CN102605082A

CN102605082A - Detection kit for systemic lupus erythematosus

Info

Publication number: CN102605082A
Application number: CN2012100867987A
Authority: CN
Inventors: 李为为; 姜丽
Original assignee: DECODING (SHANGHAI) PHARMACEUTICAL Co Ltd
Current assignee: DECODING (SHANGHAI) PHARMACEUTICAL Co Ltd
Priority date: 2012-03-28
Filing date: 2012-03-28
Publication date: 2012-07-25

Abstract

The invention provides a method, a detection probe and a detection kit for detecting gene mutation sites related to systemic lupus erythematosus (SLE). The kit comprises specific primers, the detection probe, PCR (polymerase chain reaction) components, PCR product purification components and DNA (deoxyribonucleic acid) sequencing reaction components and the like for detecting genotypes of three single nucleotide polymorphism (SNP) sites, including A49G and T-1772C on a cytotoxic T lymphocyte antigen-4 (CTLA4) gene, and C677T on methylene tetrahydrofolate reductase (MTHFR) gene. According to the invention, genotypes of three SNP sites closely related to systemic lupus erythematosus occurrence are detected by the kit, so as to estimate the risk level of the systemic lupus erythematosus from a gene level, and thus morbidity of the systemic lupus erythematosus is prevented in advance. The sampling method according to the invention is oral mucosa sampling; the sampling is painless and non-invasive; and cross infection can be avoided. The sequencing detection result is accurate and reliable; purchase of an imported special instrument at a high price is not needed; and the method is easy to popularize and promote.

Description

The systemic lupus erythematous detection kit

Technical field

The invention belongs to biology field; Relate to medical science and biotechnology; Be specifically related to a kind of gene detecting kit of systemic lupus erythematous; According to the risk class of detected result from the generation of gene aspect evaluating system property lupus erythematosus, and guidance system property lupus erythematosus high risk population prevention ahead of time and treatment.

Background technology

Systemic lupus erythematous (systemic lupus erythematosus; SLE) be a kind of comparatively common autoimmune disorder; It is characterized by the generation of multiple autoantibody and the formation of immunocomplex, final owing to the immunoreation to self causes the almost infringement of each system of whole body such as the pathology of the recycle system, urinary system, Digestive tract and cns.The cause of disease of SLE and pathogenesis are not clear fully as yet, think that at present this disease belongs to disease of multifactorial inheritance, this sick generation of inherited genetic factors and environmental factors fellowship, evolution.SLE sends out in the women well, and particularly the child-bearing period women involves a plurality of important organs of whole body such as the heart, brain, kidney.According to estimates, there are SLE patient more than 100 ten thousand people in China at present, and average morbidity occupies second in the whole world.Numerous discovering, this disease has higher genetic predisposition, with SLE closely-related gene locus takes place through the molecular diagnosis detection and just can assess the risk that the person under inspection suffers from SLE in advance, prevention ahead of time and treatment.

Cytotoxic t lymphocyte-associated antigen 4 (cytotoxic T lymphocyte associated antigen4; CTLA-4) be the expressed a kind of synexin in activating T cell surface; Activation to the T cell plays crucial negativity regulating effect, so the CTLA-4 gene becomes a candidate tumor susceptibility gene of autoimmune disorder.Data shows that CTLA-4 gene the 1st exon A49G polymorphum is except closely related with the generation of SLE, and is also relevant with Graves illness in eye, vitiligo, AITD etc.Xu An equality centering state southern area cohort study result in 2004 shows that CTLA4 gene-1 722T/C loci polymorphism is obviously relevant with SLE, and SLE patient TC genotype frequency is apparently higher than the normal control group.Meng Wei seminar discovered to Chinese southerner crowd that CTLA-4 gene-1 722T/C site was relevant with SLE in 2009, and case group and control group T/T, C/T, there were significant differences for the C/C genotype frequency, and SLE patient TT gene frequency obviously raises.Clinical study data presentation both domestic and external, CTLA-4 gene-1 722T/C loci polymorphism can also be as the independent hazard factors of multiple disease generation such as vitiligo, mammary cancer except closely related with the generation of SLE.

MTHFR (MTHFR) gene is one of key gene in the folic acid metabolism, keeps normal homocysteine level.Xu and other 2007 KUNG determination of 54 SLE patients and 62 normal controls of Hcy, VitB12 folate levels and detect polymorphism MTHFR gene 677.The result shows that disease group CC genotype frequency significantly is lower than the normal control group, and total mutation T allele frequency is significantly higher than control group.2009, people such as Feng Leiguang were to 52 routine SLE patients (male 5 examples, women 47 examples; 37～14.34 years old mean age) and 78 routine age-sexs the normal healthy controls person (male 8 examples, women 70 examples that are complementary; 34 ± 8.53 years old mean age) make case-control study.The result finds, isozygoty anomaly and T allelotrope of MTHFR 677TT significantly increases in SLE patient among the study population, and the mthfr gene 677TT variation of isozygotying is relevant with the morbidity of SLE, is the Hazard Factor of SLE.The independent hazard factor that 3 of C677T SNPs can be used as SLE on A49G and T-1772C, the mthfr gene on the CTLA4 gene carries out gene test.

