CN103205488A - Method and reagent for prediction of ankylosing spondylitis susceptibility - Google Patents
Method and reagent for prediction of ankylosing spondylitis susceptibility Download PDFInfo
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Abstract
The invention discloses a method and a reagent for prediction of ankylosing spondylitis susceptibility. According to the invention, genomic DNA of a host cell is extracted and the genotype of site + 879 in a second intron zone of the AIF1 gene of a subject is determined so as to predicate susceptibility of the subject to ankylosing spondylitis; if the genotype of the single nucleotide polymorphism site + 879 is CC, lowest ankylosing spondylitis susceptibility is obtained, and if the site + 879 contains a T allele, i.e., the genotype is CT or TT, highest susceptibility is obtained. The invention has the following advantages: correlation between the AIF1 gene polymorphism site and the ankylosing spondylitis is elaborated for the first time; the method for predicting ankylosing spondylitis susceptibility is provided; and the method is applicable to prevention, auxiliary diagnosis and treatment of ankylosing spondylitis and can also be used for research and development of a novel drug.
Description
Technical field
The present invention relates to a kind of method and reagent of predicting susceptibility of ankylosing spondylitis, say so more specifically and predict that by measuring with the polymorphism of ankylosing spondylitis genes involved AIF1 the experimenter is for the susceptibility of ankylosing spondylitis, this method can be used for auxiliary diagnosis and the new drug development of disease, belongs to biological technical field.
Background technology
(Ankylosing spondylitis is a kind of autoimmune disorder AS) to ankylosing spondylitis, is mainly in the 16-40 person between twenty and fifty in year, and the ill ratio of men and women is about 4～10: 1.Pathology Chang Shouxian betides articulatio sacroiliaca, and minority patient with severe symptoms shows as whole rigid spine.In addition, the part patient is with outer pathologies of backbone such as in various degree hip joint, eye, lung, cardiovascular, kidneys.The sickness rate of AS in the white race crowd is about 1%～3%, China's AS morbidity is about 0.2-0.6%, wherein the patient more than 60% gets involved with hip joint, cause 20% above AS patient's deformity, inflammation is mainly involved the bone attachment point of joint capsule, tendon and ligament, cause local joint accretion tetanic, limitation of activity.So far still lack the medicine that obviously to alleviate and to control disease progression clinically.AS belongs to multigenic disease, has the obvious genetic tendency, plays a leading role in the AS morbidity though it has been generally acknowledged that heredity and immune factor, and definite cause and onset of disease mechanism is still unclear.
Carrying out the research of AS inherited pathogenic factor at present, adopt SNP as the association analysis method of genomic marker more, is effectively, has obtained proof.SNP refers to the dna sequence polymorphism that single nucleotide diversity causes on the chromogene group level, frequency in the crowd needs＞1%, SNPs is biallelic marker, have 70.1% to be the conversion between the homotype base during this single base changes: as G/A or T/C, 29.1% for occurring in the transversion between purine and the pyrimidine.C (cytosine(Cyt)) is the most labile site in the human genome, because great majority are the cytosine(Cyt)s that methylate, can be converted to T (thymus pyrimidine) by spontaneous deaminizating, and SNP has comprised the 80-90% of known polymorphism, is modal heritable variation.
Because the selective pressure of existence causes the distribution of SNP in term single gene and whole genome to be ununiformity.SNPs is 4 times of coding region in the quantity of gene non-coding region, and sum can reach 300 ten thousand.SNP with its density height (average every 1kb just has 1), representative strong (SNP that is arranged in gene inside may directly influence protein structure or expression level), genetic stability good (with microsatellite polymorphism comparatively speaking), be easy to characteristics such as automated analysis (because SNP mostly is biallelic marker the crowd, can be simply with "+/-or 1/0 " direct somatotype) and become good genetic marker.
1973, Brewerton etc. at first found the human leucocyte antigen-B27 (HLA-B27) with the AS strong correlation.Along with progress of research, other tumor susceptibility genes relevant with AS such as tumor necrosis factor-alpha (TNF-α), il-1 (IL-1) are identified successively.
