcagcatc2450

cacacaggggctaatgcagcctgggaccctgtgtgccagcacttactctt2500

ttgtgcagcacatgtgacaatgaaggacggatgtatcaccttgatggttg2550

tggtgttggggtcctgatttcagcattcatgagtcagggggaaggtccctg2600

ctaaggacagacccttaggagggcagttggtccaggacccaacacttgcttt2650

cctcgtgtttcctgatcctgccttgggtctgtagtcatacttctggaaat2700

tccttttgggtccaagacgaggaggttcctctaagatctcatggccctgc2750

ttcctcccagtcccctcacaggacattttcttcccacaggtggaaaagga2800

gggagctactctcaggctgcgtgtaagtggtgggggtgggagtgtggagg2850

agctcacccaccccataattcctcctgtcccacgtctcctgcgggctctg2900

accagtcctgtttttgttctactccagccagcgacagtgcccagggctc2950

tgatgtgtctctcacagcttgaaaaggtgagattcttggggtctagagtg3000

ggcggggggggggtggggtggggagggggcagagggggaaaggcctgggta3050

atggagattctttgattgggatgttacgcgtgtgtggtgggctgtttaga3100

gtgtcatcacttaccatgactaaccagaatttgttcatgactgttgtttt3150

ctgtagcctgagacagctgtcttgtgagggactgagatgcaggatttctt3200

cactcctcccctttgtgacttcaagagcctctggcatctctttctgcaaa3250

ggcacctgaatgtgtctgcgtccctgttagcataatgtgaggaggtggag3300

agacagcccaccccccgtgtccactgtgacccct

In another aspect, the present invention provides the application of the above detection kit, that is, to type the HLA-B*59:01 gene of the sample.

For example, as shown in the examples of the present invention, the HLA-B*59:01 gene is typed using the PCR-SSO method.

Specifically, the steps for typing the HLA-B*59:01 gene are as follows:

DNA extraction from samples;

Amplification of polymorphic regions of HLA;

Isotopic or non-isotopic labeling of PCR products during amplification;

The PCR product was hybridized with the probe on the membrane, and the result was judged according to the signal by autoradiography.

Wherein, the probes are designed for PCR amplification products and fixed on the membrane.

The sample can be RNA, protein, cell or serum sample.

Most commonly, the sample is isolated peripheral blood.

The analysis results of HLA-B*59:01 gene can be used to judge SJS and TEN.

The experimental data of the present invention proves that the HLA-B*59:01 gene can be used as a marker gene for predicting SJS and TEN caused by methazolamide.

The experimental results showed that the HLA-B*59:01 positive ratio in patients with SJS and TEN induced by methazolamide was significantly different from that in the normal control group and the methazolamide-resistant group (as shown in Table 3). Show). The binding model of HLA-B*59:01 and methazolamide is shown in Figure 1 (the binding model of methazolamide and HLA-B*59:01 (Figure 1A) and the binding detail model (1B)).

Examples of the utility of the present invention are:

1) The HLA-B*59:01 gene introduced in the present invention can be used as a marker gene for predicting SJS and TEN induced by methazolamide.

2) The HLA-B*59:01 gene provided by the present invention can be used to evaluate or prepare a pre-use screening kit for methazolamide.

3) The HLA-B*59:01 molecule provided by the present invention can be used to evaluate or prepare targeted drugs for drug eruption caused by methazolamide.

The invention relates to a human leukocyte antigen gene--HLA-B*59:01 gene, which is related to the pathogenesis of SJS and TEN caused by methazolamide. HLA-B*59:01 gene can be used as a marker gene to predict SJS and TEN induced by methazolamide. After extracting DNA from the patient's peripheral blood, use the current method to detect the HLA-B*59:01 gene, such as PCR-SSO (sequence-specific oligonucleotide probe) method. The HLA-B*59:01 gene detection kit provided by the present invention can be used for screening before the use of methazolamide and for screening targeted drugs for the treatment of SJS and TEN caused by methazolamide. The former can guide clinical medication, thereby reducing the occurrence of SJS and TEN caused by methazolamide. The latter mainly targets the HLA-B*59:01 molecule, thereby blocking its interaction with methazolamide or its metabolites in pathogenesis.

Description of drawings

Figure 1 is the binding model diagram of methazolamide and HLA-B*59:01. Among them, (A) is the structure overlap diagram before and after the combination of methazolamide and HLA-B*59:01; (B) is the Methazolamide binding details to HLA-B*59:01.

