CN102146482A - IL-23R gene polymorphism detection specific primer and liquid phase chip - Google Patents
IL-23R gene polymorphism detection specific primer and liquid phase chip Download PDFInfo
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Abstract
The invention discloses an interleukin-23 receptor (IL-23R) gene polymorphism detection specific primer and an IL-23R gene polymorphism detection liquid phase chip. The liquid phase chip mainly comprises an ASPE primer, microspheres and an amplified primer, wherein the ASPE primer consists of a tag sequence of a 5' end and specific primers of a 3' end, and the specific primers are selected from more than one pair of SEQ ID NO.9 and SEQ ID NO.10 aiming at C214T, SEQ ID NO.11 and SEQ ID NO.12 aiming at C235T, SEQ ID NO.13 and SEQ ID NO.14 aiming at T82G, and SEQ ID NO.15 and SEQ ID NO.16 aiming at C53T. The IL-23R gene polymorphism detection liquid phase chip has very good signal-noise ratio, the designed probe and an anti-tag sequence basically have no cross reaction, and the selection of a tag sequence and the anti-tag sequence and the combination of the tag sequence and the specific ASPE primer can avoid cross reaction and realize parallel detection of multiple polymorphic sites.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete a kind of IL-23R gene pleiomorphism detection specificity primer and the liquid-phase chip of relating to.
Background technology
Human Inter Leukin-2's 3 acceptors (human interleukin-23receptor, hIL-23R, IL-23R) be a heterodimer cytokine of forming by IL-23p19 and IL-12p40, mainly by activatory dendritic cell and scavenger cell secretion, belong to IL-12 inflammatory factor family member, and transmit active signal by the Jak/Stat approach.The encoding gene of IL-23R is positioned on the position of No. 1 karyomit(e) 1p31.2～32.1, and 29kb base contained in the coding region, is made up of 11 exons.
Studies show that the gene pleiomorphism of IL-23R is relevant with the formation of the immunne response of multiple pulmonary disorder and disease, as: infectious diseases and lung fibrosis such as pulmonary tuberculosis, bacillus canalis capsulatus pneumonia, Whooping cough; Relevant with the immunity of various autoimmune disease and tumour in addition.
The IL-23R gene mutation site of target detect of the present invention, it is as shown in the table:
At present, the method that the IL-23R gene pleiomorphism is detected, analyzes is a lot, as: the TaqMan technology of PCR-based method, mass spectrum array technique, fluorescent quantitative PCR technique or the like, wherein the most frequently used is the TaqMan technology.But the TaqMan technology can only add a kind of fluorescent detection probe in a tube reaction, detect a kind of genotype, detect the wild-type and the mutant of a gene if desired, then need separately two tube reactions, add wild-type probe and mutant probe respectively and carry out twice independently detection.And also there is the limitation of similar detection flux in other detection method, can only detect a kind of mutation type at every turn, can not satisfy the needs of practical application.
Summary of the invention
One of purpose of the present invention provides a kind of IL-23R gene pleiomorphism and detects liquid-phase chip, and this liquid-phase chip can be used for detecting wild-type and the mutant of IL-23R gene four kinds of common genotype C214T, C235T, T82G and C53T.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind of IL-23R gene pleiomorphism detects liquid-phase chip, mainly includes:
A. wild-type and the mutant specificity ASPE primer that designs respectively at four kinds of pleomorphism sites of IL-23R gene, every kind of ASPE primer is made up of the tag sequence of 5 ' end and the specific primer sequence at the goal gene pleomorphism site of 3 ' end, and described specific primer sequence is selected from: at the SEQ ID NO.9 of C214T and SEQ ID NO.10, at the SEQ ID NO.11 of C235T and SEQ ID NO.12, at the SEQ ID NO.13 of T82G and SEQ ID NO.14, at more than a pair of among the SEQ ID NO.15 of C53T and the SEQ ID NO.16; Described tag sequence is selected from the sequence among SEQ ID NO.1～SEQ ID NO.8;
B. be coated with respectively special anti-tag sequence, have a different colours coding microball, described anti-tag sequence is selected from the sequence among SEQ ID NO.17～SEQ ID NO.24, and described anti-tag sequence can be correspondingly with A in selected tag sequence complementary pairing, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon;
C. be used for increasing have C214T, the target sequence primer of the above pleomorphism site of C235T, T82G and C53T.
Preferably, described amplimer is selected from: at the SEQ ID NO.25 of C214T and SEQ ID NO.26, at the SEQ ID NO.27 of C235T and SEQ ID NO.28, at the SEQ ID NO.29 of T82G and SEQ ID NO.30, at more than a pair of among the SEQ ID NO.31 of C53T and the SEQ ID NO.32.
