CN101812524B - Specific primer, liquid phase chip and detection method for CYP3A5 gene SNP (Single Nucleotide Polymorphism) detection - Google Patents
Specific primer, liquid phase chip and detection method for CYP3A5 gene SNP (Single Nucleotide Polymorphism) detection Download PDFInfo
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Abstract
The invention discloses a liquid phase chip for CYP3A5 gene SNP (Single Nucleotide Polymorphism) detection, which mainly comprises ASPE (Allele-Specific Primer Extension) primer pairs, microspheres and amplimers, wherein the ASPE primer pairs are respectively designed for the SNP loci of the CYP3A5 gene, and each ASPE primer comprises tag sequences on a 5' end and specific primer sequences on a 3' end to the target gene SNP loci; the microspheres are respectively coated by specific anti-tag sequences; and the amplimers are used for amplifying CYP3A5 gene target sequences with A6986G, G14690A,2713127132insT, C3699T and/or G19386A SNP loci. The invention also provides a detection method using the liquid phase chip and a specific primer for CYP3A5 gene SNP detection. The compatible rate of the detection method and a sequencing method is up to 100%. The prepared liquid phase chip for CYP3A5 gene SNP detection has favorable signal-to-noise ratio, no cross reaction exists between a designed probe and the anti-tag sequences, and parallel detection on multiple SNP loci can be realized.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete CYP3A5 gene SNP detection specific primer, liquid-phase chip and the detection method of relating to.
Technical background
(cytochrome P450 enzyme system is to participate in xenobiontics detoxifcation and the metabolic important enzyme of interior source compound system CYPs), mainly contains CYP1, CYP2, CYP3 three extended familys in cytochrome P 450 enzymes system.CYP3A5 is a kind of isozyme important in the CYP3A subtribe.Its genetic polymorphism affects many medicines through the CYP3A mediation; Like oxidative metabolisms in hepatomicrosome such as diltiazem, alfentanils; There is tangible difference between individuals in the activity of this enzyme; To pharmacological agent effect and the untoward reaction and the drug toxicity generation material impact of Different Individual, be to cause between individuality and race to one of different reason of same substrate utilization ability.It brings into play crucial effects in the interaction of medicine, and closely related with the chemotherapy of tumors resistance.
The CYP3A5 wild-type is defined as CYP3A5*1, has site 6986A and 31611C simultaneously, only carries the protein expression that the allelic individuality of at least one CYP3A5*1 just has CYP3A5.The CYP3A5 mutant is named by the morning and evening of finding in proper order, has found a plurality of mutation alleles such as CYP3A5*2, CYP3A5*3, CYP3A5*4, CYP3A5*5, CYP3A5*6, CYP3A5*7, CYP3A5*8, CYP3A5*9 so far.Wherein, (A → G) important is defined as CYP3A5*3 to be present in the SNP in the 3rd intron 6986 sites.This sudden change causes variable shearing disappearance, produces labile protein matter, thereby makes the mutant homozygote individual, and promptly genotype is that the carrier of CYP3A5*3/*3 does not express CYP3A5 albumen, the CYP3A5 enzymic activity is reduced or disappears.The occurrence frequency of CYP3A5*3 allelic mutation in each ethnic population is the highest, is the topmost polymorphum of CYP3A5 gene among the crowd, and the mutation rate of Chinese population is up to 71%-76%.There is single base mutation in 1280 sites of the 11st exon of CYP3A5, and (this site causes the tryptophane on the 398th site to become N, and produces the Tsp509 restriction enzyme site, but in asian population, does not almost find incidence for C → A), i.e. CYP3A5*2.CYP3A5*4 is the 14665A＞G sudden change that occurs in exon 7, and CYP3A5*5 is the 12952T＞C sudden change that occurs in intron 5, and both are about 1% at incidence in Chinese population; CYP3A5*6 is the 14690G＞A sudden change that occurs in the exon 7, montage is produced change, and then influence the enzyme work of CYP3A5; And CYP3A5*7 is the 27131_27132insT that occurs in the introne 3; Cause 346 reading frames to be moved; The phase shift mutation of its generation will influence the expression of CYP3A5*3, and then influence protease activity, and occur in Africa and America crowd with 10% frequency; CYP3A5*8 is the 3699C＞T sudden change on the exon 2, makes the 28th l-arginine be mutated into halfcystine (R28C); CYP3A5*9 is the 19386G＞A sudden change on the exons 10, makes the 337th alanine mutation become Threonine (A337T), and incidence is 2% in Chinese population.
