CN102559850A - ApoA5 genic mutation detection specific primer and liquid phase chip - Google Patents
ApoA5 genic mutation detection specific primer and liquid phase chip Download PDFInfo
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Abstract
The invention discloses an apoA5 genic mutation detection specific primer and a liquid phase chip, wherein the liquid phase chip mainly includes an ASPE primer formed by 5'end tag sequence and 3'end specific primers, and the specific primers are SEQ ID NO.13 and SEQ ID NO.14 for the mutation of A134G, SEQ ID NO.15 and SEQ ID NO.16 for the mutation of T63C, SEQ ID NO.17 and SEQ ID NO.18 for the mutation of C41G, SEQ ID NO.19 and SEQ ID NO.20 for the mutation of G54T, SEQ ID NO.21 and SEQ ID NO.22 for the mutation of A81G, and/or SEQ ID NO.23 and SEQ ID NO.24 for the mutation of G194A; a microsphere enveloped by anti-tag sequence; and an amplimer. The matching degree of the detection result of the apoA5 genic mutation detection liquid phase chip and the sequencing method is 100%, and the parallel detection of multiple polymorphic sites can be achieved.
Description
Technical field
The present invention bends in biology field, relates to medical science and biotechnology, concrete a kind of apoA5 gene test Auele Specific Primer and the liquid-phase chip of relating to.
Background technology
(apolipoprotein A5 gene apoA5), is the lipophorin family member to ApoA5; This assignment of genes gene mapping is in No. 11 long-armed q23 of karyomit(e) districts (11q23), with the apoA1/C3/A4 gene cluster at a distance of about 30kb, full length gene 1889bp; This gene has 4 exons, 2 introns and 4 silencers; The protein that a kind of 363 amino acid of encoding is formed, (triglyceride, TG) level has significant effects to plasma triglyceride level.The relation of plasma TG metabolism and cardiovascular disorder is very close, and NCBI has included more than 20 SNP site at present, the apoA5 gene mutation site of target detect of the present invention, and it is as shown in the table:
| Sequence number | The content of apoA5 site mutation | Write a Chinese character in simplified form |
| 1 | A → G sudden change takes place in the 134th Nucleotide of SEQ ID NO.49 | A134G |
| 2 | T → C sudden change takes place in the 63rd Nucleotide of SEQ ID NO.50 | T63C |
| 3 | C → G sudden change takes place in the 41st Nucleotide of SEQ ID NO.51 | C41G |
| 4 | G → T sudden change takes place in the 54th Nucleotide of SEQ ID NO.52 | G54T |
| 5 | A → G sudden change takes place in the 81st Nucleotide of SEQ ID NO.53 | A81G |
| 6 | G → A sudden change takes place in the 194th Nucleotide of SEQ ID NO.54 | G194A |
At present, the method that the apoA5 gene pleiomorphism is detected, analyzes is a lot, as: direct sequencing, PCR-RFLP analytical method or the like, wherein the most frequently used is the PCR-RFLP analytical method.The PCR-RFLP method is based on the change of the restriction enzyme enzyme recognition site that transgenation causes; Lose or produce novel site like the site,, use the digestion with restriction enzyme amplified production again through a certain particular segment of pcr amplification; The segmental size of electrophoresis observation; This method is used to detect the transgenation that restriction enzyme site changes, and can directly judge genotype, but this method can not be used for not producing the detection in Gene Mutation of new restriction enzyme site.Once more, these methods all exist the limitation that detects flux, 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 the object of the invention provides the apoA5 gene mutation detection liquid-phase chip.This liquid-phase chip can be used for detecting wild-type and the mutant of apoA5 gene six kinds of common genotype A134G, T63C, C41G, G54T, A81G and G194A.
