CN112553319B - Special primer for detecting anticoagulant and/or antiplatelet drug resistance related gene SNP locus and application - Google Patents
Special primer for detecting anticoagulant and/or antiplatelet drug resistance related gene SNP locus and application Download PDFInfo
- Publication number
- CN112553319B CN112553319B CN202011458539.3A CN202011458539A CN112553319B CN 112553319 B CN112553319 B CN 112553319B CN 202011458539 A CN202011458539 A CN 202011458539A CN 112553319 B CN112553319 B CN 112553319B
- Authority
- CN
- China
- Prior art keywords
- primer
- sequence
- dna molecule
- stranded dna
- molecule shown
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biology, and provides a special primer for detecting an SNP locus of a gene related to drug resistance of anticoagulant and/or antiplatelet drugs, which consists of a primer group 1 and a primer group 2, wherein the primer group 1 is a specific PCR primer consisting of 15 pairs of primers, and the primer group 2 is a single-base extension primer consisting of 15 primers. The invention also provides a preparation for detecting the SNP sites of the genes related to the drug resistance of the anticoagulant and/or antiplatelet drugs, and a method for detecting the SNP sites of the genes related to the drug resistance of the anticoagulant and/or antiplatelet drugs. The special primer provided by the invention can greatly reduce the detection time and save the cost, has very high application value for guiding the accurate medication of 6 clinical commonly used anticoagulant and/or antiplatelet drugs, and is suitable for popularization and application.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a special primer for detecting an SNP locus of a gene related to drug resistance of anticoagulant and/or antiplatelet drugs and application thereof.
Background
Aspirin, clopidogrel and ticagrelor are common antiplatelet drugs in clinic. Aspirin is a basic drug for the prevention and treatment of arterial thrombotic diseases; clopidogrel is a platelet aggregation inhibitor and is used for preventing and treating heart and brain and other arterial circulatory disorders caused by platelet high aggregation, such as thrombosis and the like; ticagrelor is a novel antiplatelet medicament like clopidogrel, directly and rapidly generates an inhibition effect on a P2Y12 receptor without metabolic activation, and can more strongly inhibit platelet aggregation compared with clopidogrel; meanwhile, the inhibition on the blood platelets is reversible, the inhibition degree reflects the plasma concentration, and all blood platelets in circulation can recover functions after drug withdrawal.
Warfarin, dabigatran etexilate and rivaroxaban are common clinical anticoagulation medicines. Warfarin is a classic oral anticoagulant used for prevention and treatment of deep vein thrombosis, pulmonary embolism, heart valve, thrombosis in the heart cavity of patients with non-valvular atrial fibrillation, perioperative treatment of surgery, and has been in use for over 50 years. Dabigatran etexilate and rivaroxaban are novel oral anticoagulant drugs, dabigatran etexilate is metabolized into dabigatran in vivo, and the dabigatran directly inhibits a thrombin (blood coagulation factor IIa) active center to play an anticoagulant role; rivaroxaban exerts an anticoagulant effect by inhibiting the formation of thrombin (factor IIa) by inhibiting factor Xa; at present, compared with warfarin, the novel oral anticoagulant drug represented by dabigatran etexilate and rivaroxaban has the advantages of quick response, short half-life period, small interaction between the drug and food, no need of laboratory monitoring, low bleeding risk and the like, and becomes the first choice for anticoagulant treatment of various thrombotic diseases at present.
However, clinically, there is a phenomenon of aspirin resistance, that is, aspirin is not effective in all patients with thrombus, and some patients cannot inhibit platelet aggregation and prevent thrombus formation even if taking aspirin, thus failing to achieve clinical therapeutic effects.
Clopidogrel is a very common clinical resistance phenomenon, and in 3 months of 2010, the FDA in the united states announces a "black box warning" of clopidogrel resistance, and recommends gene detection to remind that adverse cardiovascular events occurring after clopidogrel application are related to alleles with CYP2C19 loss of function.