The launch that has only at present a small amount of detection CYP3A5 gene pleiomorphism both at home and abroad; Mainly be to use
CYP3A5 of real-time fluorescence quantitative PCR technology, and major part still is in the experimental study stage.Reported that the method that detects the CYP3A5 polymorphum mainly contains traditional solid phase chip and real-time fluorescence quantitative PCR, wherein, traditional solid phase chip costs an arm and a leg; Susceptibility is not high; The repeatability of detected result is poor, and the real-time fluorescence quantitative PCR technology exists sensitivity low, the easy pollution of sample, the shortcoming that false positive rate is high; There is limitation owing to detect flux simultaneously, can not satisfies and detect the needs of using.
Summary of the invention
One of the object of the invention provides the CYP3A5 gene SNP detection liquid-phase chip.This liquid-phase chip can be used for detecting normal genotype and five kinds of common allele type CYP3A5*3, CYP3A5*6, CYP3A5*7, the CYP3A5*8 of CYP3A5 gene, the variation of CYP3A5*9.
The technical scheme that realizes above-mentioned purpose is following:
A kind of CYP3A5 gene SNP detection liquid-phase chip mainly includes:
(A). the SNP site difference designed ASPE primers to the CYP3A5 gene is right: every kind of ASPE primer is made up of to the specific primer sequence in goal gene SNP site the tag sequence and the 3 ' end of 5 ' end; Said specific primer sequence is respectively: to the SEQ ID NO.11 in A6986G site and SEQ ID NO.12, to the SEQ ID NO.13 in G14690A site and SEQ ID NO.14, to the SEQ ID NO.15 in 27131_27132insT site and SEQ ID NO.16, to the SEQ ID NO.17 in C3699T site and SEQ ID NO.18 and/or to the SEQ ID NO.19 and the SEQ IDNO.20 in G19386A site, said tag sequence is selected from the sequence among the SEQ ID NO.1-10;
(B). be coated with the microballoon of special anti-tag sequence respectively, above-mentioned every kind of microballoon has the different colours coding; Said anti-tag sequence is selected from the sequence among SEQ ID NO.21～SEQ ID NO.30, and said anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). be used to increase have A6986G, the amplimer of the CYP3A5 gene target sequence in G14690A, 27131_27132insT, C3699T and/or G19386A SNP site.
Preferably, said amplimer is: to the SEQ ID NO.31 in A6986G site and SEQ ID NO.32, to the SEQ ID NO.33 in G14690A site and SEQ ID NO.34, to the SEQ ID NO.35 in 27131_27132insT site and SEQ ID NO.36, to the SEQ ID NO.37 in C3699T site and SEQ ID NO.38 and/or to the SEQ ID NO.39 and the SEQ ID NO.40 in G19386A site.
Preferably, said ASPE primer is to being: the sequence that sequence of being made up of SEQ ID NO.1 and SEQ ID NO.11 and the sequence of being made up of SEQ IDNO.2 and SEQ ID NO.12, the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.13 and the sequence of being made up of SEQ IDNO.4 and SEQ ID NO.14, the sequence of being made up of SEQ ID NO.5 and SEQ ID NO.15 reach the sequence be made up of SEQ IDNO.6 and SEQ ID NO.16, be made up of SEQ ID NO.7 and SEQ ID NO.17 reaches the sequence be made up of SEQ IDNO.8 and SEQ ID NO.18 and/or reaches the sequence of being made up of SEQ ID NO.10 and SEQ ID NO.20 by the sequence that SEQ ID NO.9 and SEQ ID NO.19 form.
Preferably, also be provided with the spacerarm sequence in the middle of said anti-tag sequence is connected with microballoon.
