CN102533953A - Specific primers and liquid-phase chip for SNP (Single Nucleotide Polymorphism) detection of LPL gene - Google Patents
Specific primers and liquid-phase chip for SNP (Single Nucleotide Polymorphism) detection of LPL gene Download PDFInfo
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Abstract
The invention discloses specific primers and a liquid-phase chip for SNP (Single Nucleotide Polymorphism) detection of an LPL gene. The liquid-phase chip comprises an ASPE primer, anti-tag sequence coated microspheres, and an amplification primer, wherein the ASPF primer is composed of a 5'-terminal tag sequence and 3'-terminal specific primers for target gene mutation, and the specific primers are respectively SEQ ID NO. 9 and SEQ ID NO. 10 specific for a T161G SNP site, SEQ ID NO. 11 and SEQ ID NO. 12 specific for a C120G SNP site, SEQ ID NO. 13 and SEQ ID NO. 14 specific for a G113A SNP site, and/or SEQ ID NO. 15 and SEQ ID NO. 16 specific for an A178G SNP site. According to the invention, the coincidence rate between the detection result of the liquid-phase chip for SNP detection of the LPL gene and the detection result of a sequencing method is up to 100%, not only is the condition of single site mutation detected, but also the polymorphism conditions of multiple mutant sites can be simultaneously detected in parallel, and the detection effects are consistent.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete a kind of lpl gene SNP detection specificity primer and the liquid-phase chip of relating to.
Background technology
(lipoprteinlipase is the synthetic and a kind of gp of excretory of parenchymas such as adipocyte, myocardial cell, Skeletal Muscle Cell, mammary gland cell and scavenger cell LPL) to LPL, and molecular weight is 60ku, contains the 3%-8% glucide.The LPL physiological function is that the TG of catalysis CM and VLDL core is decomposed into lipid acid and monoglyceride, for tissue oxidizing energy supply and storage.LPL also participates in lipophorin and the conversion of phosphatide between VLDL and the HDL, and ApoC II is the indispensable cofactor of LPL, and its C holds the 61-79 amino acids to have the effect that activates LPL.
Lpl gene is positioned at the 8th the short arm of a chromosome 8p22, is about 35kb, is made up of the protein of 475 amino-acid residues of coding 10 exons and 9 introns.Research at present shows that the physiological function of LPL is to decompose the triglyceride level of lipoprotein nuclear composition; Also decompose phosphatide such as Yelkin TTS, phosphatidylethanolamine; And impel and shift SUV, phosphatide and lipophorin between the lipoprotein; Its meta-bolites free fatty acids provides energy for tissue, or resterification is TG, is stored in the fatty tissue.There is polymorphum in the lpl gene site, mainly is distributed in lpl gene intron and the flanking sequence, and the existence of these pleomorphism sites directly influences the function of lipoprotein lipase.
The lpl gene mutational site of target detect of the present invention, it is as shown in the table:
| Sequence number | The content of LPL site mutation | Write a Chinese character in simplified form |
| 1 | T → G sudden change takes place in the 161st Nucleotide of SEQ ID NO.33 | T161G |
| 2 | C → G sudden change takes place in the 120th Nucleotide of SEQ ID NO.34 | C120G |
| 3 | G → A sudden change takes place in the 113rd Nucleotide of SEQ ID NO.35 | G113A |
| 4 | A → G sudden change takes place in the 178th Nucleotide of SEQ ID NO.36 | A178G |
At present, the method that the lpl gene polymorphum is detected, analyzes mainly contains: methods such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), direct sequencing, Taqman technology, wherein the most frequently used method has 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, the detection method (PCR-RFLP) of PCR-based technology and all exist the limitation that detects flux based on the allelotrope discriminant analysis of TaqMan technology once can only be carried out a kind of detection of sudden change, can not satisfy the needs of practical application.
Summary of the invention
One of the object of the invention provides lpl gene SNP and detects liquid-phase chip.This liquid-phase chip can be used for detecting wild-type and the mutant of lpl gene four kinds of common genotype T161G, C120G, G113A and A178G.
Concrete technical scheme is following:
A kind of lpl gene SNP detects liquid-phase chip, mainly includes:
(A). the wild-type and the ASPE primer of mutant that design respectively to every kind of SNP: every kind of ASPE primer is made up of to the Auele Specific Primer in goal gene SNP site the tag sequence and the 3 ' end of 5 ' end, and the Auele Specific Primer of said wild-type and mutant is respectively: to SEQ ID NO.9 and SEQ ID NO.10, the SEQID NO.11 that is directed against C120G SNP site and SEQID NO.12, the SEQIDNO.13 that is directed against G113A SNP site and the SEQ ID NO.14 in T161G SNP site and/or be directed against the SEQ ID NO.15 and the SEQ ID NO.16 in A178G SNP site; Said tag sequence is selected from SEQ ID NO.1-8;
(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.17～SEQ IDNO.24, and said anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). be used to amplify needs primer that detect, that have the target sequence in corresponding SNP site.
