CN109536605A - The PCR primer combination and application of statins adverse reaction genotype polymorphism - Google Patents
The PCR primer combination and application of statins adverse reaction genotype polymorphism Download PDFInfo
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Abstract
This application discloses the PCR primer combinations and application of a kind of statins adverse reaction genotype polymorphism, belong to biomedical field of molecular detection.The PCR primer combination includes the primer pair 1-4 for expanding SNP site rs4149056, rs2306283, rs429358 and rs7412, and sequence is as shown in SEQ ID No.1-2,5-6,11-12,13-14；It further include the Single base extension primer combination of SNP site described in specific amplified, sequence is as shown in No.17-20 SEQ ID.Primer combination provided by the present application and detection method, solves the incompatible technical problem of multiple target spot amplification conditions, it realizes and multiple primers is added in a reacting hole, and non-interfering effect, when detecting 4 mutational site genotype on the different genes of statins safety and curative effect, minimum detection limit is up to 0.01ng/ μ L.
Description
Technical field
The invention belongs to biomedical field of molecular detection, and in particular to for detecting four SNP sites:
SLCO1B1 gene * 5c.521T > C (rs4149056), SLCO1B1 gene c.388A > G (rs2306283), ApoE gene
C.388T c.526C > C (rs429358) and ApoE gene the detection primer of the nucleotide polymorphisms of > T (rs7412) and its answer
With.
Background technique
Statins (3-hydroxy-3-methylglutaric acid-CoA-reductase inhibitors) are that one kind is used to reduce cholesterol
(LDL-C) drug, by inhibiting liver inner cholesterol to synthesize key enzyme --- 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-
CoA reductase), so that liver synthesis cholesterol reduced, removed extra cholesterol from blood.Statins remove, and there is lipid-loweringing to make
With outer, also show to reduce systemic inflammatorome, improve endothelial function and reduce the pleiotropic effects such as blood platelet high response, it can be obvious
Reduce the All-cause death wind of myocardial infarction, stroke and mortality risk and high-risk patient that all patient's cardiovascular diseases are caused
Danger, as the primary prevention medication of Silent cerebral infarction, the whole world has more than 200,000,000 people and takes such drug for a long time at present.
However, the study found that statins have following potential hazard, the adverse reaction of most serious is rhabdomyolysis,
When creatine kinase is more than 40 times of normal upper limit or more, kidney failure can be caused；It is influenced by drug dose and efficiency, Statins has
The risk of unsatisfactory curative effect or inefficacy to specific crowd.It can be seen that because individual difference uses statins potential risk
Huge, only medicine and pregnancies with conservative treatment by rule of thumb, are difficult to accomplish the individuation of therapeutic regimen and specific aim.
Using statins adverse reaction genotype detection can instruct clinician in advance to the risk assessment of patient,
Harm is layered and reasonably selects medication, formulates more suitable therapeutic scheme, and look-ahead individual patients are to statins
The sensibility of object and the occurrence risk of adverse reaction reduce medical insurance drug expenditure, reduce patient economy burden, is better to obtain
Treat income.
It is responsible in statins metabolism by SLCO1B1 gene coding organic anion transhipment polypeptide (OATP1B1)
Drug in blood is transferred in liver, directly performance drug effect or metabolic conversion are active substance.Research shows that
SLCO1B1 gene has genetic polymorphism, and wherein 388A > G, 521T > C are two kinds of common single nucleotide polymorphism, can be with shape
At 4 kinds of haplotypes: SLCO1B1*1a (388A-521T), SLCO1B1*1b (388G-521T), SLCO1B1*5 (388A-521C),
SLCO1B1*15(388G-521C).Saltant type SLCO1B1 gene causes the OATP1B1 transport protein vigor of coding to weaken, performance
Absorbing drug ability for liver reduces, and statins blood concentration is caused to rise, and increases the hair of rhabdomyolysis or myopathy
Raw risk.
ApoE (apo E) polymorphism is considered as the susceptible of hyperlipoprotememia and atherosclerotic vascular disease
Candidate gene.The mankind ApoE assignment of genes gene mapping on No. 19 chromosomes, there are mainly two types of single nucleotide polymorphism 526C > T and 388T >
C can form 3 kinds of haplotype ApoE3 (388T-526C), ApoE2 (388T-526T), ApoE4 (388C-526C).Document report
Road, the risk that ApoE4 carrier suffers from coronary heart disease want high by 40%, and statins are often bad to ApoE4 carrier's curative effect
Or inefficacy, and it is most strong to the effect for reducing fat of ApoE2 carrier.Nearest genome-wide association study (GWAS) confirmation, ApoE
Gene loci is related to the response to Rosuvastatin.