Another object of the present invention provides the method for using above-mentioned liquid-phase chip that the CYP3A5 gene SNP is detected.
Concrete technical scheme is following:
A kind of method of using above-mentioned liquid-phase chip to the detection of CYP3A5 gene SNP mainly may further comprise the steps:
(1) pcr amplification testing sample DNA;
(2) the PCR reaction product is carried out enzyme with the ExoSAP-IT test kit and is cut processing;
(3) carry out primer extension reaction with said ASPE primer, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on the reacted product band;
(4) microballoon of the anti-tag sequence that being coated with of corresponding A SPE primer is special and the product behind the above-mentioned extension carry out hybridization;
(5) product behind the hybridization and Streptavidin-phycoerythrin react;
(6) detect through fluorescence detector.
Another object of the present invention provides the Auele Specific Primer that a kind of CYP3A5 of being used for gene SNP detects.
Concrete technical scheme is following:
A kind ofly be used for the Auele Specific Primer that the CYP3A5 gene SNP detects, include: to the SEQ ID NO.11 in A6986G site and SEQ ID NO.12, to the SEQ ID NO.13 in G14690A site and SEQ ID NO.14, to the SEQ ID NO.15 in 27131_27132insT site and SEQ ID NO.16, to the SEQ ID NO.17 in C3699T site and SEQ ID NO.18 and/or to the SEQ ID NO.19 and the SEQ ID NO.20 in G19386A site.
Major advantage of the present invention is:
1. the identical rate of detection method provided by the present invention and PCR sequencing PCR is up to 100%.Prepared CYP3A5 gene SNP detection liquid-phase chip has extraordinary signal-NR, and does not have cross reaction basically between institute's designed probe and the anti-tag sequence, can realize the parallel detection in a plurality of SNP site.
2. designed ASPE Auele Specific Primer of the present invention has extraordinary specificity, can accurately distinguish the genotype of various types, can under the reaction conditions of homogeneous, carry out hybridization, and does not have non-specific binding basically between the various primer, probe.
3. detection method step of the present invention is simple; One step multiplex PCR can be accomplished five amplifications with the target sequence in SNP site; Avoid many uncertain factors of existing in the complex operations processes such as repeated multiple times PCR, thereby can improve the detection accuracy rate greatly, embodied qualitative, quantitative analysis characteristic of accurate while; Thereby the sensitivity of detection is further enhanced, and detected result more accurately and reliably.Simultaneously, the combination of multiple ASPE Auele Specific Primer makes liquid-phase chip and detection method form one and detects the intact system of effect.
Embodiment
Embodiment 1CYP3A5 gene SNP detection liquid-phase chip mainly includes:
One, ASPE primer
Five kinds of common SNP sites to CYP3A5: A6986G, G14690A, 27131_27132insT, C3699T, G19386A, the primer sequence of designs specificity respectively.The ASPE primer is made up of " Tag+ specific primer sequence ".The ASPE primer sequence is as shown in the table:
Table 1ASPE primer sequence (Tag+ specific primer sequence)
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 wild-type specific primer sequence (shown in above-mentioned table 1).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 specific primer sequence; Select the tag sequence; Reduce between the anti-tag sequence of each microballoon to greatest extent and secondary structure that tag and ASPE specific primer sequence possibly form, corresponding anti-tag sequence is as shown in table 2 on 10 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
10 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 preface 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), 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.
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 of 100ul, and [10mmol/L Tris (pH8.0), among the 1mmol/LEDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of target sequence with SNP site
Five kinds of genotypic common SNP site: CYP3A5*3 (A6986G) of target detect CYP3A5, CYP3A5*6 (G14690A), CYP3A5*7 (27131_27132insT), CYP3A5*8 (C3699T), CYP3A5*9 (G19386A) relate to 5 mononucleotide polymorphism sites.Utilize Primer5.0 design five pairs of primers (seeing table 3), amplify five target sequences respectively with SNP site.
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 embodiment 1 said CYP3A5 gene SNP detection 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:
| Reagent | The source | Final concentration | The consumption of every 250ml |
| MES(2[N-Morpholino] ethanesulfonic?acid) | Sigma?M-2933 | 0.05M | ?2.44g |
| 5MNaOH | Fisher?SS256-500 | --- | 5 |
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:
Extract the test kit explanation in a small amount with reference to AxyPrep whole blood genome, obtain DNA to be detected.