Detailed ways

Below in conjunction with specific embodiment, to further illustrate the present invention. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1

1) Collect and extract genes

Patients with SJS and TEN caused by methazolamide were from Huashan Hospital Affiliated to Fudan University, Shanghai, China;

The diagnostic criteria were proposed by Roujeau et al. (Roujeau JC. Clinical heterogeneity of drug hypersensitivity. Toxicology 2005; 209: 123-129.), that is, clinical manifestations include extensive exfoliation and necrosis of the epidermis, oral mucosal erosion, with or without severe liver and kidney function Systemic complications such as damage, among them, the area <10% is called SJS; >30% is called TEN; 10% to 30% is called SJS/TEN overlap; participate in the research of this topic under the premise of informed consent , blood was collected, and informed consent was signed; all patients were excluded from other diagnoses such as viral eruption and toxic erythema through medical history and skin pathological examination, and all patients took methazolamide orally for the first time within 2 months before the onset of rash; There were two groups of controls in the experiment, one was 283 healthy people from the human MHC database (dbMHC), and the other was a group of 30 patients from eastern China who were methazolamide-tolerant, and all of them took oral methazolamide for more than 3 months without any drug reaction; take 300ul of blood sample, extract DNA according to the kit instructions, and use UV spectrophotometer to detect DNA concentration and purity;

2) Detection of HLA genotyping

The present invention adopts PCR-SSO method (recommended SSO Kit-One Lambda, CA, USA) for HLA genotyping, the principle is to amplify the polymorphic region of HLA first, and perform isotope or non-isotope labeling on the PCR product during the amplification process, and then target PCR The amplified product is designed according to the principle of base pairing and a series of oligonucleotide probes are immobilized on the membrane. Finally, the PCR product is hybridized with the probe on the membrane, and the result is judged according to the signal by autoradiography; the HLA gene analysis is carried out according to the standard steps of the kit. Type (that is, DNA samples, substrates, Taq enzymes, and primers are mixed and mixed, added to the amplification plate, amplified according to the conditions in the kit instructions, and the amplified products are hybridized, stained, and plate read); the results are analyzed by HLA Fusion Software (One lambda, CA, USA, HLA Fusion3.0);

3) HLA-B*59:01 is associated with SJS and TEN induced by methazolamide

Statistical method: use SPSS16.0 to calculate Odds Ratio (OR) and its 95% credible interval, use Haldane’s correction if necessary, choose chi-square test for statistical analysis, and set the statistical significance level to be less than 0.05;

The results showed that: a total of 7 patients with SJS and TEN caused by methazolamide were collected, of which 6 were diagnosed as TEN, 1 was SJS (as shown in Table 1), 30 patients were in the methazolamide-resistant group; There was no statistical difference in general characteristics such as age and sex between the tolerance group and the tolerance group (as shown in Table 2); the positive proportion of HLA-B*59:01 in patients with SJS and TEN caused by acetazolamide was compared with that in the normal control group There was a statistically significant difference between the methazolamide and methazolamide-resistant groups (as shown in Table 3); the results also showed that the structural model of HLA-B*59:01 before and after combining with methazolamide (as shown in Figure 1A) , in addition, methazolamide can bind to the A-binding groove of the HLA-B*59:01 molecule (as shown in Figure 1B).

Table 1. Clinical characteristics of patients with SJS and TEN caused by methazolamide

SJS: Stevens-Johnson syndrome; TEN: toxic epidermal necrosis syndrome

Table 2. Clinical data of patients with SJS and TEN caused by methazolamide and tolerance group

Table 3. Frequency and association analysis of HLA-B*59:01, Cw*01:02 and their haplotype frequencies in SJS and TEN induced by methazolamide and the control group

95%CI: 95% credible interval.