A kind of apoA5 gene mutation detection liquid-phase chip mainly includes:
(A). the wild-type and the ASPE primer of mutant that design respectively to every kind of sudden change: every kind of ASPE primer is made up of to the Auele Specific Primer of goal gene sudden change the tag sequence and the 3 ' end of 5 ' end, and the Auele Specific Primer of said wild-type and mutant is respectively: to the SEQ ID NO.13 of A134G sudden change and SEQ ID NO.14, to the SEQ ID NO.15 of T63C sudden change and SEQ ID NO.16, to the SEQ ID NO.17 of C41G sudden change and SEQ ID NO.18, the SEQ ID NO.21 and the SEQ ID NO.22 that suddenly change to the SEQID NO.19 of G54T sudden change and SEQ ID NO.20, to A81G and/or be directed against SEQ ID NO.23 and the SEQ ID NO.24 that G194A suddenlys change; Said tag sequence is selected from SEQ ID NO.1-12;
(B). that the anti-tag sequence encapsulates, as to have different colours coding microballoon is arranged, also be provided with the spacerarm sequence in the middle of said anti-tag sequence is connected with microballoon; Said anti-tag sequence is selected from the sequence among SEQ ID NO.25～SEQ ID NO.36, and said anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). be used to amplify the primer of needs target sequence that detect, that have the mutational site.
Preferably, said amplimer is: to the SEQ ID NO.37 of A134G sudden change and SEQ ID NO.38, to the SEQ ID NO.39 of T63C sudden change and SEQ ID NO.40, to the SEQ ID NO.41 of C41G sudden change and SEQ ID NO.42, to the SEQ ID NO.43 of G54T sudden change and SEQ ID NO.44, to the SEQ ID NO.45 of A81G sudden change and SEQ IDNO.46 and/or to the SEQ ID NO.47 and the SEQ ID NO.48 of G194A sudden change.
Preferably, said ASPE primer is: the sequence that the sequence that sequence of being made up of SEQ ID NO.1 and SEQ ID NO.13 and the sequence of being made up of SEQ IDNO.2 and SEQ ID NO.14, the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.15 and the sequence of being made up of SEQ IDNO.4 and SEQ ID NO.16, the sequence of being made up of SEQ ID NO.5 and SEQ ID NO.17 reach the sequence be made up of SEQ IDNO.6 and SEQ ID NO.18, be made up of SEQ ID NO.7 and SEQ ID NO.19 reaches the sequence be made up of SEQ IDNO.8 and SEQ ID NO.20, be made up of SEQ ID NO.9 and SEQ ID NO.21 reaches the sequence be made up of SEQ IDNO.10 and SEQ ID NO.22 and/or reaches the sequence of being made up of SEQ ID NO.12 and SEQ ID NO.24 by the sequence that SEQ ID NO.11 and SEQ ID NO.23 form.
Another object of the present invention provides a kind of Auele Specific Primer of the apoA5 of being used for detection in Gene Mutation.
Concrete technical scheme is following:
A kind of Auele Specific Primer that is used for the apoA5 detection in Gene Mutation includes: to the SEQ ID NO.13 of A134G sudden change and SEQ ID NO.14, to the SEQ ID NO.15 of T63C sudden change and SEQ ID NO.16, to the SEQID NO.17 of C41G sudden change and SEQ ID NO.18, to the SEQ ID NO.19 of G54T sudden change and SEQ ID NO.20, to the SEQ ID NO.21 of A81G sudden change and SEQ ID NO.22 and/or to the SEQ ID NO.23 and the SEQ ID NO.24 of G194A sudden change.
Major advantage of the present invention is:
1. the identical rate of the detected result of apoA5 gene mutation detection liquid-phase chip provided by the present invention and PCR sequencing PCR is up to 100%, and detects the needed time well below sequencing technologies commonly used, meets the practical application needs especially.Since in very a large amount of Auele Specific Primers, through test in a large number, the reaction checking; Obtain the liquid-phase chip system of optimum combination; Prepared liquid-phase chip has extraordinary signal-NR, and does not have cross reaction basically between institute's designed probe and the anti-tag sequence, 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 mutational sites.