The blood concentration and bleeding risk of ticagrelor are related to gene polymorphism, and individual differences exist in anticoagulation treatment.
Warfarin has large individuation difference, doctors often cannot easily grasp the clinical dosage of patients, the dosage is required to be fumbled for weeks (months), and the high-incidence period of adverse events is 30-60 days. Excessive warfarin administration can easily cause bleeding risk (1-3%/year), especially for the elderly, and the patients with insufficient dosage can not achieve clinical anticoagulation effect. Warfarin gene testing was recommended by the U.S. Food and Drug Administration (FDA).
The individual differences of the anti-coagulation treatment of the dabigatran etexilate and the rivaroxaban exist generally, and the gene polymorphism in the aspects of drug metabolism and pharmacokinetics is related to the anti-coagulation effect and bleeding risk of the gene dabigatran etexilate.
Accurate medication is an important component of accurate medical treatment. The research of genetics pharmacology and pharmacogenomics proves that the single nucleotide polymorphism of genes (such as drug transport protein genes, drug metabolizing enzyme genes and DNA repair genes) carried by different patients is different, the reaction to the drugs is different, and the curative effect and the side effect of the same drug to different patients are also greatly different. Therefore, from the perspective of the curative effect and the side effect of the medicine, the gene detection is carried out to guide the accurate administration of the medicine, help a doctor to screen out an effective treatment scheme, and make the most correct treatment decision for the patient, so that the invalid medical expense of the patient is saved, the treatment effect is improved, and the side effect of the medicine to the patient is reduced.
The individual differences of the above medicines are closely related to gene polymorphisms. Although the traditional gene polymorphism detection methods such as sanger sequencing, fluorescent quantitative PCR and the like may provide better value clinically, if a plurality of SNP sites are detected simultaneously, the methods have the reasons of time consumption, high economic cost, high time cost and the like, and are not favorable for large-scale clinical popularization and application.
The matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) technology can simultaneously detect a plurality of gene polymorphic sites, has the advantages of strong compatibility, high flux, high accuracy and high cost performance, and can be used for simultaneously detecting related gene information of accurate medication of aspirin, clopidogrel, ticagrelor, warfarin, dabigatran etexilate and rivaroxaban. At present, the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technology is not applied to accurate drug gene detection of anticoagulation and/or antiplatelet drug resistance.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a special primer for detecting an SNP locus of a gene related to drug resistance of anticoagulant and/or antiplatelet drugs and application thereof.
The invention provides a special primer for detecting anticoagulant and/or antiplatelet drug resistance related gene SNP sites, which comprises a primer group 1 and a primer group 2, wherein: the primer group 1 is a specific PCR primer and comprises a primer pair 1-15; the primer group 2 is a single-base extension primer, and comprises 1-15 single-base extension primers.
In a second aspect, the present invention provides a preparation for detecting SNP sites of genes associated with drug resistance of anticoagulation and/or antiplatelet drugs, which comprises a first reagent and a second reagent; wherein:
the first reagent comprises the primer pair 1-the primer pair 15 in the primer set 1 according to the first aspect of the invention, and PCR buffer solution, MgCl 2 Dntps and DNA polymerase;
the second reagent comprises the single base extension primer 1-the single base extension primer 15 in the primer set 2 according to the first aspect of the invention, and a single base extension buffer, a single base extension enzyme, and a single base extension termination Mix.
In a third aspect, the invention provides a special primer for detecting an SNP site of a gene related to anticoagulant and/or antiplatelet drug resistance according to the first aspect of the invention, or an application of a preparation for detecting an SNP site of a gene related to anticoagulant and/or antiplatelet drug resistance according to the second aspect of the invention in preparation of a product for detecting anticoagulant and/or antiplatelet drug resistance.