Preferably, described specificity ASPE primer is selected from: the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.9 at C214T reaches the sequence of being made up of SEQ ID NO.2 and SEQ ID NO.10, at the sequence of forming by SEQ ID NO.3 and SEQ IDNO.11 of C235T and by SEQ ID NO.4 and and the sequence formed of SEQ ID NO.12, the sequence of being made up of SEQ IDNO.5 and SEQ ID NO.13 at T82G reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.14, more than a pair of in the sequence of forming at the sequence of forming by SEQ ID NO.7 and SEQ ID NO.15 of C53T and by SEQ ID NO.8 and SEQ ID NO.16.
Another object of the present invention provides the specific primer sequence that a kind of IL-23R of being used for gene pleiomorphism detects.
Concrete technical scheme is as follows:
A kind ofly be used for the specific primer sequence that the IL-23R gene pleiomorphism detects, it is selected from: at the SEQ ID NO.9 of C214T and SEQ ID NO.10, at the SEQ ID NO.11 of C235T and SEQ ID NO.12, at the SEQ ID NO.13 of T82G and SEQ ID NO.14, at more than a pair of among the SEQ ID NO.15 of C53T and the SEQ ID NO.16.
Major advantage of the present invention is:
1. the identical rate of the detected result of detection liquid-phase chip provided by the present invention and sequencing is up to 100%.And detect the needed time well below sequencing technologies commonly used, realistic especially application need.Because in very a large amount of Auele Specific Primers, through test in a large number, reaction is verified, obtains the liquid-phase chip system of optimum combination.Prepared IL-23R gene pleiomorphism detects liquid-phase chip and has extraordinary signal-noise ratio, and there is not cross reaction between designed probe and the anti-tag sequence basically, choosing of tag sequence label, anti-tag sequence label and combining of tag sequence label and concrete ASPE primer, can avoid cross reaction, realize the parallel detection in a plurality of SNP site.
2. the present invention has chosen optimum combination by the design experiences of contriver's long-term accumulation and a large amount of experimental implementation from numerous Auele Specific Primers.The genotype of various types is accurately distinguished in the mutational site that the ASPE primer specificity primer of the present invention design can sensitive recognition objective specifically detects; In same reaction system, between the different Auele Specific Primers, do not have cross reaction basically between the pcr amplification product of Auele Specific Primer and non-target detect, detection specificity is good, and cross reacting rate is lower than 3%; Except detecting single site mutation situation, also the polymorphism situation in a plurality of mutational sites of parallel detection simultaneously detects the effect unanimity.
3. detection method step of the present invention is simple, four kinds of pleomorphism sites detect and can finish four amplifications that contain the target sequence in SNP site by a step multiplex PCR, many uncertain factors of existing in the complex operations processes such as repeated multiple times PCR have been avoided, thereby can improve the detection accuracy rate greatly, embodied qualitative, quantitative analysis feature of accurate while.
4. not only to have overcome traditional solid phase chip susceptibility not high in the present invention, and the defective of the repeatability difference of detected result is improved existing liquid-phase chip technology simultaneously, makes prepared microballoon can be applicable to different test items, has very strong expansion.The fluorescent signal value that detects improves greatly, thereby makes the sensitivity that detects be further enhanced, and signal to noise ratio strengthens, and detected result more accurately and reliably.