AIF1 (allotransplantation inflammatory factor-1; Allograft Inflammatory Factor 1) gene is positioned at 6q21.3; total length 1692bp; contain 4 exons; its coded product is a kind of hormonelike cytokine; molecular weight is 17kDa; contain 146 amino-acid residues; the N end is acetylation; mainly express, secrete by the scavenger cell that activates and T lymphocyte; the assignment of genes gene mapping is in human leucocyte antigen III zone, and is closely related with generation development of organ transplant rejection, self property Immunological diseases and tumour etc.
Still do not have at present any report about being associated between AIF1 gene and the autoimmune disorder, also do not see the result of study that the AIF1 gene is associated with AS.
Summary of the invention
Main purpose of the present invention provides a kind of method that detects the susceptibility of ankylosing spondylitis gene.
Second purpose of the present invention provides a kind of reagent that detects the susceptibility of ankylosing spondylitis gene, comprises PCR primer and the test kit that contains this primer.
For achieving the above object, the present invention is by the following technical solutions:
A kind of nucleotide sequence that detects susceptibility of ankylosing spondylitis is nucleotide sequence shown in the sequence table SEQ ID No.1.
Described nucleotides sequence classifies AIF1 gene the 2nd as and includes the subarea and comprise+nucleotide fragments of 879 mononucleotide polymorphism sites.+ 879 is variant sites, indicates with letter " Y ".This nucleotide sequence is AIF1 full length gene sequence.Fig. 1 is the synoptic diagram in AIF1 gene structure and polymorphic variation site thereof, includes 4 exons in the accompanying drawing, and+879 sites are marked in the AIF1 gene map the 2nd corresponding position that includes the subarea.
When the genotype of described+879 mononucleotide polymorphism site was CC, susceptibility of ankylosing spondylitis was minimum; When containing T allelotrope, when namely genotype was CT or TT, susceptibility of ankylosing spondylitis was higher.
One group of primer that detects susceptibility of ankylosing spondylitis, can increase obtains AIF1 gene the 2nd and includes the subarea and comprise+nucleotide fragments of 879 mononucleotide polymorphism sites.
The nucleotide sequence of described primer is respectively shown in sequence table SEQ ID No.2 and the sequence table SEQ ID No.3.
A kind of detection method of susceptibility of ankylosing spondylitis gene comprises the steps:
(1) genomic dna of extracting sample, amplification AIF1 gene the 2nd include the subarea and comprise+nucleotide fragments of 879 mononucleotide polymorphism sites;
(2) detect in step (1) product+when the genotype of 879 mononucleotide polymorphism sites was CC, susceptibility of ankylosing spondylitis was minimum; Contain T allelotrope, when namely genotype was CT or TT, susceptibility of ankylosing spondylitis was higher.
Described amplification AIF1 gene the 2nd includes the subarea and comprises+nucleotide fragments of 879 mononucleotide polymorphism sites, and the nucleotide sequence of one group of primer of use is respectively shown in sequence table SEQ ID No.2 and the SEQ ID No.3.
The invention provides a kind of diagnostic kit that detects susceptibility of ankylosing spondylitis, the primer that wherein contains specific amplification AIF1 gene of the present invention+879 sites to the conventional assembly that is used for the test kit that pcr amplification detects, reagent, damping fluid etc., those skilled in the art know these conventional assembly and detection methods.Whole components, content, source and using method in the test kit of the present invention are as follows:
10 μ L, 10 * PCR damping fluid; (Pharmacia)
2 μ L 10mM dNTP mixed solutions; (Pharmacia)
2 μ L Taq archaeal dna polymerases, 2unit/ μ L; (Takara)
1 μ L F1 primer is the nucleotide sequence shown in the SEQ ID No.2, and concentration is 10pmol/ μ L;
1 μ L R1 primer is the nucleotide sequence shown in the SEQ ID No.3, and concentration is 10pmol/ μ L;
8 μ L, 10 * LC-green PLUS saturated fluorescence dyestuff; (American I daho company)
Each 0.5 μ L of 2 μ L oligonucleotide confidential reference items primers, concentration is 10pmol/ μ L, wherein low temperature confidential reference items primers F is the nucleotide sequence shown in the SEQ ID NO.4, low temperature confidential reference items primer R is the nucleotide sequence shown in the SEQ ID NO.5, high temperature confidential reference items primers F is the sequence shown in the SEQ ID NO.6, and high temperature confidential reference items primer R is the nucleotide sequence shown in the SEQ ID NO.7;
64 μ L pure water.