SEQUENCE LISTING

the

<110> Fudan University

the

<120> A detection kit for human leukocyte antigen gene

the

<160> 1

the

<170> PatentIn version 3.3

the

<210> 1

<211> 6283

<212> DNA

<213> People

the

<400> 1

gatcaggacg aagtcccatg tcccggacgg ggctctcagg gtctcaggct ccgagggccg 60

the

cgtctgcatt ggggaggcgc agcgttgggg attccccact cccacgagtt tcacttcttc 120

the

tcccaaccta tgtcgggtcc ttcttccagg atactcgtga cgcgtcccca tttcccactc 180

the

ccattgggtg tcgggtgtct agagaagcca atcagtgtcg ccggggtccc agttctaaag 240

the

tccccacgca cccacccgga ctcagagtct cctcagacac cgagatgcgg gtcacggcac 300

the

cccgaaccct cctcctgctg ctctgggggg ccctggccct gaccgagacc tgggccggtg 360

the

agtgcgggtc gggagggaaa tggcctctgt ggggaggagc gagggggaccg caggcggggg 420

the

cgcaggaccc ggggagccgc gccgggagga gggtctggcg ggtctcagcc cctcctcgcc 480

the

cccaggctcc cactccatga ggtatttcta caccgccatg tcccggcccg gccgcgggga 540

the

gccccgcttc atcgcagtgg gctacgtgga cgacacgcag ttcgtgaggt tcgacagcga 600

the

cgccgcgagt ccgagagagg agccgcgggc gccgtggata gagcaggagg ggccggagta 660

the

ttgggaccgg aacacacaga tcttcaagac caacacacag acttaccgag agaacctgcg 720

the

gatcgcgctc cgctactaca accagagcga ggccggtgag tgaccccggc ccggggcgca 780

the

ggtcacgact ccccatcccc cacgtacggc ccgggtcgcc ccgagtctcc gggtccgaga 840

the

tccgcctccc tgaggccgcg ggacccgccc agaccctcga ccggcgagag ccccaggcgc 900

the

gtttacccgg tttcattttc agttgaggcc aaaatccccg cgggttggtc ggggcggggc 960

the

ggggctcggg ggacggggct gaccgcgggg ccggggccag ggtctcacac ttggcagacg 1020

the

atgtatggct gcgacctggg gccggacggg cgcctcctcc gcgggcataa ccagttagcc 1080

the

tacgacggca aggattacat cgccctgaac gaggacctga gctcctggac cgcggcggac 1140

the

accgcggctc agatcaccca gcgcaagtgg gaggcggccc gtgtggcgga gcagctgaga 1200

the

gcctacctgg agggcacgtg cgtggagtgg ctccgcagat acctggagaa cgggaaggag 1260

the

acgctgcagc gcgcgggtac caggggcagt ggggagcctt ccccatctcc tataggtcgc 1320

the

cggggatggc ctcccacgag aagaggagga aaatgggatc agcgctagaa tgtcgccctc 1380

the

ccttgaatgg agaatggcat gagttttcct gagtttcctc tgagggcccc ctcttctctc 1440

the

taggacaatt aagggatgac gtctctgagg aaatggaggg gaagacagtc cctagaatac 1500

the

tgatcagggg tcctctttga cccctgcagc agccttggga accgtgactt ttcctctcag 1560

the

gccttgttct ctgcctcaca ctcagtgtgt ttggggctct gattccagca cttctgagtc 1620

the

actttacctc cactcagatc aggagcagaa gtccctgttc cccgctcaga gactcgaact 1680

the

ttccaatgaa taggagatta tcccaggtgc ctgcgtccag gctggtgtct gggttctgtg 1740

the

ccccttcccc accccaggtg tcctgtccat tctcaggctg gtcacatggg tggtcctagg 1800

the

gtgtcctatg agagatgcaa agcgcctgaa ttttctgact cttcccatca gaccccccaa 1860

the

agacacacgt gacccaccac cccatctctg accatgaggc caccctgagg tgctgggccc 1920

the

tgggcttcta ccctgcggag atcacactga cctggcagcg ggatggcgag gaccaaactc 1980

the

aggacactga gcttgtggag accagaccag caggagatag aaccttccag aagtgggcag 2040

the

ctgtggtggt gccttctgga gaagagcaga gatacacatg ccatgtacag catgaggggc 2100

the

tgccgaagcc cctcaccctg agatggggta aggaggggga tgaggggtca tatctcttct 2160

the

cagggaaagc aggagccctt ctggagccct tcagcagggt cagggcccct cgtcttcccc 2220

the

tcctttccca gagccatctt cccagtccac catccccatc gtgggcattg ttgctggcct 2280

the

ggctgtccta gcagttgtgg tcatcggagc tgtggtcgct actgtgatgt gtagggaggaa 2340

the

gagctcaggt agggaagggg tgaggggtgg ggtctgggtt ttcttgtccc actgggggtt 2400

the

tcaagcccca ggtagaagtg ttccctgcct cattactggg aagcagcatc cacacagggg 2460