Two, the pcr amplification of testing sample
Utilize five pairs of primers of Primer5.0 design, one step of multiplex PCR amplifies five target sequences with SNP site of CYP3A5.Wherein, mutant CYP3A5*3 (A6986G) is positioned at introne 3; Mutant CYP3A5*6 (G14690A) is positioned at exon 7; Mutant CYP3A5*7 (27131_27132insT) is positioned at introne 3; Mutant CYP3A5*8 (C3699T) is positioned at exon 2; Mutant CYP3A5*9 (G19386A) is positioned at exons 10; The product size is respectively 309bp, 448bp, 388bp, 468bp, 373bp.Primer sequence (SEQ NO.31～40) 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 NO.31～40 respectively mixes and is the multiple PCR primer working fluid in the 1.5ml Eppendorf tube.The multi-PRC reaction system is following:
10 * damping fluid (contains Mg
2+) 5ul
DNTP (each 2.5mmol/L) 4ul
Taq enzyme (5U/ul) 0.5ul
Multiple PCR primer working fluid (each 16.7pmol/mL) 6ul
Template DNA (10ng/ul) 1ul
ddH
2O 33.5ul
Be total to 50ul
The pcr amplification program is: 95 ℃ of 3min; 94 ℃ of 20s, 56 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations are subsequent use.
Three, the enzyme of PCR product is cut processing
With reference to the explanation of ExoSAP-IT test kit, detailed step is following:
1. get the reacted product of 7.5ul PCR, add 3ul ExoSAP-IT enzyme;
2.37 ℃ hatch 15min.Hatch 15min for 80 ℃, make unnecessary enzyme-deactivating.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 the locus specificity 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: the ASPE primer stock solution 10ul that respectively gets A6986G, G14690A, 2713127132insT, C3699T, G19386A corresponding wild type and mutant respectively is in the 1.5ml Eppendorf tube; Add 10mmol/LTris 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 10.ul
Be total to 20ul
The PCR program is: 96 ℃ of 2min; 94 ℃ of 30s, 58 ℃ 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 select corresponding ten kinds of optimum microballoons
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 are resuspended in microballoon in 1 * Tm hybridization buffer of 75ul, and 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.With the carrier of polystyrene microsphere, as detection platform, nucleic acid molecule is carried out high-throughout many indexs parallel detection with fluorescence detector as reaction.In the manufacturing processed of microballoon, mix the ruddiness and the infrared light staining agent of different ratios, thereby form the microballoon of 100 kinds of different colours codings of as many as.Different microballoon covalent attachment to the nucleic acid molecule of different things to be detected as probe molecule, reporter molecules is with biotin labeling, and uses high-sensitive fluorescent dyeing.These microballoons and determinand, reporter molecules, fluorescent marker just form complete microballoon detection architecture and are used for reading of Luminex system.Excitated red respectively laser of Luminex reading system and green laser are used for the detection of microsphere system, and 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 the CYP3A5 gene pleiomorphism of great amount of samples, 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 CYP3A5 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.It is thus clear that CYP3A5 gene SNP detection liquid-phase chip provided by the present invention can detect the SNP type of CYP3A5 gene exactly, and the result is reliable and stable.