Currently used gene tester has DNA direct Sequencing, nucleic acid mass-spectrometric technique, restriction fragment length polymorphism
Analysis, high-resolution solubility curve, genetic chip, liquid-phase chip, quantitative fluorescent PCR etc., it is recognized that gene mutation goldstandard be
Sequencing technologies, but testing cost is high, the period is long, flux is low, it is furthermore numerous to lab assistant technical requirements height, result judgment step
It is trivial etc., it is difficult to form commercially produced product.
Existing statins genotype detection use PCR- fluorescence probe method, principle be using Taq enzyme 5 ' →
Specific hybrid occurs for 3 ' 5 prime excision enzyme activities, the probe and DNA profiling that a fluorescent marker is added in PCR reaction system, if
Specific nucleic acid segment is amplified, fluorescence probe can hybridize therewith according to the principle of base pairing, and it is strong that instrument reads fluorescence signal
It is weak.The disadvantage is that: fluorescent quenching is difficult to thoroughly, and background is higher；The hydrolysis of probe is exo-acting dependent on the enzyme of Taq, therefore quantitative
When influenced by enzyme performance and reagent quality；Since probe is incorporated into template, certain influence is generated to amplification efficiency；It needs
Longer probe is designed, the ability relative deficiency of the workload detection point mutation of probe design is increased；To instrument, consumptive material requirement
It is high.
Summary of the invention
In view of the deficiencies in the prior art, the purpose of the present invention is to provide a kind of statins adverse reaction bases
Because of the PCR primer combination and application of type polymorphism.
The PCR primer combination retain PCR amplification high efficiency and sensitivity while, can a pipe complete it is multiple
SNP site product amplification has taken into account the accuracy of gene sequencing method detection；Sample DNA is not necessarily to measurement and diluted concentration after extracting,
Directly sample-adding carries out PCR amplification and subsequent SAP and iPLEX reaction, avoids the influence factors such as probe hybridization, saves the time, subtract
Few manual operations；Testing cost is preferably controlled, economic and social benefit is improved；The present invention facilitates MassARRAY mass spectrum
Technology is used for the inspection of clinical medicine gene, and new efficient, accurate diagnosis basis is provided for personalized medicine.
To achieve the above objectives, the technical solution adopted by the present invention is as follows:
Firstly, the present invention provides a kind of for detecting subject's statins adverse reaction genotype polymorphism
PCR primer combination, including following primer pair:
Specific amplified includes the primer pair 1 of SNP site SLCO1B1 gene * 5c.521T > C (rs4149056), described to draw
Object to 1 upstream primer as shown in SEQ ID No.1, downstream primer is as shown in SEQ ID No.2；
Specific amplified includes the primer pair 2 of SNP site SLCO1B1 gene c.388A > G (rs2306283), the primer
To 2 upstream primer as shown in SEQ ID No.5, downstream primer is as shown in SEQ ID No.6；
Specific amplified includes the primer pair 3 of SNP site ApoE gene c.388T > C (rs429358), the primer pair 3
Upstream primer is as shown in SEQ ID No.11, and downstream primer is as shown in SEQ ID No.12；
Specific amplified includes the primer pair 4 of SNP site ApoE gene c.526C > T (rs7412), the primer pair 4 it is upper
Primer is swum as shown in SEQ ID No.13, downstream primer is as shown in SEQ ID No.14.
In one embodiment, it can be used simultaneously above-mentioned primer pair 1-4, it is multiple to organize DNA progress to receptor gene
PCR, a reaction can be realized four different target sequences and (contain SNP site respectively
Rs4149056, rs2306283, rs429358 or rs7412) specific amplification, and detection sensitivity is high.