Using method:
1) pcr amplification: include subarea part fragment by the 2nd of pcr amplification AIF1 gene, preparation mixed solution: 10 * PCR reaction buffer, 1 μ L, 10mM/LdNTP0.2 μ L, TaqDNA polysaccharase 0.2 μ L, 10pM/L upstream primer 0.1 μ L, 10pM/L downstream primer 0.1 μ L, 1 * LC-Green Plus saturated fluorescence dyestuff, 0.8 μ L, oligonucleotide confidential reference items 0.2 μ L (each 0.05 μ L of high and low temperature oligonucleotide confidential reference items upstream and downstream primer) (sequence sees Table 1), genomic dna 1 μ L adds pure water to 10 μ L.The PCR reaction conditions is 95 ℃ of pre-sex change 5min, 95 ℃ of sex change 30s, and 65 ℃ of annealing 30s, 72 ℃ are extended 5s, 45 circulations altogether, 72 ℃ of total elongation 2min.Before carrying out the analysis of high resolution melting curve, carry out sex change and renaturation and handle: 95 ℃ of 30s, 25 ℃ of 2min, 94 ℃ of 30s, 24 ℃ of 4min.The paraffin oil that adds 20 μ L before the PCR in each system evaporates preventing.
2) genotype is judged: the PCR product is moved in special-purpose 96 orifice plates of HRM, carrying out HRM at Light Scanner TMHR-I96 analyzes, with Light Scanner Call IT software the curve after gathering is analyzed, judged genotype according to the difference of melting curve.
The nucleotide fragments of nucleotide sequence shown in the sequence table SEQ ID No.1 and comprising+879 mononucleotide polymorphism sites is in preparation diagnosis or the reagent for the treatment of ankylosing spondylitis or the purposes in the medicine.
AIF1 gene the 2nd includes subarea+879 mononucleotide polymorphism site in preparation diagnosis or the reagent for the treatment of ankylosing spondylitis or the purposes in the medicine.
Measuring method of the present invention has been measured the genomic dna that derives from the people, and sample source is unrestricted, as: body fluid (blood, ascites and urine etc.), histocyte (as hepatic tissue) etc.Can prepare genomic dna by extraction and these samples of purifying.Adjust the concentration of genomic dna, make it consistent as much as possible.Be template with the genomic dna, can amplify the nucleic acid fragment that contains the AIF1 gene mutation site, to obtain the great amount of samples of mensuration.Thisly contain the sample that the dna fragmentation of AIF1 genovariation point obtains by amplification, be particularly suitable for as measuring material.
When carrying out the gene auxiliary diagnosis, the present invention is preferably applied in mensuration according to the auxiliary diagnostic of the mutation type existence of AIF1 gene, auxiliary diagnostic comprises the particular agent as neccessary composition, and it is corresponding to the method that is used for measuring the AIF1 gene mutation type.Select suitable particular agent by the measuring method that adopts, as dna fragmentation and/or be used for the primer of pcr amplification step.
Advantage of the present invention is: the present invention has illustrated the dependency of AIF1 gene polymorphism sites and AS first, and a kind of method of the AS of prediction susceptibility is provided, and this method can be used for prevention, auxiliary diagnosis and the treatment of AS, can also be used for new drug development.
Below in conjunction with the drawings and specific embodiments the present invention is done further narration; so that the public has more deep understanding to summary of the invention; be not limitation of the present invention, all any this areas of doing according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.
Description of drawings
Fig. 1 is the synoptic diagram in AIF1 gene structure and polymorphic variation site thereof.
Fig. 2 be AIF1 genovariation site through the gene type figure of HRM method, transverse axis is temperature, the longitudinal axis is the fluorescent value of normalization.Unimodal curve is represented CC genotype individuality, and bimodal curve is represented CT genotype individuality.