2. the genotype of various types is accurately distinguished in the mutational site that designed ASPE primers Auele Specific Primer of the present invention 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 polymorphum 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; Six kinds of pleomorphism sites detect and can accomplish two amplifications that contain the target sequence in SNP site through 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 characteristic 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 the different detection project, has very strong expansion.The fluorescent signal value that detects improves greatly, thereby makes the sensitivity that detects be further enhanced, and SNR strengthens, and detected result more accurately and reliably.
Embodiment
Embodiment 1 said apoA5 gene mutation detection liquid-phase chip mainly includes:
One, ASPE primer
To wild-type and the mutant of six kinds of common genotype A134G, T63C, C41G, G54T, A81G and G194A of apoA5 gene, design specific primers 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 1apoA5 gene
Every the ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence to 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 that encapsulates of anti-tag sequence
According to institute's 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 possibly form, corresponding anti-tag sequence is as shown in table 2 on 12 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
12 kinds of microballoons selecting are available from U.S. Luminex company, with the anti-tag sequence encapsulate with microballoon on.Be connected with the spacerarm sequence of 5-10 T between anti-tag sequence and the microballoon, promptly before each anti-tag sequence, add the spacerarm sequence of the preceding paragraph 5-10 T, the anti-tag sequence is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Synthetic anti-tag sequence is used sterilization ddH
2O is made into the stock solution of 100nmol/ml.Said spacerarm is to be used for anti-tag and microballoon table are drawn spaced apart or anti-tag placed the sequence of hydrophilic environments.Through 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 like (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 that microballoon encapsulates is following:
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
To six kinds of common genotype A134G, T63C, C41G, G54T, A81G and G194A of apoA5 gene, design of amplification primers amplifies six 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 embodiment 1 said apoA5 gene test liquid-phase chip is to the detection of sample
The prescription of said various solution is following:
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 | Sigma?T3038 | 0.2M | 50ml |
| 5M?NaCl | 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 " method involving, obtain DNA to be detected about DNA extraction.
Two, the pcr amplification of testing sample
Design six pairs of primers; Multiplex PCR one step amplifies five kinds of common genotype A134G, T63C, C41G, G54T, A81G and G194A totally six the target sequence that contains the apoA5 gene respectively; The product size is respectively 281bp, 325bp, 185bp, 154bp, 376bp and 483bp, and primer sequence (SEQ ID NO.37-48) 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.37-48 respectively mixes and is the multiple PCR primer working fluid in the 1.5ml Eppendorf tube.The multi-PRC reaction system is following:
2 * damping fluid (contains Mg
2+) 25ul
DNTP (each 2.5mmol/L) 4ul
Taq enzyme (5U/ul) 0.2ul
Multiple PCR primer working fluid (each 8.3pmol/mL) 6ul
Template DNA (10ng/ul) 2ul
ddH
2O 12.8ul
Be total to 50ul
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 subsequent use.
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 directly is used for follow-up ASPE primer extension reaction.
Four, site-specific primer extension reaction (ASPE)
Utilize above-mentioned designed ASPE primers 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 apoA5 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 following:
10 * damping fluid 2ul
MgCl
2(50mmol/L) 0.5ul
Biotin-dCTP(400umol/L) 0.25ul
DATP, dGTP, dTTP mixed solution (each 100umol/L) 1ul
Tsp enzyme (5U/ul) 0.25ul
Blended ASPE primer working fluid (each 500nmol/L) 1ul
Enzyme is cut the pcr amplification product 5ul of processing
ddH
2O 10ul
Be total to 20ul
Response procedures is: 96 ℃ of 2min; 94 ℃ of 30s, 54 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ of preservations are subsequent use.
Five, hybridization
1. according to designed ASPE primers, (microballoon concentration is 2.5 * 10 to the corresponding 12 kinds of microballoons of every group selection
5Individual/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 through 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 confirmed threshold value (cut-off value), to divide wild-type homozygote, heterozygote and mutant homozygote.