In other embodiment, above-mentioned primer pair 1-4 can be used individually, it can also be by primer pair 1-
Any two in 4 or three kinds are applied in combination, the corresponding target sequence of PCR amplification；
Any two kinds be applied in combination can be by primer pair 1 and 2, primer pair 1 and 3, primer pair 1 and 4, primer pair 2 and 3,
Primer pair 2 and 4 or primer pair 3 and 4 are applied in combination；
Any three kinds are applied in combination and can be primer pair 1,2 and 3, primer pair 1,2 and 4, primer pair 1,3 and 4 or primer
It is applied in combination to 2,3 and 4；
The base of the amplified production of above-mentioned primer pair or combinations thereof at corresponding SNP site is detected, in conjunction with pass
In the existing research or further research of SNP site and statins adverse reaction correlation, subject's Statins can be predicted
The polymorphism of adverse drug reaction genotype.
The method detected to the base of the amplified production of above-mentioned primer pair or combinations thereof at corresponding SNP site can be with
It is PCR sequencing PCR, mass spectrography, fluorescence quantitative PCR method, multiple Single base extension SNP typing method etc..And it is above-mentioned while detecting multiple
The method of SNP site is based on multiple PCR technique.
In one embodiment, primer combination further includes the single base of the specific amplified SNP site accordingly
Extension primer, or the probe of the detection SNP site；
The amplified production of detection Single base extension primer can learn the genotype of corresponding SNP site；
The marking signal of detection probe can also learn the genotype of corresponding SNP site；
Preferably, the Single base extension primer of SNP site described in the specific amplified is as follows:
The Single base extension primer A of specific amplified SNP site SLCO1B1 gene * 5c.521T > C (rs4149056),
Sequence is SEQ ID No.17 or its complementary series；
The Single base extension primer B of specific amplified SNP site SLCO1B1 gene c.388A > G (rs2306283), sequence
It is classified as SEQ ID No.18 or its complementary series；
The Single base extension primer C of specific amplified SNP site ApoE gene c.388T > C (rs429358), sequence are
SEQ ID No.19 or its complementary series；
Single base extension the primer D, sequence SEQ of specific amplified SNP site ApoE gene c.526C > T (rs7412)
ID No.20 or its complementary series；
Single base extension primer A-D can be used simultaneously, be carried out simultaneously for the amplified production to primer pair 1-4 corresponding
The single base of SNP site expands；It can also be selected according to any combination of primer pair 1-4, and use corresponding Single base extension
Primer is combined, and expands corresponding SNP site；
Molecular weight is not identical between the amplified production of Single base extension primer A-D provided by the present application, in order to pass through matter
Spectrum distinguishes.Different Mass Spectrometer Method systems or Mass Spectrometry detection method, the minimum difference molecular weight that can be distinguished is not identical, needs
It is set according to actual conditions.
Secondly, the present invention also provides primer combinations to detect the application in following SNP site polymorphism, or making
The standby reagent for detecting following SNP site polymorphism or the application in kit:
SLCO1B1 gene * 5c.521T > C (rs4149056), SLCO1B1 gene c.388A > G (rs2306283),
ApoE gene c.388T > C (rs429358), ApoE gene c.526C > T (rs7412)；Or the SNP site is detected in preparation
Application in polymorphism product such as reagent or kit.
The present invention also provides primer combinations in detection subject's statins adverse reaction genotype polymorphism
In application, or preparation detection subject's statins adverse reaction genotype polymorphism product such as reagent or kit
In application；The statins adverse reaction genotype polymorphism is the polymorphism of following SNP site: SLCO1B1 gene *
5c.521T > C (rs4149056), SLCO1B1 gene c.388A > G (rs2306283), ApoE gene c.388T > C
(rs429358), and/or ApoE gene c.526C > T (rs7412).
The present invention protects the primer combination to detect the application in following SNP site polymorphism product: SLCO1B1 in preparation
Gene * 5c.521T > C (rs4149056), SLCO1B1 gene c.388A > G (rs2306283), ApoE gene c.388T > C
(rs429358), and/or ApoE gene c.526C > T (rs7412).
The present invention protects the primer combination to detect subject's statins adverse reaction genotype polymorphism in preparation
Application in product, the statins adverse reaction genotype polymorphism are the polymorphism of following SNP site: SLCO1B1
Gene * 5c.521T > C (rs4149056), SLCO1B1 gene c.388A > G (rs2306283), ApoE gene c.388T > C
(rs429358), and/or ApoE gene c.526C > T (rs7412).