Fig. 3 is the backward sequencing figure of AIF1 gene+879, and left figure genotype is the CC type, and right figure be CT genotype (the employing backward sequencing is so be shown as G and the A base of matching with C, T at peak figure).
Embodiment
The english abbreviation that is used for the following example expression reagent is as follows:
10 * PCR damping fluid: 10mM Tris-HCl (pH=8.3), 500mM Repone K (KCl), 10mM magnesium chloride (MgCl2), 0.01% (W/V) gelatinum
DNTP: deoxynucleoside triphosphate
EDTA: disodium ethylene diamine tetraacetate
TE：10mM?Tris-HCl(pH＝7.5)，1mM?EDTA(pH＝8.0)
Embodiment 1: the extraction of blood sample collection and genomic dna
By New York revision Case definition MethodsThe cases enrolled in 1984, choose AS patient's 118 examples from the Jilin Area consanguinity-less relation (age: 14-44 year, average 24 years old) altogether, with normal healthy controls volunteer's 148 examples in area (age: 39-72 year, average 46 years old).All persons under inspection are Han nationality and sign written Informed Consent Form, this research obtains Beijing Hospital, the approval of ethics audit committee of Gerontological Research Center institute of the Ministry of Health meets " World Medical Association's Declaration of Helsinki ": the ethic principle of human medical research.
According to following method, the preparation human gene group DNA.1. at first add 1000 μ L erythrocyte cracked liquids in the 1.5mLEP pipe of label, the back adds 400 μ LEDTA anticoagulations (anticoagulation is put upside down mixing 3-5 time before adding), puts upside down mixing, and room temperature left standstill 10 minutes; 2. 13000rpm removes supernatant liquor after centrifugal 30 seconds; 3. add 480 μ L nucleic acid lysates in gained precipitation, the attack tube wall fully adds 20 μ L Proteinase Ks (with splitting 20 times of diluted protein enzyme K of karyolymph dilution) behind the mixing, put upside down mixing, 65 ℃ of incubators 10 minutes, (during frequently about mixing, guaranteeing does not have grumeleuse); 4. be down to room temperature after taking out, add 300 μ L albumen precipitation liquid, fully put upside down mixing, left standstill centrifugal 2 minutes of 13000rpm 10 minutes; 5. supernatant liquor is moved in the new EP pipe, add the Virahol of 670 μ L precoolings, fully put upside down mixing (more than 10 times), visible linear DNA forms little agglomerate, centrifugal 2 minutes of 13000rpm gradually; 6. abandon supernatant liquor and guarantee the precipitation stay in the EP pipe, add 670 μ L70% ethanol, the mixing that turns upside down, centrifugal 2 minutes of 13000rpm; 7. abandon supernatant, make the interior ethanol volatilization of pipe clean; 8. add TE solution (400 μ L), fully dissolving is carried out the analysis of concentration and purity to the genomic dna that extracts, and draws the part dna solution as working fluid, and concentration correction is to 20ng/ μ L, place 4 ℃ standby, the residue genomic dna is put-20 ℃ of refrigerators and is preserved.
The identification of embodiment 2 SNP is identified
The present invention adopt PCR-high resolving power melting curve (HRM) analytical method and PCR sequencing technologies simultaneously to the 2nd of AIF1 gene include the subarea+genotype of 879 sites (its loci is C/T) detects.Fig. 2 is that AIF1 genovariation site is the sequencer map in AIF1 genovariation site through gene type figure, Fig. 3 of HRM method.
