CN104313140A - Reagent and method for predicting susceptibility to ankylosing spondylitis - Google Patents

Abstract

The invention discloses a reagent and a method for predicting susceptibility to of ankylosing spondylitis, and belongs to the technical field of medical biology. Susceptibility of a subject to ankylosing spondylitis is predicted by extracting genome DNA of a host cell and testing genotype of a polymorphic site of HSPA1B gene of the subject on 31795536 location. The method disclosed by the invention has the advantages that correlation between the polymorphic site of the HSPA1B gene on the 31795536 location and ankylosing spondylitis is explained for the first time; the method for predicting susceptibility to ankylosing spondylitis is provided, which is applied to prevention, auxiliary diagnosis and treatment of ankylosing spondylitis, and can be also used for new drug research and development.

Description

A kind of reagent and method predicting susceptibility of ankylosing spondylitis

Technical field

The present invention relates to a kind of reagent and the method for predicting susceptibility of ankylosing spondylitis, predict the susceptibility of experimenter for ankylosing spondylitis by measuring with the polymorphism of AS genes involved HSPA1B in particular, the method can be used for the auxiliary diagnosis of disease, treatment and new drug development, belongs to biological technical field.

Background technology

Ankylosing spondylitis (Ankylosing spondylitis, AS) is a kind of autoimmune disorder, and be mainly in the person between twenty and fifty in 16-40 year, 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, some patients is with the outer pathology of the backbones such as hip joint in various degree, eye, lung, cardiovascular, kidney.The sickness rate of AS in white race crowd is about 1% ~ 3%, China's AS morbidity is about 0.2-0.6%, wherein the patient of more than 60% gets involved with hip joint, cause more than 20% AS patient disabilities, inflammation mainly involves the bone attachment point of joint capsule, tendon and ligament, cause local joint accretion tetanic, limitation of activity.So far the medicine can obviously alleviating and control disease progression is still lacked clinically.AS belongs to multigenic disease, has obvious genetic predisposition, although it has been generally acknowledged that heredity and immune factor play a leading role in AS falls ill, definite cause and onset of disease mechanism is still unclear.

Carry out the research of AS inherited pathogenic factor at present, the SNP that adopts, as the association analysis method of genomic marker, is effective, is proven more.SNP refers to the DNA sequence polymorphism that in chromogene group level, single nucleotide diversity causes, frequency in crowd needs >1%, SNPs is biallelic marker, 70.1% is had for the conversion between homotype base: as G/A or T/C, 29.1% for occurring in the transversion between purine and pyrimidine in this single base change.C (cytosine(Cyt)) is the most labile site in human genome, because great majority are methylated cytosines, can be converted to T (thymus pyrimidine) by spontaneous deaminizating, SNP contains 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 high (average every 1kb just have 1), representative strong (SNP being arranged in gene internal may directly affect protein structure or expression level), genetic stability good (with microsatellite polymorphism comparatively speaking), be easy to the features such as automated analysis (because SNP mostly is biallelic marker crowd, can simply with " +/-or 1/0 " direct somatotype) and become good genetic marker.

1973, first Brewerton etc. found and the Human leukocyte antigen-B27 of AS strong correlation (HLA-B27).Along with progress of research, other tumor susceptibility genes relevant to AS such as tumor necrosis factor-alpha (TNF-α), il-1 (IL-1) are identified successively.

HSPA1B (heat shock 70kDa protein 1B, heat shock protein 70 Kda-1B) gene is positioned at 6q21.3, total length 8069bp.This assignment of genes gene mapping, in human leucocyte antigen III region, occurs to develop etc. closely related with immune response and self property Immunological diseases.

The research that HSPA1B gene is associated with AS there is no report in recent years.

Summary of the invention

Main purpose of the present invention is to provide a kind of reagent predicting susceptibility of ankylosing spondylitis, comprises PCR primer and the test kit containing this primer.

Second object of the present invention is to provide a kind of method detecting susceptibility of ankylosing spondylitis gene.