The present invention also provides a kind of for detecting the kit of multiple SNP site polymorphisms, including the primer simultaneously
Combination；Preferably, in the kit further include: PCR reaction reagent and/or alkaline phosphatase and its reaction buffer, and/
Or single base extension reagent and/or desalination resin；
The PCR reaction reagent includes: dNTP, Mg2+, PCR reaction premixed liquid, and/or archaeal dna polymerase；
The single base extension reagent includes: ddNTP, single base extension buffer, single base extension
Terminate liquid, single base extension enzyme.
Multiple SNP site polymorphisms are detected simultaneously the present invention also provides one kind or detection subject's statins are bad
The method of response gene type polymorphism, includes the following steps:
S1, the genomic DNA for taking subject in vitro；
S2, using DNA described in step S1 as template, use the primer pair 1-4, carry out multiplexed PCR amplification, obtain first
Amplified production；
S3, first amplified production is detected, determines the genotype of each SNP site；
The multiple SNP site are as follows: SLCO1B1 gene * 5c.521T > C (rs4149056), SLCO1B1 gene are c.388A
> G (rs2306283), ApoE gene c.388T > C (rs429358) and ApoE gene c.526C > T (rs7412).
Preferably, the step S3 includes the following steps:
S31, using first amplified production as template, using the Single base extension primer carry out Single base extension, obtain
To the second amplified production；
S32, second amplified production is detected, determines the genotype of each SNP site.
Preferably, the method for detection described in the step S32 is Mass Spectrometer Method.
Beneficial effects of the present invention are as follows:
The combination of primer pair 1-4 provided by the invention cleverly solves the incompatible technology of multiple target spot amplification conditions and asks
Topic is realized and multiple primers is added in a reacting hole, and non-interfering effect, Single base extension primer A-D Combination Design are closed
Reason, meeting nucleic acid mass spectral analysis requirement, (molecular weight difference of single base extension product is greater than 20Da, to guarantee that molecular weight can be distinguished
Open), and reaction sample volume needed for nucleic acid mass-spectrometric technique is small, operating procedure is simple, is a kind of quick, easy, economic, practical
Technical system.Primer combination provided by the invention and detection method, detection receptor gene organize statins safety in DNA
And on the different genes of curative effect when 4 mutational site genotype, minimum detection limit is up to 0.01ng/ μ L.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present application, constitutes part of this application, this Shen
Illustrative embodiments and their description please are not constituted an undue limitation on the present application for explaining the application.In the accompanying drawings:
Fig. 1 is the mass spectrogram of detection site SLCO1B1 gene * 5c.521T > C (rs4149056).
Fig. 2 is the mass spectrogram of detection site SLCO1B1 gene c.388A > G (rs2306283).
Fig. 3 is the mass spectrogram of detection site ApoE gene c.388T > C (rs429358).
Fig. 4 is the mass spectrogram of detection site ApoE gene c.526C > T (rs7412).
Wherein, the abscissa in Fig. 1-4 is molecular weight, and ordinate is intensity.
Fig. 5 is the result figure of PCR fluorescence probe method detection site SLCO1B1 gene * 5c.521T > C (rs4149056).
Fig. 6 is the result figure of PCR fluorescence probe method detection site SLCO1B1 gene c.388A > G (rs2306283).
Fig. 7 is the result figure of PCR fluorescence probe method detection site ApoE gene c.388T > C (rs429358).
Fig. 8 is the result figure of PCR fluorescence probe method detection site ApoE gene c.526C > T (rs7412).
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1, multiple PCR primer design and screening
C.388A from NCBI retrieval SNP site SLCO1B1 gene * 5c.521T > C (rs4149056), SLCO1B1 gene
The sequence letter of > G (rs2306283), ApoE gene c.388T > C (rs429358) and ApoE gene c.526C > T (rs7412)
Breath compares by bioinformatics and analyzes, respectively finds one section of highly conserved region for separately including one of above-mentioned SNP site,
3-5 specific primers are separately designed to (totally 4 for each highly conserved region (totally 4) comprising corresponding SNP site
Group) and 3-5 Single base extension primers (totally 4 groups) for corresponding SNP site, pass through DNAstar etc. different sequences and analyzes
Software carries out interference assay to the combination of primer pair between 4 groups and the combination of Single base extension primer respectively, finally, from every
Selection evaluates two pairs of best primer pairs (as shown in table 1) after combining with other each group primer pairs respectively in group primer pair；And from
A Single base extension primer is selected in every group respectively, the possible amplified production of the primer draws with Single base extension in other each groups
Object may amplified production mass difference 20Da or more and with evaluated most after the combination of Single base extension primer in other each groups
Good (as shown in table 2).