Embodiment
Embodiment 1IL-23R gene pleiomorphism detects liquid-phase chip, mainly includes:
One, ASPE primer
Wild-type and mutant at IL-23R gene four kinds of common genotype C214T, C235T, T82G and C53T design specific primer sequence respectively.The ASPE primer is made up of " Tag sequence+specific primer sequence ".The ASPE primer sequence is as shown in the table:
The ASPE primer sequence (Tag sequence+specific primer sequence) of table 1IL-23R gene
Every the ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence at anti-tag sequence on the corresponding microballoon, and 3 ' end is mutant or the special primer fragment (shown in above-mentioned table 1) of wild-type.All ASPE primers are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Two, the microballoon of anti-tag sequence bag quilt
According to designed ASPE Auele Specific Primer fragment, select the tag sequence, reduce between the anti-tag sequence of each microballoon to greatest extent and secondary structure that tag and ASPE Auele Specific Primer fragment may form, corresponding anti-tag sequence is as shown in table 2 on 8 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
8 kinds of microballoons selecting are available from U.S. Luminex company, with anti-tag sequence bag by on microballoon.Be connected with the spacerarm sequence of 5-10 T between anti-tag sequence and the microballoon, promptly add the spacerarm sequence of the preceding paragraph 5-10 T before each anti-tag sequence, the anti-tag sequence is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.With synthetic anti-tag sequence sterilization ddH
2O is made into the stock solution of 100nmol/ml.Described spacerarm is to be used for anti-tag and microsphere surface is spaced apart or anti-tag is placed the sequence of hydrophilic environments.By the spacerarm sequence of suitable length is set between anti-tag sequence and microballoon, can reduce sterically hinderedly, improve the efficient of hybridization and the specificity of hybridization.Common spacerarm sequence comprises poly dT, i.e. poly (dT), and oligomerization four polyoxyethylene glycol and (CH2) n spacerarm (n 〉=3) are as (CH2) 12, (CH2) 18 etc.In addition, if exist poly (dA) to disturb, can also use poly (TTG) as spacerarm.Spacerarm of the present invention is preferably 5-10 T, and the process of microballoon bag quilt is as follows:
Get 5 * 10 respectively
6The carboxylated microballoon of individual above-mentioned numbering (available from Luminex company) is suspended in the MES solution of 50ul 0.1mol/L (pH4.5), adds 10ul synthetic anti-tag molecule (100nmol/ml).EDC (N-(3-Dimethylaminopropyl-N-ethylcarbodiimide) (available from the Pierce Chemical company) working fluid of preparation 10ng/ml.Add the EDC working fluid of 2.5ul in the microballoon suspension, constant temperature was hatched 30 minutes, added the EDC working fluid of 2.5ul again, and constant temperature was hatched 30 minutes again.After reaction finished, the Tween-20 washing with 0.02% was once washed once with 0.1% SDS liquid again.The microballoon that is coated with the anti-tag sequence after the washing is resuspended in the Tris-EDTA solution [10mmol/L Tris (pH8.0)] of 100ul, and among the 1mmol/LEDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains the mutational site
At IL-23R gene four kinds of common genotype C214T, C235T, T82G and C53T, design of amplification primers amplifies four target sequences that contain pleomorphism site respectively to (seeing Table 3).
Table 3 amplifies the primer of the target sequence with pleomorphism site
All primers are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Embodiment 2 utilization IL-23R gene test liquid-phase chips are to the detection of sample
The prescription of described various solution is as follows:
MES damping fluid (pH5.0) prescription (250ml) of 50mM:
2 * Tm hybridization buffer
| Reagent | The source | Final concentration | The consumption of every 250ml |
| 1MTris-HCl，pH8.0 | SigmaT3038 | 0.2M | 50ml |
| 5MNaCl | Sigma?S5150 | 0.4M | 20ml |
| Triton?X-100 | Sigma?T8787 | 0.16％ | 0.4ml |
Be stored in 4 ℃ after the filtration.
The ExoSAP-IT test kit is available from U.S. USB company.
Biotin labeled dCTP is available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
With reference to " molecular cloning " methods involving, obtain DNA to be detected about DNA extraction.
Two, the pcr amplification of testing sample
Design four pairs of primers, one step of multiplex PCR amplifies contains IL-23R gene four kinds of common genotype C214T, C235T, T82G and C53T target sequence of totally four respectively, the product size is respectively 412bp, 480bp, 345bp and 343bp, and primer sequence (SEQID NO.25-32) is seen shown in the above-mentioned table 3.