the

ctaatgcagc ctgggaccct gtgtgccagc acttactctt ttgtgcagca catgtgacaa 2520

the

tgaaggacgg atgtatcacc ttgatggttg tggtgttggg gtcctgattt cagcattcat 2580

the

gagtcagggg aaggtccctg ctaaggacag accttaggag ggcagttggt ccaggaccca 2640

the

cacttgcttt cctcgtgttt cctgatcctg ccttgggtct gtagtcatac ttctggaaat 2700

the

tccttttggg tccaagacga ggaggttcct ctaagatctc atggccctgc ttcctcccag 2760

the

tcccctcaca ggacattttc ttcccacagg tggaaaagga gggagctact ctcaggctgc 2820

the

gtgtaagtgg tgggggtggg agtgtggagg agctcaccca ccccataatt cctcctgtcc 2880

the

cacgtctcct gcgggctctg accagtcct gtttttgttc tactccagcc agcgacagtg 2940

the

cccagggctc gatcaggacg aagtcccatg tcccggacgg ggctctcagg gtctcaggct 3000

the

ccgagggccg cgtctgcatt ggggaggcgc agcgttgggg attccccact cccacgagtt 3060

the

tcacttcttc tcccaaccta tgtcgggtcc ttcttccagg atactcgtga cgcgtcccca 3120

the

tttcccactc ccattgggtg tcgggtgtct agagaagcca atcagtgtcg ccggggtccc 3180

the

agttctaaag tccccacgca cccacccgga ctcagagtct cctcagacac cgagatgcgg 3240

the

gtcacggcac cccgaaccct cctcctgctg ctctgggggg ccctggccct gaccgagacc 3300

the

tgggccggtg agtgcgggtc gggagggaaa tggcctctgt ggggaggagc gagggggaccg 3360

the

caggcggggg cgcaggaccc ggggagccgc gccgggga gggtctggcg ggtctcagcc 3420

the

cctcctcgcc cccaggctcc cactccatga ggtatttcta caccgccatg tcccggcccg 3480

the

gccgcgggga gccccgcttc atcgcagtgg gctacgtgga cgacacgcag ttcgtgaggt 3540

the

tcgacagcga cgccgcgagt ccgagagagg agccgcgggc gccgtggata gagcaggagg 3600

the

ggccggagta ttgggaccgg aacacacaga tcttcaagac caacacacag acttaccgag 3660

the

agaacctgcg gatcgcgctc cgctactaca accagagcga ggccggtgag tgaccccggc 3720

the

ccggggcgca ggtcacgact ccccatcccc cacgtacggc ccgggtcgcc ccgagtctcc 3780

the

gggtccgaga tccgcctccc tgaggccgcg ggacccgccc agaccctcga ccggcgagag 3840

the

ccccaggcgc gtttacccgg tttcattttc agttgaggcc aaaatccccg cgggttggtc 3900

the

ggggcggggc ggggctcggg ggacggggct gaccgcgggg ccggggccag ggtctcacac 3960

the

ttggcagacg atgtatggct gcgacctggg gccggacggg cgcctcctcc gcgggcataa 4020

the

ccagttagcc tacgacggca aggattacat cgccctgaac gaggacctga gctcctggac 4080

the

cgcggcggac accgcggctc agatcaccca gcgcaagtgg gaggcggccc gtgtggcgga 4140

the

gcagctgaga gcctacctgg agggcacgtg cgtggagtgg ctccgcagat acctggagaa 4200

the

cgggaaggag acgctgcagc gcgcgggtac caggggcagt ggggagcctt ccccatctcc 4260

the

tataggtcgc cggggatggc ctcccacgag aagaggagga aaatgggatc agcgctagaa 4320

the

tgtcgccctc ccttgaatgg agaatggcat gagttttcct gagtttcctc tgagggcccc 4380

the

ctcttctctc taggacaatt aagggatgac gtctctgagg aaatggaggg gaagacagtc 4440

the

cctagaatac tgatcagggg tcctctttga cccctgcagc agccttggga accgtgactt 4500

the

ttcctctcag gccttgttct ctgcctcaca ctcagtgtgt ttggggctct gattccagca 4560

the

cttctgagtc actttacctc cactcagatc aggagcagaa gtccctgttc cccgctcaga 4620

the

gactcgaact ttccaatgaa taggagatta tcccaggtgc ctgcgtccag gctggtgtct 4680

the

gggttctgtg ccccttcccc accccaggtg tcctgtccat tctcaggctg gtcacatggg 4740

the

tggtcctagg gtgtcctatg agagatgcaa agcgcctgaa ttttctgact cttcccatca 4800

the

gaccccccaa aagacacacgt gacccaccac cccatctctg accatgaggc caccctgagg 4860

the

tgctgggccc tgggcttcta ccctgcggag atcacactga cctggcagcg ggatggcgag 4920

the

gaccaaactc aggacactga gcttgtggag accagaccag caggagatag aaccttccag 4980

the

aagtgggcag ctgtggtggt gccttctgga gaagagcaga gatacacatg ccatgtacag 5040