Table 4 pattern detection result (MFI)
| Sequence number NO. | A6986G -w | A6986G -m | G14690 A-w | G14690 A-m | 27131_2 7132ins | 27131_2 7132ins | C3699T- w | C3699T- m | G19386 A-w | G19386 A-m |
| ? | ? | ? | ? | ? | T-w | T-m | ? | ? | ? | ? |
| Negative to clear | 12 | 11 | 12 | 14 | 12 | 10 | 17 | 16 | 11 | 13 |
| 1 | 3433 | 147 | 3453 | 144 | 3062 | 226 | 3093 | 222 | 2906 | 227 |
| 2 | 35 | 3148 | 2815 | 228 | 3350 | 122 | 2814 | 260 | 3343 | 136 |
| 3 | 2493 | 2755 | 2697 | 215 | 2572 | 220 | 2876 | 249 | 3042 | 218 |
| 4 | 1400 | 1996 | 3022 | 224 | 2557 | 2563 | 2052 | 226 | 3284 | 148 |
| 5 | 35 | 2494 | 3193 | 141 | 2033 | 229 | 3295 | 131 | 2402 | 2032 |
| 6 | 3209 | 140 | 3213 | 239 | 2362 | 316 | 2652 | 224 | 3206 | 232 |
| 7 | 1952 | 1336 | 2494 | 238 | 3455 | 236 | 3273 | 242 | 2536 | 123 |
| 8 | 2555 | 141 | 20 | 2102 | 2458 | 228 | 2969 | 303 | 2942 | 241 |
| 9 | 2335 | 3381 | 2558 | 231 | 2727 | 231 | 2559 | 229 | 2423 | 125 |
| 10 | 2292 | 245 | 3110 | 244 | 3325 | 189 | 2380 | 246 | 2918 | 224 |
| 11 | 2410 | 248 | 3159 | 127 | 2602 | 275 | 2026 | 368 | 2058 | 234 |
| 12 | 42 | 3141 | 2610 | 247 | 3484 | 326 | 3192 | 228 | 2942 | 224 |
| 13 | 2861 | 3106 | 3343 | 227 | 2190 | 254 | 2654 | 189 | 46 | 2257 |
| 14 | 3071 | 2985 | 2132 | 328 | 2376 | 241 | 2991 | 245 | 2857 | 247 |
| 15 | 1215 | 1315 | 2900 | 319 | 2352 | 248 | 19 | 2910 | 3439 | 238 |
| 16 | 2165 | 231 | 2579 | 240 | 3224 | 216 | 3373 | 219 | 2677 | 247 |
| 17 | 3150 | 216 | 3228 | 318 | 2053 | 239 | 3144 | 189 | 3036 | 220 |
| 18 | 3117 | 2595 | 2871 | 127 | 3371 | 315 | 2404 | 225 | 3113 | 218 |
| 19 | 2750 | 246 | 2576 | 332 | 2658 | 218 | 3481 | 245 | 2511 | 127 |
| 20 | 2144 | 1799 | 2428 | 225 | 3336 | 241 | 3217 | 346 | 2425 | 249 |
Table 5 sample CYP3A5 Polymorphism Analysis result
Table 6 sample CYP3A5 Polymorphism Analysis result
The liquid-phase chip of the ASPE primer that embodiment 3 is different detects the detection of transgenation to the CYP3A5 gene SNP
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
Detection liquid-phase chip with CYP3A5 gene C YP3A5*6 (G14690A) site mutation is an example; To the wild-type of G14690A and the specific primer sequence of mutant design ASPE primer 3 ' end; The Tag sequence of ASPE primer 5 ' end then is selected from 6 among the SEQ ID NO.1-SEQ ID NO.10; Accordingly, encapsulate anti-tag sequence selection SEQ ID NO.21-SEQ ID NO.30 on microballoon and corresponding tag sequence complementary pairing.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 (MFI) and Polymorphism Analysis
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 equivalence of the present invention and implement or change, all should be contained in the claim of the present invention.
Claims (6)
1. a CYP3A5 gene SNP detection liquid-phase chip is characterized in that, mainly includes:
(A). the SNP site difference designed ASPE primers to the CYP3A5 gene is right: every kind of ASPE primer is made up of to the specific primer sequence in goal gene SNP site the tag sequence and the 3 ' end of 5 ' end; Said specific primer sequence is respectively: to the SEQ ID NO.11 in A6986G site and SEQ ID NO.12, to the SEQ ID NO.13 in G14690A site and SEQID NO.14, to the SEQ ID NO.15 in 27131_27132insT site and SEQ ID NO.16, to the SEQ ID NO.17 in C3699T site and SEQ ID NO.18 and/or to the SEQ ID NO.19 and the SEQ ID NO.20 in G19386A site, said tag sequence is selected from the sequence among the SEQ ID NO.1-SEQ ID NO.10;
(B). be coated with the microballoon of special anti-tag sequence respectively, above-mentioned every kind of microballoon has the different colours coding; Said anti-tag sequence is selected from the sequence among SEQ ID NO.21～SEQ ID NO.30, and said anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). be used to increase have A6986G, the amplimer of the CYP3A5 gene target sequence in G14690A, 27131_27132insT, C3699T and/or G19386A SNP site.