Use present method to detect 20 increments apoA5 gene polymorphism sites originally, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Being provided with as follows of threshold value (cut-off value): the sudden change ratio range is regarded as the wild-type homozygote at 0%-20%; 30%-70% is regarded as heterozygote; 80%-100% is regarded as the anomaly homozygote.Compare with the liquid-phase chip result with the PCR sequencing PCR detection, calculate the identical rate of classifying method detected result provided by the present invention.Present method detects 20 increments apoA5 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.Can detect apoA5 gene polymorphism sites type exactly it is thus clear that apoA5 gene pleiomorphism provided by the present invention detects liquid-phase chip, and the result is reliable and stable.
One of table 4 pattern detection result (MFI)
Table 5 pattern detection result's two (MFI)
Table 6 sample apoA5 transgenation ratio (%)
Table 7 sample apoA5 gene mutation type analytical results
| Catalogue number(Cat.No.) | The liquid-phase chip detected result | Sequencing result |
| 1 | 54TT | 54TT |
| 2 | Wild-type | Wild-type |
| 3 | 134AG | 134AG |
| 4 | Wild-type | Wild-type |
| 5 | 63GG | 63GG |
| 6 | 81GG | 81GG |
| 7 | Wild-type | Wild-type |
| 8 | 134GG | 134GG |
| 9 | Wild-type | Wild-type |
| 10 | 41GG | 41GG |
| 11 | 54TT、194GA | 54TT、194GA |
| 12 | Wild-type | Wild-type |
| 13 | Wild-type | Wild-type |
| 14 | 41CG | 41CG |
| 15 | 134GG | 134GG |
| 16 | Wild-type | Wild-type |
| 17 | Wild-type | Wild-type |
| 18 | Wild-type | Wild-type |
| 19 | 81GG | 81GG |
| 20 | Wild-type | Wild-type |
The liquid-phase chip of the ASPE primer that embodiment 3 is different is to the detection of apoA5 gene polymorphism sites
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
Detecting liquid-phase chip with apoA5 Gene A 134G and C41G site mutation is example; Respectively to the wild-type of A134G and C41G 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.12; Accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing that encapsulates on microballoon is selected from SEQ ID NO.25-SEQ ID NO.36.Specifically design shown in following table (table 8).It is said like embodiment 1 and embodiment 2 that synthetic, the anti-tag sequence of ASPE primer encapsulates microballoon, amplimer, detection method.
The design of table 8 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned designing and preparing, by embodiment 2 said testing processes and method sample 21-40 is detected, detected result is following:
Table 9 sample A134G detected result and Polymorphism Analysis
Table 10 sample C41G detected result and Polymorphism Analysis
Other is to 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 (SNR is better) is referring to present embodiment test group 1 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.
More than be to 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 apoA5 gene mutation detection liquid-phase chip is characterized in that, mainly includes:
(A). the wild-type and the ASPE primer of mutant that design respectively to every kind of sudden change: every kind of ASPE primer is made up of to the Auele Specific Primer of goal gene sudden change the tag sequence and the 3 ' end of 5 ' end, and the Auele Specific Primer of said wild-type and mutant is respectively: to the SEQ ID NO.13 of A134G sudden change and SEQ ID NO.14, to the SEQ ID NO.15 of T63C sudden change and SEQID NO.16, to the SEQ ID NO.17 of C41G sudden change and SEQ ID NO.18, the SEQ ID NO21 and the SEQ ID NO.22 that suddenly change to the SEQ IDNO.19 of G54T sudden change and SEQ ID NO.20, to A81G and/or be directed against SEQ ID NO.23 and the SEQ ID NO.24 that G194A suddenlys change; Said tag sequence is selected from SEQ ID NO.1-12;
(B). that the anti-tag sequence encapsulates, as to have different colours coding microballoon is arranged, also be provided with the spacerarm sequence in the middle of said anti-tag sequence is connected with microballoon; Said anti-tag sequence is selected from the sequence among SEQ ID NO.25～SEQ ID NO.36, and said anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). be used to amplify the primer of needs target sequence that detect, that have the mutational site.