the

catgaggggc tgccgaagcc cctcaccctg agatggggta aggaggggga tgaggggtca 5100

the

tatctcttct cagggaaagc aggagccctt ctggagccct tcagcagggt cagggcccct 5160

the

cgtcttcccc tcctttccca gagccatctt cccagtccac catccccatc gtgggcattg 5220

the

ttgctggcct ggctgtccta gcagttgtgg tcatcggagc tgtggtcgct actgtgatgt 5280

the

gtagggaggaa gagctcaggt agggaagggg tgaggggtgg ggtctgggtt ttcttgtccc 5340

the

actgggggtt tcaagcccca ggtagaagtg ttccctgcct cattactggg aagcagcatc 5400

the

cacacagggg ctaatgcagc ctgggaccct gtgtgccagc acttactctt ttgtgcagca 5460

the

catgtgacaa tgaaggacgg atgtatcacc ttgatggttg tggtgttggg gtcctgattt 5520

the

cagcattcat gagtcagggg aaggtccctg ctaaggacag accttaggag ggcagttggt 5580

the

ccaggaccca cacttgcttt cctcgtgttt cctgatcctg ccttgggtct gtagtcatac 5640

the

ttctggaaat tccttttggg tccaagacga ggaggttcct ctaagatctc atggccctgc 5700

the

ttcctcccag tcccctcaca ggacattttc ttcccacagg tggaaaagga gggagctact 5760

the

ctcaggctgc gtgtaagtgg tgggggtggg agtgtgggagg agctcaccca ccccataatt 5820

the

cctcctgtcc cacgtctcct gcgggctctg accagtcct gtttttgttc tactccagcc 5880

the

agcgacagtg cccagggctc tgatgtgtct ctcacagctt gaaaaggtga gattcttggg 5940

the

gtctagagtg ggcggggggg gggtggggtg gggaggggggc agggggaaa ggcctggggta 6000

the

atggagattc tttgattggg atgttacgcg tgtgtggtgg gctgtttaga gtgtcatcac 6060

the

ttaccatgac taaccagaat ttgttcatga ctgttgtttt ctgtagcctg agacagctgt 6120

the

cttgtgaggg actgagatgc aggatttctt cactcctccc ctttgtgact tcaagagcct 6180

the

ctggcatctc tttctgcaaa ggcacctgaa tgtgtctgcg tccctgttag cataatgtga 6240

the

ggaggtggag agacagccca cccccgtgtc cactgtgacc cct 6283

the

Claims (10)

1. a detection kit of human leukocyte antigen gene, it is characterized in that, the reagent that contains the nucleic acid of detection human leukocyte antigen gene or albumen in described test kit; Described human leukocyte antigen gene is HLA-B*59: 01 gene. 2. The detection kit according to claim 1, characterized in that, said kit contains amplification primers for human leukocyte antigen gene; or labeled probes. 3. The detection kit according to claim 1, characterized in that, the kit contains specific antibodies to human leukocyte antigen genes. 4. The application of the detection kit according to claim 1 in typing the HLA-B*59:01 gene of the sample. 5. The application according to claim 4, characterized in that, the HLA-B*59:01 gene is typed by using the PCR-SSO method. 6. application as claimed in claim 4, is characterized in that, the step to the typing of HLA-B*59:01 gene is as follows: (1) extract the DNA in the sample; (2) Amplify the polymorphic region of HLA; (3) Isotopic or non-isotopic labeling of PCR products during the amplification process; (4) Hybridize the PCR product with the probe on the membrane, perform autoradiography, and judge the result according to the signal. 7. The application according to claim 6, wherein the probes are designed for PCR amplification products and fixed on the membrane. 8. The application according to claim 4, wherein the sample is RNA, protein, cell or serum sample. 9. The application according to claim 4, wherein the sample is isolated peripheral blood. 10. The application according to claim 4, characterized in that Stevens-Johnson syndrome and toxic epidermal necrolysis are judged by using the analysis results of HLA-B*59:01 gene.