Therefore, set up simply, systemic lupus erythematous tumor susceptibility gene detection method fast, the high risk population that examination in time goes out to be prone to suffer from systemic lupus erythematous carries out health guidance targetedly, has very important significance for guarantee human security health.

Summary of the invention

3 SNPs loci gene types based on C677T on A49G and T-1772C, the mthfr gene on the CTLA4 gene can be used to evaluating system property lupus erythematosus onset risk rank, and the present invention provides a kind of systemic lupus erythematous gene detecting kit.

This test kit comprises:

Detect on the CTLA4 gene on the A49G and T-1772C, mthfr gene the Auele Specific Primer of 3 SNPs loci gene types of C677T to reaching the dna sequencing primer;

PCR reaction component (comprising Taq enzyme, dNTP mixed solution, reaction buffer, ddH2O etc.);

PCR product purification assembly (comprising SAP enzyme, ExoI enzyme, ddH2O etc.);

Dna sequencing reaction assembly (comprising BigDye mix, EDTA solution, 100% ethanolic soln, 70% ethanolic soln, HIDI solution, ddH2O etc.).

The component and the content of test kit of the present invention comprise:

PCR reaction system: 10 * PCR reaction buffer 2.5ul; 25mM dNTP mixed solution 0.2ul; 5U/ul Taq archaeal dna polymerase 0.125ul; The 20uM Auele Specific Primer is to (every each 0.25ul of primer); DdH2O19.175ul.

PCR product purification system: 1U/ul SAP enzyme 0.75ul; 10U/ul ExoI enzyme 0.375ul; DdH2O3.875ul.

Sequencing reaction system: 25%BigDye mix 1ul; 3.2uM dna sequencing primer 1ul; 125mMEDTA solution 1ul; 100% ethanolic soln 15ul; 70% ethanolic soln 30ul; HIDI solution 8ul; DdH2O2ul.

This test kit supplies a person-portion to detect use, and the storage temperature of test kit is-20 ℃.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.

Only if definition separately, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.

The use of embodiment 1. detection kit

1, extracting dna profiling

Scrape and get person under inspection's oral mucosa epithelial cell, with phenol chloroform method extracting genomic dna.

2, pcr amplification reaction

Use the PCR reaction component in the detection kit, wherein, it is right to contain following primer:

(1) CTLA4 (A49G) forward primer: 5 '-CTGAACACCGCTCCCATAAA-3 '

CTLA4 (A49G) reverse primer: 5 '-GAATACAGAGCCAGCCAAGC-3 '

(2) CTLA4 (T-1772C) forward primer: 5 '-CACAGAAGCAAAGAACCCAT-3 '

CTLA4 (T-1772C) reverse primer: 5 '-TGTATGTGGGTAGGAGATGGA-3 '

(3) MTHFR (C677T) forward primer: 5 '-AGGGAGGCTTCAACTACGC-3 '

MTHFR (C677T) reverse primer: 5 '-GCGGTTGAGGGTGTAGAAGT-3 '

The reaction system of pcr amplification is: 10 * PCR reaction buffer, 2.5 μ l; The dNTP mixed solution 0.2 μ l of 25mM, 5U/ul Taq enzyme 0.125 μ l, dna profiling 1 μ l (about 12-15ng), each 0.25 μ l of 20uM forward primer reverse primer, ddH2O 19.175 μ l;

Reaction conditions is: 94 ℃ of sex change in 4 minutes and enzyme activate, 94 ℃ of sex change in 30 seconds, and 55 ℃ of annealing in 30 seconds, 72 ℃ prolonged in 1 minute, circulates 28 times, and last 72 ℃ of prolongations are more than 5 minutes.

3, pcr amplification product purifying

Use the PCR product purification assembly in the detection kit, reaction system is TV 25ul, comprises PCR product 20ul, 1U/ul SAP enzyme 0.75ul, 10U/ul ExoI enzyme 0.375ul, deionized water 3.875ul.

On ABI2720 type pcr amplification appearance, react, reaction conditions is 37 ℃, 15min, 72 ℃, 20min.

4, dna sequencing reaction

Use the sequencing reaction assembly in the detection kit, wherein, contain following dna sequencing primer:

(1) CTLA4 (A49G) sequencing primer: 5 '-CTGAACACCGCTCCCATAAA-3 '

(2) CTLA4 (T-1772C) sequencing primer: 5 '-CACAGAAGCAAAGAACCCAT-3 '

(3) MTHFR (C677T) sequencing primer: 5 '-AGGGAGGCTTCAACTACGC-3 '

The system of reaction is TV 5ul, comprises PCR purified product 1ul, 25%Bigdye mix1ul, 3.2uMDNA sequencing primer 1ul, deionized water 2ul.

On ABI2720 type pcr amplification appearance, react, reaction conditions is 98 ℃ of 2min, carries out 96 ℃ of 30s of 25 round-robin, 55 ℃ of 30s, 60 ℃ of 4min.