1) the PCR-HRM primer determines
Look into from Genebank and get+near 879 DNA base sequence (SEQ ID No.1), design of primers is finished under Oligo7.0 software.The purpose fragment is positioned at AIF1 gene the 2nd and includes the subarea, total length 58bp, determined positive-sense strand F1 (+848bp-+867bp) with antisense strand R1 (+887bp-+905bp), specific primer sequence is as follows:
F1：5’-GGTGTGCAGGACTAAGAAGA-3’(SEQ?ID?No.2)
R1：5’-CAAATCGTGAGGAATGGAG-3’(SEQ?ID?No.3)
2) PCR-HRM reaction system and condition
Include subarea part fragment by pcr amplification AIF1 gene the 2nd, the PCR reaction system is: 10 * PCR reaction buffer, 1 μ L, 10mM/LdNTP0.2 μ L, TaqDNA polysaccharase 0.2 μ L, 10pM/L upstream primer 0.1 μ L, 10pM/L downstream primer 0.1 μ L, 10 * LC-Green Plus saturated fluorescence dyestuff, 0.8 μ L, oligonucleotide confidential reference items 0.2 μ L (each 0.05 μ L of high and low temperature oligonucleotide confidential reference items upstream and downstream primer) (sequence sees Table 1), genomic dna 1 μ L adds deionized water to 10 μ L.In each system, add 20 μ L paraffin oils during PCR, prevent from evaporating.The PCR reaction conditions is 95 ℃ of pre-sex change 5min, 95 ℃ of sex change 30s, and 65 ℃ of annealing 30s, 72 ℃ are extended 5s, 45 circulations altogether, 72 ℃ of total elongation 2min.Before carrying out the analysis of high resolution melting curve, carry out sex change and renaturation and handle: 95 ℃ of 30s, 25 ℃ of 2min, 94 ℃ of 30s, 24 ℃ of 4min.
Table 1 high and low temperature oligonucleotide confidential reference items primer sequence, annealing temperature and product sheet segment length
3) HRM judges genotype
The PCR product is moved in special-purpose 96 orifice plates of HRM, carrying out HRM at Light Scanner TMHR-I 96 analyzes: since 40 ℃, slope with 0.3 ℃/s is gathered melting curve, to 98 ℃ of end, with Light Scanner CallIT software the curve (Fig. 2) after gathering is analyzed, judged genotype.
4) sequence verification
From the individuality of the different genotype of gained, randomly draw 3 routine samples respectively and carry out sequence verification.The order-checking sample carries out pcr amplification again, and the sequencing primer sequence is: F2:5 '-GGCAACCCCTTCCTCAGTC-3 ' (SEQID No.8), R2:5 '-GTTTCTCCAGCATTCGTTTC-3 ' (SEQ ID No.9), the long 349bp of amplified fragments.The PCR reaction totally is 30 μ L, comprises: genomic dna 2 μ L, and 10 * PCRBuffer3 μ L, 10mMdNTP0.5 μ L, TaqDNA polysaccharase (5U/ μ L) 0.5 μ L, each 0.5 μ L of upstream and downstream primer (10pM/ μ L), pure water is supplemented to cumulative volume 30 μ L.The PCR reaction conditions is: 95 ℃ of pre-sex change 5min are laggard to go into major cycle, 95 ℃ of sex change 30s, and 64 ℃ of annealing 30s, 72 ℃ are extended 20s, 35 circulations, 72 ℃ are extended 2min.The PCR product detects through 8% polyacrylamide gel electrophoresis, send the big gene sequencing portion of China to carry out sequence verification (Fig. 3) after the gel imaging system observation is qualified.
The dependency of embodiment 3 embodiment, 3 gene SNPs and AS
Statistical method: colony's representativeness of using Hardy-Weinberg balance check research sample.Utilize the Pearson chi square test calculating AIF1 gene+allelotrope of 879 pleomorphism sites, the distribution frequency of genotype between AS case group and normal control group in the SPSS11.0 software, ill risk OR value and the 95%CI credibility interval thereof of AS are significance of difference standard with P＜0.05.
The result: the genotype and the distribution of gene frequency between case and control group that are positioned at SNP+879 polymorphic site on the AIF1 gene in 6p21.3 zone see table 2 for details.
Genotype and the distribution of gene frequency between the case-control group of table 2AIF1 gene+879C/T polymorphic site
Annotate: OR: odds ratio; CI: credibility interval.* T allelotrope is the risk allelotrope of easily suffering from AS.The experimenter is divided into risk allelotrope (CT) carrier and non-risk allelotrope (CC) carrier of AS.