For achieving the above object, the present invention is by the following technical solutions:

Detect a nucleotide sequence for susceptibility of ankylosing spondylitis, have the base sequence shown in sequence table SEQ ID No.1, namely its 31795536 position is variant sites, indicates with letter " R ".This nucleotide sequence is HSPA1B Gene Partial sequence.Fig. 1 is the schematic diagram in HSPA1B gene structure and polymorphic variation site thereof, and 31795536 are marked on the corresponding position in HSPA1B gene map.

A kind of method detecting susceptibility of ankylosing spondylitis, by extracting the genomic dna of host cell, measure the genotype of the HSPA1B gene 31795536 position pleomorphism site of experimenter, experimenter is to the susceptibility of ankylosing spondylitis in prediction: when the genotype of HSPA1B gene 31795536 position is GG, the susceptibility of experimenter is minimum; When carrying A allelotrope, the susceptibility of experimenter raises.

The primer of one group of detection susceptibility of ankylosing spondylitis, the nucleotide sequence of primer is respectively shown in sequence table SEQ ID No.2 and sequence table SEQ ID No.3, length is respectively 25bp and 24bp, and can amplify the product including 31795536 positions in sequence shown in sequence table SEQ ID No.1 specifically.

The invention provides a kind of diagnostic kit detecting susceptibility of ankylosing spondylitis, the general components, reagent, damping fluid etc. of the primer pair wherein containing specific amplification HSPA1B gene 31795536 position of the present invention and the test kit for pcr amplification detection, those skilled in the art know these general components and detection method.Whole components in test kit of the present invention, content, source and using method are as follows:

Detect a test kit for ankylosing spondylitis tumor susceptibility gene, be made up of following reagent:

(1) 10 μ L 10 × PCR damping fluid;

(2) 2 μ L 10mMdNTP mixed solutions;

(3) 2 μ L Taq DNA polymerase, 2unit/ μ L;

(4) 1 μ L F1 primers, be the nucleotide sequence shown in SEQ ID NO.2, concentration is 10pmol/ μ L;

(5) 1 μ L R1 primers, be the nucleotide sequence shown in SEQ ID NO.3, concentration is 10pmol/ μ L;

(6) 8 μ L 10 × LC-Green Plus saturated fluorescence dyestuffs;

(7) 2 μ L oligonucleotide internal references, be made up of 4 kinds of each 0.5 μ L of internal reference primer, concentration is 10pmol/ μ L, wherein low temperature oligonucleotide internal reference upstream primer F is the nucleotide sequence shown in SEQ ID NO.4, low temperature oligonucleotide internal reference downstream primer R is the nucleotide sequence shown in SEQ ID NO.5, high temperature oligonucleotide internal reference upstream primer F is the nucleotide sequence shown in SEQ ID NO.6, and high temperature oligonucleotide internal reference downstream primer R is the nucleotide sequence shown in SEQ ID NO.7;

(8) 64 μ L pure water.

Using method:

(1) pcr amplification: by pcr amplification HSPA1B Gene Partial fragment, prepare mixed solution: 10 × PCR reaction buffer 1 μ L, 10mM/LdNTP0.2 μ L, Taq DNA polymerase 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 internal reference 0.2 μ L (each 0.05 μ L of high and low temperature oligonucleotide internal reference upstream and downstream primer) (sequence is in table 1), genomic dna 1 μ L, adds pure water to 10 μ L.PCR reaction conditions is 95 DEG C of denaturation 3min, 95 DEG C of sex change 30s, and 69 DEG C of annealing 30s, 72 DEG C extend 4s, altogether 45 circulations, 72 DEG C of total elongation 2min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature process: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 2min.In each system, the paraffin oil of 20 μ L is added, to prevent from evaporating before PCR.

(2) genotype judges: PCR primer moved in special 96 orifice plates of HRM, Light Scanner TMHR-I96 carries out HRM analysis, analyze the curve after collection with Light Scanner Call IT software, the difference according to melting curve judges genotype.

The purposes of HSPA1B gene 31795536 position pleomorphism site in the reagent preparing diagnosing ankylosing spondylitis.