Preferably, said amplimer is: to the SEQ ID NO.25 in T161G SNP site and SEQ ID NO.26, to the SEQID NO.27 in C 120G SNP site and SEQID NO.28, to the SEQIDNO.29 in G113A SNP site and SEQID NO.30 and/or to the SEQID NO.31 and the SEQID NO.32 in A178G SNP site.
Preferably, said ASPE primer is: the sequence that sequence of being made up of SEQ ID NO.1 and SEQ ID NO.9 and the sequence of being made up of SEQ IDNO.2 and SEQID NO.10, the sequence of being made up of SEQID NO.3 and SEQID NO.11 reach the sequence be made up of SEQID NO.4 and SEQID NO.12, be made up of SEQID NO.5 and SEQID NO.13 reaches the sequence be made up of SEQID NO.6 and SEQID NO.14 and/or reaches the sequence of being made up of SEQ ID NO.8 and SEQ ID NO.16 by the sequence that SEQID NO.7 and SEQID NO.15 form.
Another object of the present invention provides a kind of Auele Specific Primer that lpl gene SNP detects that is used for.
Concrete technical scheme is following:
A kind of Auele Specific Primer that is used for lpl gene SNP detection; Include, to the SEQID NO.9 in T161G SNP site and SEQID NO.10, to the SEQID NO.11 in C 120G SNP site and SEQID NO.12, to the SEQ ID NO.13 in G113A SNP site and SEQ ID NO.14 and/or to the SEQID NO.15 and the SEQID NO.16 in A178G SNP site.
Major advantage of the present invention is:
1. the detected result that lpl gene SNP provided by the present invention detects liquid-phase chip and the identical rate of PCR sequencing PCR be up to 100%, and detect the needed time well below the sequencing technologies of using always, 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 SNP site.
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; Four kinds of SNPs detect and can accomplish four 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 1LPL gene SNP detection liquid-phase chip mainly includes:
One, ASPE primer
To wild-type and the mutant of four kinds of common genotype T161G, C 120G, G113A and the A178G of lpl 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 1LPL 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/LTris 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 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 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 microsphere surface is 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), oligomerization four polyoxyethylene glycol and (CH
2)
nSpacerarm (n>=3) is like (CH
2)
12, (CH
2)
18Deng.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 50ul0.1mol/L (pH4.5), adds 10ul synthetic anti-tag molecule (100nmol/mL).EDC [1-Ethyl-(3-dimethylaminopropyl) carbodiimide] (available from PierceChemical 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/L EDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains the mutational site
To 4 kinds of common genotype T161G, C 120G, G113A and the A178G of lpl gene, design of amplification primers amplifies 4 target sequences that contain the SNP site respectively to (seeing table 3) respectively.
Table 3 amplifies the primer of the target sequence with SNP 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 lpl genes detect the detection of liquid-phase chip to 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 | SigmaT3038 | 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 four pairs of primers, multiplex PCR one step amplifies four kinds of common genotype T 161G, C 120G, G113A and A178G totally four the target sequence that contains lpl gene respectively, and the sequence fragment size is respectively 278bp, 299bp, 227bp, 282bp.Primer sequence (SEQ ID 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 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: 4 kinds of common genotype T 161G that get lpl gene to be detected respectively; C 120G; G113A; A178G corresponding wild type and mutant ASPE primer stock solution 10ul add 10mmol/L Tris Buffer and mend to 200ul in the 1.5ml Eppendorf tube, mix and are ASPE mix primer working fluid.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, (every kind of microballoon concentration is 2.5 * 10 to the corresponding 8 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 lpl gene SNP site 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 lpl gene type detected result and the sequencing result rate of coincideing originally and reaches 100%.It is thus clear that lpl gene SNP provided by the present invention detects the SNP type that liquid-phase chip can detect LPL exactly, and the result is reliable and stable.
