CN103710447B - Method and reagent for predicting susceptibility of ankylosing spondylitis - Google Patents

Abstract

The invention discloses a method and reagent for predicting susceptibility of ankylosing spondylitis, belonging to the technical field of medical biology. The method comprises the following steps: extracting a genome DNA (deoxyribonucleic acid) of a host cell, determining the genotype of the SNP (single-nucleotide polymorphism) site of the SKIV2L gene of the testee, and predicting the susceptibility of ankylosing spondylitis of the testee. The invention elaborates the relationship between SKIV2L gene polymorphism site and ankylosing spondylitis, and provides a method for predicting susceptibility of ankylosing spondylitis. The method can be used for prevention, auxiliary diagnosis and treatment of ankylosing spondylitis, and can also be used for research and development of new drugs.

Description

A kind of method and reagent predicting susceptibility of ankylosing spondylitis

Technical field

The present invention relates to a kind of method and the reagent of predicting susceptibility of ankylosing spondylitis, predict the susceptibility of experimenter for ankylosing spondylitis by measuring with the polymorphism of AS genes involved SKIV2L 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)) be the most labile site in human genome because great majority are methylated cytosines, can spontaneous deaminizating be converted to T(thymus pyrimidine), 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.

SKIV2L(superkiller viralicidic activity2-like, antivirally superly killing and wounding activity factor 2) gene is positioned at 6p21.3, total length 1056bp, containing 21 exons, its coded product is a kind of albumen, 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.

There is no any result of study be associated with AS about SKIV2L gene at present.

Summary of the invention

Main purpose of the present invention is to provide a kind of method predicting susceptibility of ankylosing spondylitis.

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

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

Detecting the nucleotide sequence of susceptibility of ankylosing spondylitis, is base sequence shown in sequence table SEQ ID No.1.Namely its+438 be variant sites, indicates with letter " R ".This nucleotide sequence is SKIV2L full length gene sequence.Fig. 1 is the schematic diagram in SKIV2L gene structure and polymorphic variation site thereof, includes 3 exons in accompanying drawing, and rs144147284 site is marked on the corresponding position of the 16th exon 1 in SKIV2L gene map.

Detect a method for mandatory spondylitis susceptibility, detect the genotype in rs144147284 site, experimenter SKIV2L gene the 16th exon 1, when genotype 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, can increase and obtain the nucleotide sequence SEQ ID No.1 of described 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.

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 SKIV2L gene rs144147284 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 are as follows:

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

(1) 10 μ L10 × PCR damping fluid;

(2) 2 μ L10mMdNTP 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 L10 × 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 is as follows:

(1) pcr amplification: by the 16th exon 1 Partial Fragment of pcr amplification SKIV2L gene, 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, 1 × LC-Green Plus saturated fluorescence dyestuff 0.8 μ L, the each 0.05 μ L of oligonucleotide internal reference 0.2 μ L(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 5min, 95 DEG C of sex change 30s, and 65 DEG C of annealing 30s, 72 DEG C extend 5s, 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 4min.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 SKIV2L gene the 16th exon 1 rs144147284 pleomorphism site in the reagent preparing diagnosis or treatment ankylosing spondylitis or medicine.

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 SKIV2L gene mutation site can be amplified, to obtain the great amount of samples of mensuration.This sample obtained containing the DNA fragmentation of SKIV2L 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 SKIV2L gene, auxiliary diagnostic comprises the particular agent as neccessary composition, and it corresponds to the method for measuring SKIV2L 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 SKIV2L gene polymorphism sites and AS first, provides a kind of method predicting AS susceptibility, and 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 is the schematic diagram in SKIV2L gene structure and polymorphic variation site thereof

Fig. 2 is the gene type figure of SKIV2L genovariation site through HRM method

Fig. 3 is the sequencer map in SKIV2L 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 altogether from Jilin Area consanguinity-less relation AS patient 136 example (age: 14-44 year, average 24 years old), with area normal healthy controls volunteer 148 example (age: 39-72 year, average 46 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, add 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, putting 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 2SNP

The present invention adopts PCR-high resolving power melting curve (HRM) analytical method and PCR sequencing technologies to be G/A to its loci of rs144147284(of the 16th exon 1 of SKIV2L gene simultaneously) genotype detect.