The purposes of HSPA1B gene 31795536 position pleomorphism site in the medicine of preparation treatment ankylosing spondylitis.

Measuring method of the present invention determines the genomic dna deriving from people, and sample source is unrestricted, as: body fluid (blood, ascites and urine etc.), histocyte (as hepatic tissue) etc.Genomic dna can be prepared by these samples of Isolation and purification.The concentration of adjustment genomic dna, makes it consistent as much as possible.Take genomic dna as template, the nucleic acid fragment containing HSPA1B gene mutation site can be amplified, to obtain the great amount of samples of mensuration.This sample obtained containing the DNA fragmentation of HSPA1B genovariation point by amplification, is particularly suitable for as mensuration material.

When carrying out gene auxiliary diagnosis, the present invention is preferably applied in the auxiliary diagnostic measuring and exist according to the mutation type of HSPA1B gene, auxiliary diagnostic comprises the particular agent as neccessary composition, and it corresponds to the method for measuring HSPA1B gene mutation type.Suitable particular agent is selected, as DNA fragmentation and/or the primer for pcr amplification step by the measuring method adopted.

Advantage of the present invention is: the present invention illustrates the dependency of HSPA1B gene 31795536 position pleomorphism site and AS first, provide a kind of method predicting AS susceptibility, the method can be used for the prevention of AS, auxiliary diagnosis and treatment, can also be used for new drug development.

Below in conjunction with the drawings and specific embodiments, the present invention is further described; so that the public has a better understanding summary of the invention; not limitation of the present invention, the equivalent replacement of all any this areas of doing according to the disclosure of invention, all belongs to protection scope of the present invention.

Accompanying drawing explanation

Fig. 1 HSPA1B gene 31795536 position view

Fig. 2 is the gene type figure of HSPA1B genovariation site through HRM method

Fig. 3 is the sequencer map in HSPA1B genovariation site

Embodiment

As follows for representing the english abbreviation of reagent in the following example:

10 × PCR damping fluid: 10mM Tris-HCI (pH=8.3), 500mM Repone K (KCl), 10mM magnesium chloride (MgCl ₂), 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:

One. case is selected in:

By the New York Revised diagnostic criteria MethodsThe cases enrolled of 1984, choose the AS patient 283 example (age: 8-71 year from Ningxia, China consanguinity-less relation altogether, average 27 years old), with area normal healthy controls volunteer 259 example (age: 18-21 year, average 19 years old).All persons under inspection are Han nationality and signature Written informed consent, this research obtains Beijing Hospital, the accreditation 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.

Two. according to following method, preparation human gene group DNA.

1. in the 1.5mLEP pipe of label, add 1000 μ L erythrocyte cracked liquids, after add 400 μ LEDTA anticoagulations (anticoagulation puts upside down mixing 3-5 time before adding), put upside down mixing, standing 10 minutes of room temperature;

2.13000rpm, after centrifugal 30 seconds, removes supernatant liquor;

3. in gained precipitation, adding 480 μ L nucleic acid cleavage liquid, attack tube wall, fully adding 20 μ L Proteinase Ks (diluting 20 times of diluted protein enzyme K with splitting karyolymph) after mixing, put upside down mixing, 65 DEG C of incubators 10 minutes, (period mixes frequently up and down, guarantees without grumeleuse);

4. be down to room temperature after taking out, add 300 μ L albumen precipitation liquid, fully put upside down mixing, leave standstill 10 minutes, centrifugal 2 minutes of 13000rpm;

5. moved to by supernatant liquor in 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 gradually, centrifugal 2 minutes of 13000rpm;

6. abandon supernatant liquor and guarantee that precipitation is stayed in EP pipe, adding 670 μ L70% ethanol, mixing of turning upside down, centrifugal 2 minutes of 13000rpm;

7. abandon supernatant, make ethanol volatilization in pipe clean;

8. add TE solution (400 μ L), fully dissolve, the genomic dna extracted is carried out to the analysis of concentration and purity, draw part DNA solution as working fluid, concentration correction to 20ng/ μ L, be placed in 4 DEG C for subsequent use, residue genomic dna put-20 DEG C of Refrigerator stores.