2. CYP3A5 gene SNP detection liquid-phase chip according to claim 1; It is characterized in that said amplimer is: to the SEQ ID NO.31 in A6986G site and SEQ ID NO.32, to the SEQ ID NO.33 in G14690A site and SEQID NO.34, to the SEQ ID NO.35 in 27131_27132insT site and SEQ ID NO.36, to the SEQ ID NO.37 in C3699T site and SEQ ID NO.38 and/or to the SEQ ID NO.39 and the SEQ ID NO.40 in G19386A site.
3. CYP3A5 gene SNP detection liquid-phase chip according to claim 1 and 2; It is characterized in that said ASPE primer is to being: the sequence that sequence of being made up of SEQ ID NO.1 and SEQ ID NO.11 and the sequence of being made up of SEQ ID NO.2 and SEQ ID NO.12, the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.13 and the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.14, the sequence of being made up of SEQ ID NO.5 and SEQ ID NO.15 reach the sequence be made up of SEQ ID NO.6 and SEQ ID NO.16, be made up of SEQ ID NO.7 and SEQ ID NO.17 reaches the sequence be made up of SEQ ID NO.8 and SEQ ID NO.18 and/or reaches the sequence of being made up of SEQ ID NO.10 and SEQ ID NO.20 by the sequence that SEQ ID NO.9 and SEQ ID NO.19 form.
4. CYP3A5 gene SNP detection liquid-phase chip according to claim 1 is characterized in that, also is provided with the spacerarm sequence in the middle of said anti-tag sequence is connected with microballoon.
5. CYP3A5 gene SNP detection liquid-phase chip according to claim 4 is characterized in that, said spacerarm sequence is 5-10 T.
6. one kind is used for the Auele Specific Primer that the CYP3A5 gene SNP detects, and includes: to the SEQ ID NO.11 in A6986G site and SEQ ID NO.12, to the SEQ ID NO.13 in G14690A site and SEQ ID NO.14, to the SEQ ID NO.15 in 27131_27132insT site and SEQ ID NO.16, to the SEQ ID NO.17 in C3699T site and SEQ ID NO.18 and/or to the SEQ ID NO.19 and the SEQ ID NO.20 in G19386A site.
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| CN103361433A (en) * | 2013-07-26 | 2013-10-23 | 天津市秀鹏生物技术开发有限公司 | Primer group and kit for detecting genetic typing of human cytochrome P450 enzyme system 3A5(CYP3A5) |
| CN103436631B (en) * | 2013-09-22 | 2015-11-25 | 刘辉 | A kind of test kit and method detecting CYP3A5 gene pleiomorphism |
| CN105274221A (en) * | 2015-10-14 | 2016-01-27 | 北京晋祺生物科技有限公司 | CYP3A5*3 detection kit |
| CN108179180A (en) * | 2017-10-25 | 2018-06-19 | 广州和康医疗技术有限公司 | A kind of method and kit that genotype detection is carried out to CYP3A5*3 sites |
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| CN101067149A (en) * | 2006-12-13 | 2007-11-07 | 上海生物芯片有限公司 | CYP3A detecting chip and its application |
| CN101481736A (en) * | 2009-02-06 | 2009-07-15 | 中山大学 | DNA micro-array chip, detection method thereof and uses in CYP3A4, CYP3A5 and MDR1 gene polymorphism detection |
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| CN101067149A (en) * | 2006-12-13 | 2007-11-07 | 上海生物芯片有限公司 | CYP3A detecting chip and its application |
| CN101481736A (en) * | 2009-02-06 | 2009-07-15 | 中山大学 | DNA micro-array chip, detection method thereof and uses in CYP3A4, CYP3A5 and MDR1 gene polymorphism detection |
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