The determination of one .PCR-HRM primer

From Genebank, look into the DNA base sequence (SEQ ID No.1) got near rs144147284, design of primers completes under Oligo7.0 software.Object fragment is positioned at SKIV2L gene the 16th exon 1, total length 48bp, positive-sense strand F1 and antisense strand R1 specific primer sequence as follows:

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

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

Two .PCR reaction system and reaction conditionss

By pcr amplification SKIV2L gene the 16th exon 1 Partial Fragment, PCR reaction system is: 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, the each 0.05 μ L of oligonucleotide internal reference 0.2 μ L(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 5min, 95 DEG C of sex change 30s, and 65 DEG C of annealing 30s, 72 DEG C extend 5s, 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 4min.

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-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.Fig. 2 is the gene type figure of SKIV2L genovariation site through HRM method.

Four. order-checking judges genotype

From the individuality of the different genotype of gained, randomly draw 3 routine samples respectively carry out sequence verification.Order-checking sample re-starts pcr amplification, sequencing primer sequence is: F2:5 '-GGGAAATTACCTGGAAATGCGACTGA-3 ' (SEQ ID No.8), R2:5 '-GAGACCAGCGGGTGAGAAGAGGAG-3 ' (SEQ ID No.9), the long 270bp of amplified fragments.PCR reaction is totally 30 μ L, and comprise: 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 20s, 60 DEG C of annealing 20s, 72 DEG C extend 20s, 35 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.Fig. 3 is the sequencer map in SKIV2L genovariation site.

The dependency of embodiment 3 gene SNP and mandatory spondylitis (AS)

One. statistical method:

Use the populational representation of 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 in SKIV2L gene rs144147284 site, the risk OR value of AS and 95%CI credibility interval thereof take P<0.05 as significance of difference standard.

Two. result: on the SKIV2L gene being positioned at 6p21.3 region, the genotype in rs144147284 site and the distribution of gene frequency between case and control group refer to table 2.

The genotype in table 2SKIV2L gene rs144147284 site 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 risk allelotrope (AG) carrier of AS, and non-risk allelotrope (GG) carrier.

From table 2, the A allelotrope of SKIV2L gene rs144147284, namely be T allelotrope on its DNA complementary strand, distribution frequency in PATIENT POPULATION is significantly higher than its genotypes distribution and allele frequencies (4.8%vs.0.3%) in healthy normal population, there is significant difference (P<0.001), and the OR value in this site is 14.81,95%CI:1.92-113.96; In 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 SKIV2L gene rs144147284 site 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 SKIV2L gene rs144147284, 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 L10 × PCR damping fluids (Pharmacia)

2 μ L10mMdNTP mixed solutions (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 L10 × LC-Green Plus saturated fluorescences 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.

This test kit detects through PCR-HRM, can detect SKIV2L gene the 16th exon 1 rs144147284 polymorphism.

Adopt kit measurement AS patient of the present invention and normal people SKIV2L gene the 16th exon 1 rs144147284 loci polymorphism.Randomly draw AS patient gene and organize DNA and each 10 examples of normal people's genomic dna.

One. method:

1.PCR increases: 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, 1 × LC-Green Plus saturated fluorescence dyestuff 0.8 μ L, each 0.05 μ L of oligonucleotide internal reference 0.2 μ L(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 5min, 95 DEG C of sex change 30s, and 65 DEG C of annealing 30s, 72 DEG C extend 5s, 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 4min.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.

Two. result:

Result shows, and AS clinical samples SKIV2L gene the 16th exon 1 rs144147284 loci polymorphism genotype is 7 examples of 3 examples of GA, GG; Control group genotype GA type 1 example, GG type 9 example.Test kit of the present invention effectively can detect the genotype in rs144147284 site, experimenter SKIV2L gene the 16th exon 1, and when 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 SKIV2L gene pleiomorphism of the present invention can be used for the T allelotrope of the rare variant sites on the SKIV2L gene in analyst's euchromosome 6p21.3 district, namely be A 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 in SKIV2L gene rs144147284 site, 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 SKIV2L to express, promote AS new drug development.

The nucleotide sequence of detection SKIV2L 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 in SKIV2L gene rs144147284 site 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 SKIV2L Gene A S-phase is closed, and provide a kind of method measuring HCP 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 (2)

1. 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 2.SKIV2L gene the 13rd exon 1 rs144147284 pleomorphism site in the reagent preparing diagnosing ankylosing spondylitis or medicine.