The identification qualification of embodiment 2:SNP

The present invention adopts PCR-high resolving power melting curve (HRM) analytical method and PCR sequencing technologies to detect the genotype of HSPA1B gene 31795536 position (its loci is G/A) simultaneously.Fig. 2 is HSPA1B genovariation site is the sequencer map in HSPA1B genovariation site through gene type figure, Fig. 3 of HRM method.

The determination of one .PCR-HRM primer

From Genebank, look into the DNA base sequence (SEQ ID No.1) got near HSPA1B gene 31795536 position, design of primers completes under Oligo7.0 software.Object fragment is positioned at HSPA1B gene, total length 51bp, and specific primer sequence is as follows:

F1：5’-TTTGAAAACGGCCAGCCTGAGGAGC-3’(SEQ?ID?No.2)

R1：5’-GAAAGACGAAGCAGACCCTCGCAG-3’(SEQ?ID?No.3)

Two .PCR reaction system and reaction conditionss

By pcr amplification HSPA1B Gene Partial fragment, PCR reaction system is: 10 × PCR reaction buffer 1 μ L, 10mM/LdNTP 0.2 μ L, Taq DNA polymerase 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 internal reference 0.2 μ L (each 0.05 μ L of high and low temperature oligonucleotide internal reference upstream and downstream primer) (sequence is in 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.PCR reaction conditions is 95 DEG C of denaturation 3min, 95 DEG C of sex change 30s, and 69 DEG C of annealing 30s, 72 DEG C extend 4s, altogether 45 circulations, 72 DEG C of total elongation 2min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature process: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 2min.

Table 1: high and low temperature oligonucleotide internal reference primer sequence, annealing temperature and product sheet segment length

Three .HRM judge genotype

PCR primer is moved in special 96 orifice plates of HRM, Light Scanner TMHR-I 96 carries out HRM analysis: from 40 DEG C, melting curve is gathered with the slope of 0.3 DEG C/s, to 98 DEG C of end, with Light Scanner Call IT software, the curve after collection is analyzed, judge genotype.

Four. order-checking judges genotype

From the individuality of the different genotype of gained, randomly draw 10 routine samples respectively carry out sequence verification.Order-checking sample re-starts pcr amplification, sequencing primer sequence is: F2:5 '-AATATTCCCGACCTGGCAGCCT-3 ' (SEQ ID No.8), R2:5 '-AAGAGCTCAGTCCTTCGGAACG-3 ' (SEQ ID No.9), the long 249bp of amplified fragments.PCR reaction is totally 30 μ L, comprises: genomic dna 2 μ L, 10 × PCRBuffer3 μ L, 10mMdNTP0.5 μ L, Taq DNA polymerase (5U/ μ L) 0.5 μ L, upstream and downstream primer (10pM/ μ L) each 0.5 μ L, pure water is supplemented to cumulative volume 30 μ L.PCR reaction conditions is: 95 DEG C of denaturation 5min laggard enter major cycle, 95 DEG C of sex change 30s, 67 DEG C of annealing 30s, 72 DEG C extend 20s, 45 circulations, 72 DEG C of extension 2min.PCR primer detects through 8% polyacrylamide gel electrophoresis, send Hua Da gene sequencing portion to carry out sequence verification after gel imaging system observation is qualified.

Embodiment 3: the dependency of gene SNP and ankylosing spondylitis (AS)

One. statistical method:

Statistical method: the populational representation using Hardy-Weinberg balance check research sample.Pearson chi square test in SPSS11.0 software is utilized to calculate allelotrope, the distribution frequency of genotype between AS case group and Normal group of HSPA1B gene 31795536 position, the risk OR value of AS and 95%CI credibility interval thereof take P<0.05 as significance of difference standard.

Two. result: the genotype and the distribution of gene frequency between case and control group that are positioned at HSPA1B gene 31795536 position in 6p21.3 region refer to table 2.