Table 1, multiple PCR primer design
Table 2, Single base extension design of primers
| SNP site | Primer sequence (5 ' -3 ') | Molecular weight Da |
| rs4149056 | A:GGTCATACATGTGGATATATG(SEQ ID No.17) | 6537 |
| rs2306283 | B:CCTAATCTAAAGAAACTAATATC(SEQ ID No.18) | 7018 |
| rs429358 | C:GGAGGACGAGTGCGGCCGC(SEQ ID No.19) | 5959 |
| rs7412 | D:CTCCCGATCACCTGCAGAAG(SEQ ID No.20) | 6085 |
Embodiment 2, Quadruple- PCR primer Combinatorial Optimization (sensitivity technique experiment)
By four groups of primer pair random combines shown in table 1 (totally 24Kind combination) after, sensitivity technique is carried out as follows:
The purified genes group DAN sample for the blood sample for taking an adult EDTA anticoagulant, extremely with sterile water gradient dilution
10ng/μL、1ng/μL、0.5ng/μL、0.1ng/μL、0.05ng/μL、0.025ng/μL、0.01ng/μL、0.005ng/μL、
0.0025ng/ μ L draws 10 μ L as template respectively, and is combined with primer pair shown in table 3 carry out Quadruple- PCR detection respectively, will
Each amplified production successively carries out SAP reaction, single base extension (using the combination of four kinds of Single base extension primers shown in table 2)
And Mass Spectrometer Method, counting the testing result that the combination of each primer pair is directed to each sample concentration, experiment sets water as negative control, and eight
The substance PCR of a primer pair is positive control, and each different amplified reaction is all provided with 3 repetitions, by four mesh in each repetition
Mark amplification SNP site base is that positive result is denoted as the positive, is otherwise denoted as feminine gender, is the minimum of the positive by 3 repetitions
The minimal detectable concentration that template concentrations are combined as each primer pair, the results are shown in Table 3.
The minimal detectable concentration that table 3, Quadruple- PCR primer pair combine
Note: through detecting, negative control is without amplified production, and without Mass Spectrometer Method peak.
Table 3 the result shows that, the sensitivity expanded to template using primer pair combination 1-1/2-1/3-2/4-1 is most
Low, minimal detectable concentration is 0.01ng/ μ L.
The specific method is as follows for above-mentioned detection:
Detect sample: the EDTA anticoagulant blood sample of an adult；The sample rs4149056, rs2306283,
(Sanger PCR sequencing PCR) known to the genotype in the site rs429358 and rs7412；
Detecting instrument: MassARRAY Genetic Analysis System DNA mass spectrum gene alaysis system
(SEQUENOM Inc. Signode Corp., the U.S.)；AB-Vii A7 fluorescence quantitative PCR instrument (Applied biosystems
(ABI))；Centrifuge (TL-5.0W, Shanghai)；Rotary oscillator (Shanghai).
Detection method:
1, using the genomic DNA in poba gene group extracts kit difference purifying blood sample；
2, multiplexed PCR amplification: at the same use 4 primer pairs, using step 1 obtain genomic DNA as template, while set with
Water is the negative control of template, carries out multiplexed PCR amplification, and reaction system is as follows:
The reaction condition of multiplexed PCR amplification are as follows: 95 DEG C of initial denaturation 3min；Then 45 times: the 95 DEG C of denaturation of following circulation are carried out
30s and 72 DEG C of 20s, 60 DEG C of annealing extension 20s；Last 72 DEG C of extensions 3min.
3, SAP reacts
5 μ L of liquid, addition 2 μ L SAP reaction solutions progress SAP reaction after PCR amplification in above-mentioned steps 2 are taken respectively, wherein
It is as follows that SAP reacts formula of liquid:
| Reagent component | Dosage |
| Deionization H2O | 1.53μL |
| SAP buffer | 0.17μL |
| SAP enzyme (1.7U/ μ L) | 0.30μL |
| It is total | 2.00μL |
SAP reaction condition is as follows: 37 DEG C 40 minutes, 85 DEG C 5 minutes, 4 DEG C preservation.