By table 2 as seen, the T allelotrope of AIF1 gene+879, be A allelotrope at its DNA complementary strand namely, distribution frequency in patient colony is significantly higher than its The genotypes distribution and allele frequencies (7.2%vs.2.0%) in healthy normal population, has significant difference (P=0.004), and the OR value in T site is 3.752,95%CI:1.455-9.673; In risk allelotrope (CT) carrier and non-risk allelotrope (CC) carrier of AS, the distribution frequency of risk genes type in the case group is significantly higher than (P＜0.05) in the control group, all show AIF1 gene+879 sites and AS is ill is proportionate, the risk that increases the AS morbidity may be arranged.
Embodiment 4 detection kit
Preparation detects the test kit of AS relevant risk, and it is right to include the primer that can amplify AIF1 gene SNP+879 sites, and other PCR-HRM corresponding reagent.Test kit of the present invention detects for 10 person-portions and uses, and keeps in Dark Place in-20 ℃, and its component, content and source comprise:
10 μ L, 10 * PCR damping fluids (Pharmacia),
2 μ L 10mM dNTP mixed solutions (Pharmacia),
2 μ L Taq archaeal dna polymerases (2unit/ μ L) (Takara),
1μL?F1(SEQ?ID?NO.2)(10pmol/μL)，
1 μ L R1 (SEQ ID NO.3) (10pmol/ μ L) primer, (it is synthetic that the worker is given birth in Shanghai)
8 μ L, 10 * LC-green PLUS saturated fluorescence dyestuff (American I daho company),
2 μ L oligonucleotide confidential reference items (10pmol/ μ L) (each 0.5 μ L of high and low temperature oligonucleotide confidential reference items upstream and downstream primer), (sequence sees Table 1),
64 μ L pure water.
After PCR-HRM detects, can detect the polymorphism that AIF1 gene the 2nd includes subarea SNP+879 easily.
Proof test: adopt this test kit, picked at random AS patient sample 10 examples, control group sample 10 examples detect the single nucleotide polymorphism that AIF1 gene the 2nd includes subarea+879 sites through PCR-HRM.
One. method
1) pcr amplification: include subarea part fragment by the 2nd of pcr amplification AIF1 gene, preparation mixed solution: 10 * PCR reaction buffer, 1 μ L, 10mM/LdNTP0.2 μ L, TaqDNA polysaccharase 0.2 μ L, 10pM/L upstream primer 0.1 μ L, 10pM/L downstream primer 0.1 μ L, 1 * LC-Green Plus saturated fluorescence dyestuff, 0.8 μ L, oligonucleotide confidential reference items 0.2 μ L (each 0.05 μ L of high and low temperature oligonucleotide confidential reference items upstream and downstream primer) (sequence sees Table 1), genomic dna 1 μ L adds pure water to 10 μ L.The PCR reaction conditions is 95 ℃ of pre-sex change 5min, 95 ℃ of sex change 30s, and 65 ℃ of annealing 30s, 72 ℃ are extended 5s, 45 circulations altogether, 72 ℃ of total elongation 2min.Before carrying out the analysis of high resolution melting curve, carry out sex change and renaturation and handle: 95 ℃ of 30s, 25 ℃ of 2min, 94 ℃ of 30s, 24 ℃ of 4min.The paraffin oil that adds 20 μ L before the PCR in each system evaporates preventing.
2) genotype is judged: the PCR product is moved in special-purpose 96 orifice plates of HRM, carrying out HRM at Light Scanner TMHR-I96 analyzes, with Light Scanner Call IT software the curve after gathering is analyzed, judged genotype according to the difference of melting curve.
Two. the result:
The result shows that the CT genotype frequency that AS patient's sample AIF1 gene the 2nd includes subarea+879 sites is significantly higher than the CT genotype in control group sample+879 sites.
The present invention has the illustration of practicality:
The detection method of AIF1 gene pleiomorphism of the present invention can be used for the T allelotrope of the rare variant sites on the AIF1 gene in analyst's euchromosome 6p21.3 district, be A allelotrope at its DNA complementary strand namely, be applied to the individual ill risk of AS of the complementary diagnosis of AS and assessment how to be beneficial to carry out early intervention and the treatment of AS.