2. apoA5 gene mutation detection liquid-phase chip according to claim 1; It is characterized in that said amplimer is: to the SEQ ID NO.37 of A134G sudden change and SEQ ID NO.38, to the SEQ ID NO.39 of T63C sudden change and SEQ IDNO.40, to the SEQ ID NO.41 of C41G sudden change and SEQ ID NO.42, to the SEQ ID NO.43 of G54T sudden change and SEQ ID NO.44, to the SEQ ID NO.45 of A81G sudden change and SEQ ID NO.46 and/or to the SEQ ID NO.47 and the SEQ ID NO.48 of G194A sudden change.
3. apoA5 gene mutation detection liquid-phase chip according to claim 1; It is characterized in that said ASPE primer is: the sequence that the sequence that sequence of being made up of SEQ ID NO.1 and SEQ ID NO.13 and the sequence of being made up of SEQ ID NO.2 and SEQ ID NO.14, the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.15 and the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.16, the sequence of being made up of SEQ ID NO.5 and SEQ ID NO.17 reach the sequence be made up of SEQ ID NO.6 and SEQ ID NO.18, be made up of SEQ ID NO.7 and SEQ ID NO.19 reaches the sequence be made up of SEQ ID NO.8 and SEQ ID NO.20, be made up of SEQ ID NO.9 and SEQ ID NO.21 reaches the sequence be made up of SEQ ID NO.10 and SEQ ID NO.22 and/or reaches the sequence of being made up of SEQ ID NO.12 and SEQ ID NO.24 by the sequence that SEQ ID NO.11 and SEQ ID NO.23 form.
4. apoA5 gene mutation detection liquid-phase chip according to claim 1 is characterized in that,
(A). said ASPE primer is: the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.13 reaches the sequence of being made up of SEQ ID NO.2 and SEQ ID NO.14; The sequence of being made up of SEQ ID NO.3 and SEQ ID NO.15 reaches the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.16; The sequence of being made up of SEQ ID NO.5 and SEQ ID NO.17 reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.18; The sequence of being made up of SEQ ID NO.7 and SEQ ID NO.19 reaches the sequence of being made up of SEQ ID NO.8 and SEQ ID NO.20; The sequence of being made up of SEQ ID NO.9 and SEQ ID NO.21 reaches the sequence of being made up of SEQ ID NO.10 and SEQ ID NO.22; Reach the sequence of forming by SEQ IDNO.12 and SEQ ID NO.24 with the sequence of forming by SEQ ID NO.11 and SEQ ID NO.23;
(B). that the anti-tag sequence encapsulates, as to have different colours coding microballoon is arranged, also be provided with the spacerarm sequence in the middle of said anti-tag sequence is connected with microballoon; Said anti-tag sequence is selected from the sequence among SEQ ID NO.25～SEQ ID NO.36, and said anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). said amplimer is: to the SEQ ID NO.37 of A134G sudden change and SEQ ID NO.38, to the SEQ ID NO.39 of T63C sudden change and SEQ ID NO.40, to the SEQ ID NO.41 of C41G sudden change and SEQ ID NO.42, to the SEQ ID NO.43 of G54T sudden change and SEQ ID NO.44, to the SEQ ID NO.45 of A81G sudden change and SEQ IDNO.46 with to the SEQ ID NO.47 and the SEQ ID NO.48 of G194A sudden change.
5. according to each described apoA5 gene mutation detection liquid-phase chip of claim 1-4, it is characterized in that said spacerarm is 5-10 T.
6. Auele Specific Primer that is used for the apoA5 detection in Gene Mutation; It is characterized in that, include: to the SEQ ID NO.13 of A134G sudden change and SEQ ID NO.14, to the SEQ ID NO.15 of T63C sudden change and SEQ ID NO.16, to the SEQ ID NO.17 of C41G sudden change and SEQ ID NO.18, to the SEQ ID NO.19 of G54T sudden change and SEQ ID NO.20, to the SEQ ID NO.21 of A81G sudden change and SEQ ID NO.22 and/or to the SEQ ID NO.23 and the SEQID NO.24 of G194A sudden change.
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