4, single base extension
It is separately added into the extension liquid of 2 μ L into the liquid reacted by step 3, carries out single base extension,
In, extension system is as follows:
| Component | Dosage |
| Deionization H2O | 0.619μL |
| IPLEX buffer | 0.2μL |
| IPLEX terminates mix | 0.2μL |
| The mixture (0.5 μM) of each Single base extension primer isoconcentration | 0.94μL |
| IPLEX enzyme | 0.041μL |
| It is total | 2μL |
The condition of extension is as follows:
5, resin desalination
It is centrifuged after 41 μ L water are added in the extension fluid apertures that each has sample.15mg cleaning resin is added
(Resin): gently by sample plane reversion high up in the air, being placed on the dimple plate for having put resin, then by dimple plate
(two allegros are not horizontally moveable in the process) is inverted together with sample plane, resin is allowed to drop in hole.Plate is sealed with film, is placed on rotation
Turn oscillation on device to shake up 15 minutes.Plate is centrifuged 5 minutes with 3200g (4000rpm of on-gauge plate centrifuge).
6, SpectroCHIP chip loading
Use MassARRAYTM1000 auto sample applicator of RS carries out volume test to find out suitable point sample speed.Point sample
Suitable volumes are 8-12nL ± 25%.Volume test is carried out with the sample on plate, then uses MassARRAYTMRS 1000 is automatic
Point sample instrument will be on the sample point after reaction to 96 point SpectroCHIP (chip).
7, Mass Spectrometer Method
Mass Spectrometer Method is carried out using MassARRAY Genetic Analysis System DNA mass spectrum gene alaysis system
And analysis, determine that multiplex PCR produces according to the difference of the molecular weight at Mass Spectrometer Method peak and the molecular weight of corresponding single base amplimer
The base type of corresponding SNP site in object.
Embodiment 3, the detection of blind sample
It is combined using the primer pair combination 1-1/2-1/3-2/4-1 in table 3 and the Single base extension primer in table 2, according to
In embodiment 2 method of step 1-7 detect respectively 10 parts of unknown samples (adult EDTA anticoagulant blood) rs4149056,
The genotype in the site rs2306283, rs429358 and rs7412, wherein the concentration of template DNA solution is 10ng/ μ L in step 2
(excess).
Control 1: it is individually detected using genotype of traditional PCR fluorescence probe method to each SNP site.
Control 2: product that step 2 obtains is detected to the genotype of each SNP site using Sanger PCR sequencing PCR.
As a result: above-mentioned three kinds of method testing results are identical.One of pattern detection result is as follows:
Mass Spectrometer Method result is as shown in Fig. 1-4, as the result is shown: the genotype in the site rs4149056 of detected sample is
It is the genotype in the site TT, rs7412 is CC that the genotype in the site TT, rs2306283, which is the genotype in the site GG, rs429358,.
Control 1: for result as shown in Fig. 5-8, the result and the result of Fig. 1-4 are completely the same.
Control 2: the genotype results of each SNP site and the result that Fig. 1-4 is obtained are completely the same, and sequencing result also confirms
The specificity of primer pair combination 1-1/2-1/3-2/4-1 is good, and characteristic expands nothing but.
Result above proves: the primer pair combination 1-1/2-1/3-2/4-1 in table 1 and the Single base extension primer in table 2
The method of combination and step 1-7 is accurately and reliably to subject's statins adverse reaction genotype is detected simultaneously.
The content being not described in detail in this specification belongs to the prior art well known to professional and technical personnel in the field.More than
Described is only embodiments herein, is not intended to limit this application.To those skilled in the art, the application can
To there is various modifications and variations.All any modification, equivalent replacement, improvement and so within the spirit and principles of the present application,
It should be included within the scope of the claims of this application.