Utilize the present invention to set forth the nucleotide variation in AIF1 gene+879 sites, as one of biomarker, the screening of the molecular target of useful as drug design is regulated the bioactive molecule that AIF1 expresses to help to seek to have, and promotes the AS new drug development.
Nucleotide sequence and the AS related locus of the detection AIF1 gene pleiomorphism that the present invention sets up, but highly sensitive, the specific test kit that is applied to AS gene auxiliary diagnosis.
As above tell, reach a conclusion, the polymorphism in AIF1 gene+879 sites and AS tool significant correlation.Therefore, measure this polymorphism according to the present invention, can be used for the gene auxiliary diagnosis of AS.
The present invention has narrated the relevant new mutant site of AIF1 Gene A S, and a kind of method of the AIF1 of mensuration gene pleiomorphism is provided, and, according to the present invention, only need a small amount of DNA sample just to be enough to measure the polymorphism of AIF1 gene.
The invention provides a kind of gene aided diagnosis method of the AS of mensuration related gene polymorphism.
Claims (10)
1. a nucleotide sequence that detects susceptibility of ankylosing spondylitis is characterized in that: be nucleotide sequence shown in the sequence table SEQ IDNo.1.
2. the nucleotide sequence of detection susceptibility of ankylosing spondylitis according to claim 1 is characterized in that: described nucleotides sequence classifies AIF1 gene the 2nd as and includes the subarea and comprise+and the nucleotide fragments of 879 mononucleotide polymorphism sites.
3. the nucleotide sequence of detection susceptibility of ankylosing spondylitis according to claim 2, it is characterized in that: when the genotype of described+879 mononucleotide polymorphism site was CC, susceptibility of ankylosing spondylitis was minimum; When containing T allelotrope, when namely genotype was CT or TT, the susceptibility of ankylosing spondylitis significance raise.
4. one group of primer that detects susceptibility of ankylosing spondylitis is characterized in that: can increase obtains AIF1 gene the 2nd and includes the subarea and comprise+nucleotide fragments of 879 mononucleotide polymorphism sites.
5. one group of primer that detects susceptibility of ankylosing spondylitis according to claim 4, it is characterized in that: the nucleotide sequence of described primer is respectively shown in sequence table SEQ ID No.2 and the sequence table SEQ ID No.3.
6. the detection method of a susceptibility of ankylosing spondylitis gene is characterized in that: comprise the steps:
(1) genomic dna of extracting sample, amplification AIF1 gene the 2nd include the subarea and comprise+nucleotide fragments of 879 mononucleotide polymorphism sites;
(2) detect in step (1) product+when the genotype of 879 mononucleotide polymorphism sites was CC, susceptibility of ankylosing spondylitis was minimum; Contain T allelotrope, when namely genotype was CT or TT, susceptibility of ankylosing spondylitis was higher.
7. the detection method of susceptibility of ankylosing spondylitis gene according to claim 6, it is characterized in that: described amplification AIF1 gene the 2nd includes the subarea and comprises+nucleotide fragments of 879 mononucleotide polymorphism sites, the nucleotide sequence of one group of primer of use is respectively shown in sequence table SEQ ID No.2 and the SEQ ID No.3.
8. test kit that detects the ankylosing spondylitis tumor susceptibility gene is characterized in that being made up of following reagent:
10 μ L, 10 * PCR damping fluid;
2 μ L 10mM dNTP mixed solutions;
2 μ L Taq archaeal dna polymerases, 2unit/ μ L;
1 μ L F1 primer is the nucleotide sequence shown in the SEQ ID No.2, and concentration is 10pmol/ μ L;
1 μ L R1 primer is the nucleotide sequence shown in the SEQ ID No.3, and concentration is 10pmol/ μ L;
8 μ L, 10 * LC-green PLUS saturated fluorescence dyestuff;
Each 0.5 μ L of 2 μ L oligonucleotide confidential reference items primers, concentration is 10pmol/ μ L, wherein low temperature confidential reference items primers F is the nucleotide sequence shown in the SEQ ID NO.4, low temperature confidential reference items primer R is the nucleotide sequence shown in the SEQ ID NO.5, high temperature confidential reference items primers F is the sequence shown in the SEQ ID NO.6, and high temperature confidential reference items primer R is the nucleotide sequence shown in the SEQ ID NO.7;
64 μ L pure water.