The genotype of table 2HSPA1B gene 31795536 position and the distribution of gene frequency between case-control group

Note: OR: odds ratio; CI: credibility interval.* A allelotrope is the risk allelotrope of easily suffering from AS.Experimenter is divided into the risk allelotrope AG carrier of AS, and non-risk allelotrope GG carrier.Genotypic chi-square value is drawn by more non-risk allelotype GG and risk allelotype AG.

From table 2, the A allelotrope of HSPA1B gene 31795536 position, namely be T allelotrope on its DNA complementary strand, distribution frequency in PATIENT POPULATION is significantly higher than its genotypes distribution and allele frequencies (2.7%vs.0.6%) in healthy normal population, there is significant difference (P=7.7 × 10-3), and the OR value in A site is 4.67,95%CI:1.35-16.24; In the risk allelotrope AG carrier and non-risk allelotrope GG carrier of AS, the distribution frequency of risk genotype in case group is significantly higher than (P < 0.05) in control group, all show HSPA1B gene 31795536 position and AS is ill is proportionate, the risk increasing AS morbidity may be had.

Implement 4: detection kit

Preparation detects the test kit of AS relevant risk, includes the primer pair that can amplify HSPA1B gene 31795536 position, and other PCR-HRM corresponding reagent.Test kit of the present invention detects application for 10 person-portions, and keep in Dark Place in-20 DEG C, its component, content and source comprise:

10 μ L 10 × PCR damping fluid (Pharmacia),

2 μ L 10mMdNTP mixed solution (Pharmacia),

2 μ L Taq DNA polymerase (2unit/ μ L) (Takara)

1μL?F1(SEQ?ID?No.2)(10pM/μL)

1 μ L R1 (SEQ ID No.3) (10pM/ μ L) primer,

8 μ L 10 × LC-Green Plus saturated fluorescence dyestuff (American I daho company),

2 μ L oligonucleotide internal references, be made up of 4 kinds of each 0.5 μ L of internal reference primer, concentration is 10pmol/ μ L, wherein low temperature oligonucleotide internal reference upstream primer F is the nucleotide sequence shown in SEQ ID NO.4, low temperature oligonucleotide internal reference downstream primer R is the nucleotide sequence shown in SEQ ID NO.5, high temperature oligonucleotide internal reference upstream primer F is the nucleotide sequence shown in SEQ ID NO.6, and high temperature oligonucleotide internal reference downstream primer R is the nucleotide sequence shown in SEQ ID NO.7;

64 μ L pure water.

After PCR-HRM detects, HSPA1B gene 31795536 position polymorphism can be detected easily.

Adopt kit measurement AS patient of the present invention and normal people HSPA1B gene 31795536 position polymorphism, randomly draw AS patient gene and organize DNA and each 10 examples of normal people's genomic dna.

One. method:

1.PCR increases: by pcr amplification HSPA1B Gene Partial fragment, PCR reaction system is: 10 × PCR reaction buffer 1 μ L, 10mM/LdNTP 0.2 μ L, Taq DNA polymerase 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 internal reference 0.2 μ L (each 0.05 μ L of high and low temperature oligonucleotide internal reference upstream and downstream primer) (sequence is in 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.PCR reaction conditions is 95 DEG C of denaturation 3min, 95 DEG C of sex change 30s, and 69 DEG C of annealing 30s, 72 DEG C extend 4s, altogether 45 circulations, 72 DEG C of total elongation 2min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature process: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 2min.

2. genotype judges: PCR primer moved in special 96 orifice plates of HRM, Light Scanner TMHR-I96 carries out HRM analysis: from 40 DEG C, melting curve is gathered with the slope of 0.3 DEG C/s, to 98 DEG C of end, with Light Scanner Call IT software, the curve after collection is analyzed, judge genotype.

Two. result:

Result shows, and the genotype of AS patient HSPA1B gene 31795536 position polymorphism is 2 examples of AG, 8 examples of GG; 10 routine control group genotype are GG.Present method effectively can determine HSPA1B gene 31795536 position polymorphism, and when HSPA1B gene 31795536 position pleomorphism site genotype is GG, the susceptibility of experimenter is minimum; When carrying A allelotrope, the susceptibility of experimenter raises.