At first prepare the multiple PCR primer working fluid: the primer stock solution 100ul that respectively gets SEQ ID NO.25-32 respectively mixes and is the multiple PCR primer working fluid in the 1.5ml Eppendorf tube.The multi-PRC reaction system is as follows:
The pcr amplification program is: 95 ℃ of 3min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 40s, 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations are standby.
Three, the enzyme of PCR product is cut processing
1. get the reacted product of 7.5ul PCR, add 1ul 10 * SAP damping fluid, 1ul SAP enzyme and 0.5ul Exo-I enzyme;
2.37 ℃ hatch 15min, hatch 15min for 80 ℃, the enzyme that deactivation is unnecessary.The product that enzyme is cut after the processing is directly used in follow-up ASPE primer extension reaction.
Four, site-specific primer extension reaction (ASPE)
Utilize the ASPE primer of above-mentioned design to carry out primer extension reaction, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on the reacted product band.
At first prepare blended ASPE primer working fluid: get IL-23R gene corresponding wild type to be detected and mutant ASPE primer stock solution 10ul respectively in the 1.5ml Eppendorf tube, add 10mmol/L Tris Buffer and mend, mix and be ASPE mix primer working fluid to 200ul.The system of ASPE reaction is as follows:
Response procedures is: 96 ℃ of 2min; 94 ℃ of 30s, 54 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ of preservations are standby.
Five, hybridization
1. according to the ASPE primer of design, the corresponding 8 kinds of microballoons of every group selection (every kind of microballoon concentration be 2.5 * 105/ml);
2. the microballoon of getting every kind of numbering of 1ul respectively is in the Eppendorf tube of 1.5ml;
3. microballoon is in 〉=centrifugal the 1-2min of 10000g;
4. supernatant discarded, microballoon is resuspended in 2 * Tm hybridization buffer of 100ul, the vortex mixing;
5. get the above-mentioned microballoon suspension of 25ul in the corresponding hole of 96 hole filter plates, control wells adds the ddH of 25ul
2O;
6. the ASPE reaction solution of getting 5-25ul is used ddH in corresponding hole
2O complements to 50ul;
7. encase 96 orifice plates with lucifuge with masking foil, 95 ℃ of 60s, 37 ℃ of 15min are hatched hybridization;
8. the microballoon after the hybridization is in 〉=centrifugal the 2-5min of 3000g;
9. remove supernatant, microballoon is resuspended in 1 * Tm hybridization buffer of 75ul;
10. microballoon is in 〉=centrifugal the 2-5min of 3000g;
11. microballoon is resuspended in 1 * Tm hybridization buffer of 75ul, adding 15ul concentration is Streptavidin-phycoerythrin (SA-PE) of 10ug/ml;
12.37 ℃ hatch 15min, on the Luminex instrument, detect.
Six, the result detects and data analysis
The reaction after product is by Luminex serial analysis instrument detecting.Detected result is shown in table 4, table 5 and table 6.
Fluorescent value (MFI) and data processing there are following requirement:
1. each site need have an allelotrope MFI at least greater than 300 and greater than 10 * PCR negative control MFI;
2.NET MFI=sample MFI-PCR negative control MFI (NET MFI less than 0 with 0 the expression);
3. satisfy the data of above two conditions, calculate sudden change ratio by following formula:
Sudden change ratio=mutant NET MFI ÷ (mutant NET MFI+ wild-type NET MFI)
4. rule of thumb the sudden change ratio of each detection site is determined threshold value (cut-off value), to divide wild-type homozygote, heterozygote and mutant homozygote.
Use present method to detect 20 increments IL-23R gene polymorphism sites originally, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Being provided with of threshold value (cut-off value) is as follows: the sudden change ratio range is considered as the wild-type homozygote at 0%-20%; 30%-70% is considered as heterozygote; 80%-100% is considered as the anomaly homozygote.Compare with the liquid-phase chip result with the sequencing detection, calculate the identical rate of classifying method detected result provided by the present invention.Present method detects 20 increments IL-23R genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.As seen IL-23R gene pleiomorphism provided by the present invention detects liquid-phase chip can detect IL-23R gene polymorphism sites type exactly, and the result is reliable and stable.