Reaction finishes the back and adds 125mM EDTA solution 1ul and 100% ethanolic soln 15ul, in room temperature settle 15min; At 4 ℃, the centrifugal 30min of the rotating speed of 3600rpm/min removes supernatant gently; Add 70% ethanolic soln 30ul, the centrifugal 15min of 3600rpm/min removes supernatant gently; Room temperature adds HIDI solution 8ul after placing 20min, puts into sequenator.

5, gene type assay

The those skilled in the art that are familiar with the dna sequencing technology can be through the genotype in the definite SNP site of being detected of identification dna sequencing collection of illustrative plates.

Embodiment 2. carries out the service of systemic lupus erythematous gene test for the crowd

1. sample and extracting DNA

Instructing the person under inspection to use the oral cavity sampling to wipe away by the hospital laboratory doctor carries out the mouth epithelial cells sampling, adopts phenol chloroform method to carry out the DNA extracting of mouth epithelial cells.

2. genotype detection

Use test kit provided by the invention, dna sequencing is carried out in 3 SNPs sites of C677T on A49G and T-1772C, the mthfr gene on the CTLA4 gene of person under inspection's genomic dna respectively, confirm the genotype in these 3 SNPs sites.

3. systemic lupus erythematous gene high risk population risk assessment is analyzed

Through to the genotypic analysis of person under inspection SNPs, provide systemic lupus erythematous risk assessment analysis report list.The SNP locus gene that has specified C677T on A49G and T-1772C on the person under inspection CTLA4 gene, the mthfr gene in the report detects information and genetic risk assessment result.In addition, according to person under inspection's risk class, and specify to the person under inspection by the doctor and to conciliate read apparatus property lupus erythematosus gene test report.

Claims

1. a screening systematic lupus erythematosus high risk population gene does not have the wound detection kit, it is characterized in that: 3 genotypic Auele Specific Primers of mononucleotide polymorphism site and the DNA detection probe, Taq enzyme, dNTP mixed solution, reaction buffer, SAP enzyme, ExoI enzyme, BigDye mix, EDTA solution, 70% ethanolic soln, 100% ethanolic soln, HIDI solution, the ddH that detect C677T on A49G and T-1772C on the CTLA4 gene, the mthfr gene ₂O etc.

2. test kit according to claim 1; It is characterized in that: described Auele Specific Primer is to being meant 3 SNPs (SNPs) site to C677T on A49G on the CTLA4 gene and T-1772C, the mthfr gene, and it is right that the ability specific amplification goes out to comprise the primer of dna fragmentation in these 3 SNPs sites.

3. test kit according to claim 1; It is characterized in that: described dna sequencing primer is to design to 3 SNPs sites of C677T on A49G on the CTLA4 gene and T-1772C, the mthfr gene, can go out the dna sequencing primer of above-mentioned 3 SNPs loci gene types through the technological specific detection of dna sequencing.

4. according to the test kit shown in the claim 1, it is characterized in that 3 pairs of contained specific primer sequences are following:

(1) CTLA4 (A49G) forward primer: 5 '-CTGAACACCGCTCCCATAAA-3 '

CTLA4 (A49G) reverse primer: 5 '-GAATACAGAGCCAGCCAAGC-3 '

(2) CTLA4 (T-1772C) forward primer: 5 '-CACAGAAGCAAAGAACCCAT-3 '

CTLA4 (T-1772C) reverse primer: 5 '-TGTATGTGGGTAGGAGATGGA-3 '

(3) MTHFR (C677T) forward primer: 5 '-AGGGAGGCTTCAACTACGC-3 '

MTHFR (C677T) reverse primer: 5 '-GCGGTTGAGGGTGTAGAAGT-3 '.

5. test kit according to claim 1 is characterized in that, 3 contained dna sequencing primer sequences are following:

(1) CTLA4 (A49G) sequencing primer: 5 '-CTGAACACCGCTCCCATAAA-3 '

(2) CTLA4 (T-1772C) sequencing primer: 5 '-CACAGAAGCAAAGAACCCAT-3 '

(3) MTHFR (C677T) sequencing primer: 5 '-AGGGAGGCTTCAACTACGC-3 '.

6. test kit according to claim 1 is characterized in that the component of test kit and content comprise:

(1) PCR reaction system: 10 * PCR reaction buffer 2.5ul, 25mM dNTP mixed solution 0.2ul, 5U/ul Taq archaeal dna polymerase 0.125ul, the 20uM Auele Specific Primer is right, every each 0.25ul of primer, ddH ₂O 19.175ul.

(2) PCR product purification system: 1U/ul SAP enzyme 0.75ul, 10U/ul ExoI enzyme 0.375ul, ddH ₂O 3.875ul.

(3) sequencing reaction system: 25% BigDye mix1ul, 3.2uM dna sequencing primer 1ul, 125mMEDTA solution 1ul, 100% ethanolic soln 15ul, 70% ethanolic soln 30ul, HIDI solution 8ul, ddH ₂O2ul.

This test kit supplies a person-portion to detect application, and the storage temperature of test kit is-20 ℃.