Sequence table
<110>Shenzhen City Second People's Hospital
<120>PCR primer combination and application of statins adverse reaction genotype polymorphism
<130> JH-SDHZ-PI-20180319X
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 1
ggaatctggg tcatacatgt gga 23
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 2
tcatcaatgt aagaaagccc ca 22
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 3
ggttgtttaa aggaatctgg gtca 24
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 4
tgtaagaaag ccccaatggt 20
<210> 5
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 5
tcagtgatgt tcttacagtt acaggt 26
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 6
ccactatctc aggtgatgct ct 22
<210> 7
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 7
agtgatgttc ttacagttac aggtatt 27
<210> 8
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 8
accttttccc actatctcag gtg 23
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 9
tggacgagac catgaaggag 20
<210> 10
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 10
cagctcctcg gtgctctg 18
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 11
gacgagacca tgaaggagtt ga 22
<210> 12
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 12
ctcctcggtg ctctggcc 18
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 13
gatgccgatg acctgcagaa 20
<210> 14
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 14
ctgggcccgc tcctgtag 18
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 15
gatgacctgc agaagcgcct 20
<210> 16
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 16
ctcctgtagc ggctggcc 18
<210> 17
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 17
ggtcatacat gtggatatat g 21
<210> 18
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 18
cctaatctaa agaaactaat atc 23
<210> 19
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 19
ggaggacgag tgcggccgc 19
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 20
ctcccgatca cctgcagaag 20
Claims (10)
1. a kind of for detecting the PCR primer combination of subject's statins adverse reaction genotype polymorphism, feature exists
In: including following primer pair:
Specific amplified includes the primer pair 1 of SNP site SLCO1B1 gene * 5c.521T > C (rs4149056), the primer pair 1
Upstream primer as shown in SEQ ID No.1, downstream primer is as shown in SEQ ID No.2；
Specific amplified includes the primer pair 2 of SNP site SLCO1B1 gene c.388A > G (rs2306283), the primer pair 2
Upstream primer is as shown in SEQ ID No.5, and downstream primer is as shown in SEQ ID No.6；
Specific amplified includes the primer pair 3 of SNP site ApoE gene c.388T > C (rs429358), the upstream of the primer pair 3
Primer is as shown in SEQ ID No.11, and downstream primer is as shown in SEQ ID No.12；
Specific amplified includes the primer pair 4 of SNP site ApoE gene c.526C > T (rs7412), and the upstream of the primer pair 4 is drawn
Object is as shown in SEQ ID No.13, and downstream primer is as shown in SEQ ID No.14.
2. primer combination as described in claim 1, it is characterised in that: further include the single base of SNP site described in specific amplified
Extension primer, or the probe of the detection SNP site；
Preferably, the Single base extension primer of SNP site described in the specific amplified is as follows:
The Single base extension primer A of specific amplified SNP site SLCO1B1 gene * 5c.521T > C (rs4149056), sequence
For SEQ ID No.17 or its complementary series；
And/or the Single base extension primer B of specific amplified SNP site SLCO1B1 gene c.388A > G (rs2306283),
Sequence is SEQ ID No.18 or its complementary series；
And/or the Single base extension primer C of specific amplified SNP site ApoE gene c.388T > C (rs429358), sequence
For SEQ ID No.19 or its complementary series；
And/or the Single base extension primer D of specific amplified SNP site ApoE gene c.526C > T (rs7412), sequence are
SEQ ID No.20 or its complementary series.
3. primer combination as claimed in claim 1 or 2 is detecting the application in following SNP site polymorphism: SLCO1B1 gene *
5c.521T > C (rs4149056), SLCO1B1 gene c.388A > G (rs2306283), ApoE gene c.388T > C
(rs429358), and/or ApoE gene c.526C > T (rs7412).
4. primer combination as claimed in claim 1 or 2 is in detection subject's statins adverse reaction genotype polymorphism
Using the statins adverse reaction genotype polymorphism is the polymorphism of following SNP site: SLCO1B1 gene *
5c.521T > C (rs4149056), SLCO1B1 gene c.388A > G (rs2306283), ApoE gene c.388T > C
(rs429358), and/or ApoE gene c.526C > T (rs7412).
5. primer combination as claimed in claim 1 or 2 detects the application in following SNP site polymorphism product: SLCO1B1 in preparation
Gene * 5c.521T > C (rs4149056), SLCO1B1 gene c.388A > G (rs2306283), ApoE gene c.388T > C
(rs429358), and/or ApoE gene c.526C > T (rs7412).
6. primer combination as claimed in claim 1 or 2 detects subject's statins adverse reaction genotype polymorphism in preparation
Application in product, the statins adverse reaction genotype polymorphism are the polymorphism of following SNP site: SLCO1B1
Gene * 5c.521T > C (rs4149056), SLCO1B1 gene c.388A > G (rs2306283), ApoE gene c.388T > C
(rs429358), and/or ApoE gene c.526C > T (rs7412).