Table 4 pattern detection result (MFI)
Table 5 sample lpl gene sudden change ratio (%)
Table 6 sample lpl gene mutation type analytical results
| Catalogue number(Cat.No.) | The liquid-phase chip detected result | Sequencing result |
| 1 | Wild-type | Wild-type |
| 2 | 1421CG | 1421CG |
| 3 | Wild-type | Wild-type |
| 4 | Wild-type | Wild-type |
| 5 | 953GG | 953GG |
| 6 | Wild-type | Wild-type |
| 7 | 1322+483GG | 1322+483GG |
| 8 | Wild-type | Wild-type |
| 9 | Wild-type | Wild-type |
| 10 | 1421GG | 1421GG |
| 11 | Wild-type | Wild-type |
| 12 | Wild-type | Wild-type |
| 13 | Wild-type | Wild-type |
| 14 | Wild-type | Wild-type |
| 15 | 1322+483TG | 1322+483TG |
| 16 | 106AA | 106AA |
| 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 in lpl gene SNP site
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
Detecting liquid-phase chip with lpl gene C 120G site mutation is example; Respectively to the wild-type of C 120G 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, the anti-tag sequence with corresponding tag sequence complementary pairing that encapsulates on microballoon is selected from SEQ ID NO.17-SEQ ID NO.24.Specifically design shown in following table (table 7).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 7 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 8 pattern detection 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 2.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. a lpl gene SNP detects liquid-phase chip, it is characterized in that, mainly includes:
(A). the wild-type and the ASPE primer of mutant that design respectively to every kind of SNP: every kind of ASPE primer is made up of to the Auele Specific Primer in goal gene SNP site the tag sequence and the 3 ' end of 5 ' end, and the Auele Specific Primer of said wild-type and mutant is respectively: to SEQ ID NO.9 and SEQ ID NO.10, the SEQID NO.11 that is directed against C 120G SNP site and SEQID NO.12, the SEQIDNO.13 that is directed against G113A SNP site and the SEQID NO.14 in T161G SNP site and/or be directed against the SEQID NO.15 and the SEQID NO.16 in A178G SNP site; Said tag sequence is selected from SEQ ID NO.1-8;
(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.17～SEQ IDNO.24, and said anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). be used to amplify needs primer that detect, that have the target sequence in corresponding SNP site.
2. lpl gene SNP according to claim 1 detects liquid-phase chip; It is characterized in that said amplimer is: to the SEQID NO.25 in T161G SNP site and SEQID NO.26, to the SEQIDNO.27 in C 120G SNP site and SEQID NO.28, to the SEQID NO.29 in G113A SNP site and SEQID NO.30 and/or to the SEQID NO.31 and the SEQID NO.32 in A178G SNP site.
3. lpl gene SNP according to claim 1 detects liquid-phase chip; It is characterized in that said ASPE primer is: the sequence that sequence of being made up of SEQID NO.1 and SEQID NO.9 and the sequence of being made up of SEQID NO.2 and SEQID NO.10, the sequence of being made up of SEQID NO.3 and SEQID NO.11 reach the sequence be made up of SEQID NO.4 and SEQID NO.12, be made up of SEQID NO.5 and SEQID NO.13 reaches the sequence be made up of SEQID NO.6 and SEQID NO.14 and/or reaches the sequence of being made up of SEQID NO.8 and SEQID NO.16 by the sequence that SEQID NO.7 and SEQID NO.15 form.
4. lpl gene SNP according to claim 1 detects liquid-phase chip, it is characterized in that,
(A). said ASPE primer is: the sequence of being made up of SEQID NO.1 and SEQID NO.9 reaches the sequence of being made up of SEQID NO.2 and SEQID NO.10; The sequence of being made up of SEQID NO.3 and SEQID NO.11 reaches the sequence of being made up of SEQIDNO.4 and SEQID NO.12; The sequence of being made up of SEQID NO.5 and SEQID NO.13 reaches the sequence of being made up of SEQID NO.6 and SEQID NO.14; Reach the sequence of forming by SEQID NO.8 and SEQID NO.16 with the sequence of forming by SEQID NO.7 and SEQID NO.15;
(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.17～SEQ IDNO.24, 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.25 in T161G SNP site and SEQ ID NO.26, to the SEQID NO.27 in C120G SNP site and SEQID NO.28, to the SEQIDNO.29 in G113A SNP site and SEQID NO.30 with to the SEQID NO.31 and the SEQID NO.32 in A178G SNP site.
5. according to each described apoC3 gene SNP detection liquid-phase chip of claim 1-4, it is characterized in that said spacerarm is 5-10 T.
6. one kind is used for the Auele Specific Primer that lpl gene SNP detects; It is characterized in that, include: to the SEQID NO.9 in T161G SNP site and SEQID NO.10, to the SEQID NO.11 in C 120G SNP site and SEQIDNO.12, to the SEQ ID NO.13 in G113A SNP site and SEQ ID NO.14 and/or to the SEQID NO.15 and the SEQID NO.16 in A178G SNP site.
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| CN101173310A (en) * | 2006-11-03 | 2008-05-07 | 上海主健生物工程有限公司 | Reagent kit for detecting hyperlipemia disease susceptibility with LPL |
| US20100130600A1 (en) * | 2007-03-30 | 2010-05-27 | Cedars-Sinai Medical Center | Lipoprotein lipase and its effect on statin treatments |
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| CN101130809A (en) * | 2006-08-25 | 2008-02-27 | 上海主健生物工程有限公司 | Reagent kit for detecting hyperlipemia susceptibility |
| CN101173310A (en) * | 2006-11-03 | 2008-05-07 | 上海主健生物工程有限公司 | Reagent kit for detecting hyperlipemia disease susceptibility with LPL |
| US20100130600A1 (en) * | 2007-03-30 | 2010-05-27 | Cedars-Sinai Medical Center | Lipoprotein lipase and its effect on statin treatments |
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| CN104531876A (en) * | 2014-12-31 | 2015-04-22 | 上海鼎晶生物医药科技有限公司 | Primer and kit for detecting human APOC3 gene mutation |
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