Table 4 pattern detection result (MFI)
Table 5 sample IL-23R transgenation ratio (%)
Table 6 sample IL-23R gene mutation type analytical results
| Catalogue number(Cat.No.) | The liquid-phase chip detected result | Sequencing result |
| 1 | Wild-type | Wild-type |
| 2 | Wild-type | Wild-type |
| 3 | Wild-type | Wild-type |
| 4 | Wild-type | Wild-type |
| 5 | 82GG | 82GG |
| 6 | Wild-type | Wild-type |
| 7 | Wild-type | Wild-type |
| 8 | 214TT | 214TT |
| 9 | Wild-type | Wild-type |
| 10 | Wild-type | Wild-type |
| 11 | 53CT | 53CT |
| 12 | Wild-type | Wild-type |
| 13 | Wild-type | Wild-type |
| 14 | Wild-type | Wild-type |
| 15 | 235CT | 235CT |
| 16 | 53TT | 53TT |
| 17 | Wild-type | Wild-type |
| 18 | Wild-type | Wild-type |
| 19 | Wild-type | Wild-type |
| 20 | Wild-type | Wild-type |
The liquid-phase chip of the ASPE primer that embodiment 3 is different is to the detection of IL-23R gene polymorphism sites
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
Detecting liquid-phase chip with IL-23R gene C 235T and T82G site mutation is example, respectively at the wild-type of C235T and T82G and the specific primer sequence of mutant design ASPE primer 3 ' end, the Tag sequence of ASPE primer 5 ' end then is selected from SEQ ID NO.1-SEQ ID NO.8, accordingly, bag is selected from SEQ ID NO.17-SEQ ID NO.24 by the anti-tag sequence with corresponding tag sequence complementary pairing on microballoon.Specific design is shown in following table (table 7).Synthetic, the anti-tag sequence bag of ASPE primer is described like embodiment 1 and embodiment 2 by microballoon, amplimer, detection method.
The design of table 7 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned design preparation, by embodiment 2 described testing processes and method sample 21-40 is detected, detected result is as follows:
Table 8 pattern detection result and Polymorphism Analysis
Table 9 pattern detection result and Polymorphism Analysis
Other is at the liquid-phase chip in different mutational sites, and the ASPE primer uses different Tag sequences, and its result is still reliable and stable, and concrete data are omitted.And the ASPE primer is when selecting among the embodiment 1 collocation of tag sequence and specific primer sequence for use, and effect better (signal to noise ratio is better) is referring to present embodiment test group 2 and test group 5.Other different tag sequences and specific primer sequence collocation, with coming to the same thing of embodiment 2 and present embodiment, concrete data are omitted.
The selection of embodiment four IL-23R gene pleiomorphism detection specificity primer sequences
One, the design (selection of wild-type and mutant specific primer sequence) of liquid-phase chip preparation
Detecting liquid-phase chip with IL-23R gene C 214T and C53T site mutation is example, respectively at the wild-type of C214T and C53T and the specific primer sequence of mutant design ASPE primer 3 ' end, comprise preferred specific primer sequence and two alternative specific primer sequences in the embodiment of the invention 1, as shown in table 10.Wherein,
Interior base is a pleomorphism site.
Table 10 specific primer sequence
Detecting liquid-phase chip with IL-23R gene C 214T and C53T site mutation is example, respectively at the wild-type of C214T and C53T and the specific primer sequence of mutant design ASPE primer 3 ' end, the Tag sequence of ASPE primer 5 ' end then is fixed as the best effect sequence among the embodiment 1, and select corresponding with it anti-tag sequence for use, specific design is shown in following table (table 11).Synthetic, the anti-tag sequence bag of ASPE primer is described like embodiment 1 and embodiment 2 by microballoon, amplimer, detection method.
Two of the design of table 11 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned design preparation, by embodiment 2 described testing processes and method sample 41-60 is detected, detected result is as follows:
Table 12 pattern detection result and Polymorphism Analysis
Table 13 pattern detection result and Polymorphism Analysis
Other is at the liquid-phase chip in different mutational sites, and the ASPE primer uses different specific primer sequences, and its result is still reliable and stable, and concrete data are omitted.And the ASPE primer is when selecting among the embodiment 1 collocation of specific primer sequence and tag sequence for use, and effect better (signal to noise ratio is better) is referring to present embodiment test group 7 and test group 10.Other different specific primer sequences and the collocation of tag sequence, with coming to the same thing of embodiment 2 and present embodiment, concrete data are omitted.