The present invention has the illustration of practicality:

The detection method of HSPA1B gene pleiomorphism of the present invention can be used for the A allelotrope of the rare variant sites on the HSPA1B gene in analyst's euchromosome 6p21.3 district, namely be T allelotrope on its DNA complementary strand, be applied to the complementary diagnosis of AS and the individual AS risk of assessment how, be beneficial to early intervention and the treatment of carrying out AS.

Utilize the present invention to set forth the nucleotide variation of HSPA1B gene 31795536 position, as one of biomarker, can be used as the screening of the molecular target of medicinal design, to help to find the bioactive molecule having and regulate HSPA1B genetic expression, promote AS new drug development.

The nucleotide sequence of detection HSPA1B gene pleiomorphism that the present invention sets up and AS related locus, can highly sensitive, the specific test kit being applied to AS gene auxiliary diagnosis.

As above told, reached a conclusion, the polymorphism of HSPA1B gene 31795536 position and AS tool significant correlation.Therefore, measure this polymorphism according to the present invention, can be used for the gene auxiliary diagnosis of AS.

Invention describes the new mutant site that HSPA1B Gene A S-phase is closed, and provide a kind of method measuring HSPA1B gene pleiomorphism, and, according to the present invention, only need a small amount of DNA sample to be just enough to measure gene polynorphisms.As a result, the invention provides a kind of gene aided diagnosis method measuring AS related gene polymorphism.

Claims (6)

1. one kind is detected the method for susceptibility of ankylosing spondylitis, it is characterized in that: by extracting the genomic dna of host cell, measure the genotype of the HSPA1B gene 31795536 position pleomorphism site of experimenter, experimenter is to the susceptibility of ankylosing spondylitis in prediction.

2. a kind of method detecting susceptibility of ankylosing spondylitis according to claim 1, is characterized in that: when the genotype of HSPA1B gene 31795536 position is GG, the susceptibility of experimenter is minimum; When carrying A allelotrope, the susceptibility of experimenter raises.

3. one group is detected the primer of susceptibility of ankylosing spondylitis, it is characterized in that: the nucleotide sequence of primer is respectively shown in sequence table SEQ ID No.2 and sequence table SEQ ID No.3.

4. detect a test kit for ankylosing spondylitis tumor susceptibility gene, it is characterized in that being made up of following reagent:

(1) 10 μ L 10 × PCR damping fluid;

(2) 2 μ L 10mMdNTP mixed solutions;

(3) 2 μ L Taq DNA polymerase, 2unit/ μ L;

(4) 1 μ L F1 primers, be the nucleotide sequence shown in SEQ ID NO.2, concentration is 10pmol/ μ L;

(5) 1 μ L R1 primers, be the nucleotide sequence shown in SEQ ID NO.3, concentration is 10pmol/ μ L;

(6) 8 μ L 10 × LC-Green Plus saturated fluorescence dyestuffs;

(7) 2 μ L oligonucleotide internal references, be made up of 4 kinds of each 0.5 μ L of internal reference primer, concentration is 10pmol/ μ L, wherein low temperature oligonucleotide internal reference upstream primer F is the nucleotide sequence shown in SEQ ID NO.4, low temperature oligonucleotide internal reference downstream primer R is the nucleotide sequence shown in SEQ ID NO.5, high temperature oligonucleotide internal reference upstream primer F is the nucleotide sequence shown in SEQ ID NO.6, and high temperature oligonucleotide internal reference downstream primer R is the nucleotide sequence shown in SEQ ID NO.7;

(8) 64 μ L pure water.

The purposes of 5.HSPA1B gene 31795536 position pleomorphism site in the reagent preparing diagnosing ankylosing spondylitis.

The purposes of 6.HSPA1B gene 31795536 position pleomorphism site in the medicine of preparation treatment ankylosing spondylitis.