7. a kind of kit for detecting subject's statins adverse reaction genotype polymorphism, it is characterised in that: including power
Benefit requires the 1 or 2 primer combinations；Preferably, in the kit further include: PCR reaction reagent and/or alkaline phosphatase
And its reaction buffer and/or single base extension reagent and/or desalination resin；
The PCR reaction reagent includes: dNTP, Mg2+, PCR reaction premixed liquid and archaeal dna polymerase；
The single base extension reagent includes: ddNTP, single base extension buffer, single base extension termination
Liquid, single base extension enzyme.
8. a kind of detect multiple SNP site polymorphisms or detection subject's statins adverse reaction genotype polymorphism simultaneously
Method, include the following steps:
S1, the genomic DNA for taking subject in vitro；
S2, using DNA described in step S1 as template, using the primer pair 1-4 in claims 1 or 22 or 7, carry out multiple
PCR amplification obtains the first amplified production；
S3, first amplified production is detected, determines the genotype of each SNP site；
The statins adverse reaction genotype polymorphism is the polymorphism of following SNP site: SLCO1B1 gene *
5c.521T > C (rs4149056), SLCO1B1 gene c.388A > G (rs2306283), ApoE gene c.388T > C
(rs429358), ApoE gene c.526C > T (rs7412).
9. method according to claim 8, it is characterised in that: the step S3 includes the following steps:
S31, using first amplified production as template, use the Single base extension primer in claim 2 or 7 to carry out single
Base extension obtains the second amplified production；
S32, second amplified production is detected, determines the genotype of each SNP site.
10. method as claimed in claim 9, it is characterised in that: the method for detection described in the step S32 is mass spectrum inspection
It surveys.
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| CN110951859A (en) * | 2019-12-17 | 2020-04-03 | 苏州天隆生物科技有限公司 | Extraction-free kit for detecting polymorphism of human APOE and SLCO1B1 genes |
| CN111154860A (en) * | 2020-01-17 | 2020-05-15 | 深圳会众生物技术有限公司 | Primer, probe composition, kit and method for detecting SLCO1B1 and APOE gene polymorphism |
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| CN110016503A (en) * | 2019-04-22 | 2019-07-16 | 南京格致医学检验有限公司 | For instructing the SNP site detection kit and its application method of reducing blood lipid medication |
| CN112210598A (en) * | 2019-07-11 | 2021-01-12 | 合肥中科普瑞昇生物医药科技有限公司 | Primer group and kit for detecting polymorphism of gene related to metabolism of hyperglycemia drug and application of primer group and kit |
| CN110257523A (en) * | 2019-07-22 | 2019-09-20 | 上海市胸科医院 | A kind of primer sets and detection method detecting chemotherapeutical medicine curative effect and side effect related SNP |
| CN110951859A (en) * | 2019-12-17 | 2020-04-03 | 苏州天隆生物科技有限公司 | Extraction-free kit for detecting polymorphism of human APOE and SLCO1B1 genes |
| CN110951859B (en) * | 2019-12-17 | 2023-09-15 | 苏州天隆生物科技有限公司 | Extraction-free kit for detecting human APOE and SLCO1B1 gene polymorphism |
| CN111154860A (en) * | 2020-01-17 | 2020-05-15 | 深圳会众生物技术有限公司 | Primer, probe composition, kit and method for detecting SLCO1B1 and APOE gene polymorphism |
| CN113528637A (en) * | 2021-02-22 | 2021-10-22 | 北京市理化分析测试中心 | Primer group for amplifying anti-infective drug gene SNP locus, detection primer group and application thereof |
| CN113528637B (en) * | 2021-02-22 | 2023-09-22 | 北京市理化分析测试中心 | Primer group for amplifying SNP locus of anti-infective drug gene, detection primer group and application thereof |
| CN113584146A (en) * | 2021-06-15 | 2021-11-02 | 湖南菲思特精准医疗科技有限公司 | Detection kit for statin metabolic marker, detection method and application thereof |
| CN114525326A (en) * | 2021-12-16 | 2022-05-24 | 深圳市第二人民医院(深圳市转化医学研究院) | Multi-SNP locus genotyping method based on nMALDI-TOF technology |
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