9. the nucleotide fragments of nucleotide sequence shown in the sequence table SEQ ID No.1 and comprising+879 mononucleotide polymorphism sites is in preparation diagnosis or the reagent for the treatment of ankylosing spondylitis or the purposes in the medicine.
10.AIF1 gene the 2nd includes subarea+879 mononucleotide polymorphism site in preparation diagnosis or the reagent for the treatment of ankylosing spondylitis or the purposes in the medicine.
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| CN103710448A (en) * | 2013-12-20 | 2014-04-09 | 卫生部北京医院 | Method and kit for predicting susceptibility of ankylosing spondylitis |
| CN103710446A (en) * | 2013-12-20 | 2014-04-09 | 卫生部北京医院 | Reagent and method for predicting ankylosing spondylitis susceptibility |
| CN103710447A (en) * | 2013-12-20 | 2014-04-09 | 卫生部北京医院 | Method and reagent for predicting susceptibility of ankylosing spondylitis |
| CN103882110A (en) * | 2013-12-20 | 2014-06-25 | 卫生部北京医院 | Reagent of detecting susceptibility of ankylosing spondylitis |
| CN103882112A (en) * | 2013-12-20 | 2014-06-25 | 卫生部北京医院 | Reagent and method for predicting ankylosing spondylitis susceptibility |
| CN104293959A (en) * | 2014-10-16 | 2015-01-21 | 卫生部北京医院 | Kit for predicating susceptibility to ankylosing spondylitis |
| CN104313140A (en) * | 2014-10-16 | 2015-01-28 | 卫生部北京医院 | Reagent and method for predicting susceptibility to ankylosing spondylitis |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710448A (en) * | 2013-12-20 | 2014-04-09 | 卫生部北京医院 | Method and kit for predicting susceptibility of ankylosing spondylitis |
| CN103710446A (en) * | 2013-12-20 | 2014-04-09 | 卫生部北京医院 | Reagent and method for predicting ankylosing spondylitis susceptibility |
| CN103710447A (en) * | 2013-12-20 | 2014-04-09 | 卫生部北京医院 | Method and reagent for predicting susceptibility of ankylosing spondylitis |
| CN103882110A (en) * | 2013-12-20 | 2014-06-25 | 卫生部北京医院 | Reagent of detecting susceptibility of ankylosing spondylitis |
| CN103882112A (en) * | 2013-12-20 | 2014-06-25 | 卫生部北京医院 | Reagent and method for predicting ankylosing spondylitis susceptibility |
| CN103710446B (en) * | 2013-12-20 | 2015-05-13 | 卫生部北京医院 | Reagent and method for predicting ankylosing spondylitis susceptibility |
| CN103710448B (en) * | 2013-12-20 | 2015-05-13 | 卫生部北京医院 | Method and kit for predicting susceptibility of ankylosing spondylitis |
| CN103882110B (en) * | 2013-12-20 | 2015-09-09 | 卫生部北京医院 | A kind of reagent detecting susceptibility of ankylosing spondylitis |
| CN103882112B (en) * | 2013-12-20 | 2016-02-10 | 卫生部北京医院 | A kind of reagent and method predicting susceptibility of ankylosing spondylitis |
| CN104293959A (en) * | 2014-10-16 | 2015-01-21 | 卫生部北京医院 | Kit for predicating susceptibility to ankylosing spondylitis |
| CN104313140A (en) * | 2014-10-16 | 2015-01-28 | 卫生部北京医院 | Reagent and method for predicting susceptibility to ankylosing spondylitis |
| CN104313140B (en) * | 2014-10-16 | 2016-06-08 | 卫生部北京医院 | A kind of reagent predicting susceptibility of ankylosing spondylitis and method |
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