More than be at the specifying of possible embodiments of the present invention, but this embodiment is not in order to limiting claim of the present invention, does not allly break away from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the claim of the present invention.
Claims (6)
1. an IL-23R gene pleiomorphism detects liquid-phase chip, it is characterized in that, mainly includes:
A. wild-type and the mutant specificity ASPE primer that designs respectively at the different pleomorphism sites of IL-23R gene, every kind of ASPE primer is made up of the tag sequence of 5 ' end and the specific primer sequence at the goal gene pleomorphism site of 3 ' end, and described specific primer sequence is selected from: at the SEQ ID NO.9 of C214T and SEQ ID NO.10, at the SEQ ID NO.11 of C235T and SEQ ID NO.12, at the SEQ ID NO.13 of T82G and SEQ ID NO.14, at more than a pair of among the SEQ ID NO.15 of C53T and the SEQ ID NO.16; Described tag sequence is selected from the sequence among SEQ ID NO.1～SEQ IDNO.8;
B. be coated with respectively special anti-tag sequence, have a different colours coding microball, described anti-tag sequence is selected from the sequence among SEQ IDNO.17～SEQ ID NO.24, and described anti-tag sequence can be correspondingly with A in selected tag sequence complementary pairing, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon;
C. be used for increasing have C214T, the primer of the target sequence of the above pleomorphism site of C235T, T82G and C53T.
2. IL-23R gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that described amplimer is selected from: at the SEQ ID NO.25 of C214T and SEQ ID NO.26, at the SEQ ID NO.27 of C235T and SEQ IDNO.28, at the SEQ ID NO.29 of T82G and SEQ ID NO.30, at more than a pair of among the SEQ ID NO.31 of C53T and the SEQID NO.32.
3. IL-23R gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that described specificity ASPE primer is selected from: the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.9 at C214T reaches the sequence of being made up of SEQ ID NO.2 and SEQID NO.10, at the sequence of forming by SEQ ID NO.3 and SEQ ID NO.11 of C235T and by SEQID NO.4 and and the sequence formed of SEQ ID NO.12, the sequence of being made up of SEQ ID NO.5 and SEQ ID NO.13 at T82G reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.14, more than a pair of in the sequence of forming at the sequence of forming by SEQ ID NO.7 and SEQ ID NO.15 of C53T and by SEQ ID NO.8 and SEQ ID NO.16.
4. IL-23R gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that,
A. described ASPE primer is: the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.9 at C214T reaches the sequence of being made up of SEQ IDNO.2 and SEQ ID NO.10, at the sequence of forming by SEQ ID NO.3 and SEQ ID NO.11 of C235T and by SEQ ID NO.4 and and the sequence formed of SEQ ID NO.12, the sequence of being made up of SEQ ID NO.5 and SEQ ID NO.13 at T82G reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.14, reach the sequence of forming by SEQ ID NO.8 and SEQ ID NO.16 with the sequence of forming by SEQ ID NO.7 and SEQ ID NO.15 at C53T.
B. be coated with respectively special anti-tag sequence, have a different colours coding microball, described anti-tag sequence is selected from the sequence among SEQ IDNO.17～SEQ ID NO.24, and described anti-tag sequence can be correspondingly with A in selected tag sequence complementary pairing, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon;
C. described amplimer is: at the SEQ ID NO.25 of C214T and SEQ ID NO.26, at the SEQ ID NO.27 of C235T and SEQ ID NO.28, at the SEQ ID NO.29 of T82G and SEQ ID NO.30 with at SEQ ID NO.31 and the SEQ ID NO.32 of C53T.
5. detect liquid-phase chip according to each described IL-23R gene pleiomorphism of claim 1-4, it is characterized in that described spacerarm sequence is 5-10 T.
6. one kind is used for the specific primer sequence that the IL-23R gene pleiomorphism detects, it is characterized in that described specific primer sequence is selected from: at the SEQ ID NO.9 of C214T and SEQ ID NO.10, at the SEQ ID NO.11 of C235T and SEQ ID NO.12, at the SEQ ID NO.13 of T82G and SEQ ID NO.14, at more than a pair of among the SEQ ID NO.15 of C53T and the SEQ ID NO.16.
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