In a fourth aspect, the present invention provides a method for detecting SNP sites of genes associated with drug resistance to anticoagulation and/or antiplatelet drugs, the method comprising the steps of:
1) carrying out PCR amplification by using the primer pair 1-the primer pair 15 in the primer set 1 according to the first aspect of the invention and using the genomic DNA of a sample to be detected as a template to obtain a PCR amplification product;
2) carrying out alkaline phosphatase digestion on the PCR amplification product to obtain a digestion product;
3) subjecting the digestion product to a single base extension reaction using the single base extension primer 1-the single base extension primer 15 in the primer set 2 according to the first aspect of the invention to obtain a single base extension reaction product;
4) and purifying the single base extension reaction product, and performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection to obtain the SNP locus information of the related genes related to the drug resistance of the anticoagulant and/or antiplatelet drugs in the sample to be detected.
By using the special primer for detecting the SNP locus of the gene related to the drug resistance of the anticoagulant and/or antiplatelet drug, accurate medication prediction of 6 clinical commonly used anticoagulant and/or antiplatelet drugs can be efficiently completed at the same time through one-time detection; by adopting the special primer and the method for detecting the drug resistance related gene SNP locus of the heart failure drug, the detection time is greatly reduced, the cost is saved, and the special primer and the method have very high application value for guiding the accurate medication of 6 clinical commonly used anticoagulation and/or antiplatelet drugs, and are suitable for popularization and application.
Drawings
Fig. 1 shows the detection result of rs730012 in embodiment 2 of the present invention.
Fig. 2 shows the detection result of rs4986893 in example 2 of the present invention.
Fig. 3 shows the detection result of rs4148738 in example 2 of the present invention.
Fig. 4 shows the detection result of rs6065 in embodiment 2 of the present invention.
Fig. 5 shows a detection result of rs1045642 in embodiment 2 of the present invention.
Fig. 6 is a detection result of rs113681054 in embodiment 2 of the present invention.
Fig. 7 shows the detection result of rs1330344 in embodiment 2 of the present invention.
Fig. 8 shows the detection result of rs4149056 in embodiment 2 of the present invention.
Fig. 9 shows the detection result of rs4244285 in example 2 of the present invention.
Fig. 10 shows the detection result of rs28399504 in embodiment 2 of the present invention.
Fig. 11 is a result of rs1057910 detection in embodiment 2 of the present invention.
Fig. 12 shows the detection result of rs2244613 in embodiment 2 of the present invention.
Fig. 13 shows the detection result of rs2032582 in embodiment 2 of the present invention.
Fig. 14 shows a detection result of rs9923231 of embodiment 2 of the present invention.
Fig. 15 shows the detection result of rs8192935 in embodiment 2 of the present invention.
Detailed Description
In order to make the technical solution, objects and advantages of the present invention clearer, the present invention is further described in detail by the following specific embodiments. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
The first aspect of the invention provides a special primer for detecting an SNP locus of an anticoagulant and/or antiplatelet drug resistance related gene, which comprises a primer group 1 and a primer group 2; wherein:
the primer group 1 is a specific PCR primer, and comprises a primer pair 1, a primer pair 2, a primer pair 3, a primer pair 4, a primer pair 5, a primer pair 6, a primer pair 7, a primer pair 8, a primer pair 9, a primer pair 10, a primer pair 11, a primer pair 12, a primer pair 13, a primer pair 14 and a primer pair 15;
the primer pair 1 consists of a single-stranded DNA molecule shown in a sequence 1 and a single-stranded DNA molecule shown in a sequence 2;
the primer pair 2 consists of a single-stranded DNA molecule shown in a sequence 3 and a single-stranded DNA molecule shown in a sequence 4;
the primer pair 3 consists of a single-stranded DNA molecule shown in a sequence 5 and a single-stranded DNA molecule shown in a sequence 6;
the primer pair 4 consists of a single-stranded DNA molecule shown in a sequence 7 and a single-stranded DNA molecule shown in a sequence 8;
the primer pair 5 consists of a single-stranded DNA molecule shown in a sequence 9 and a single-stranded DNA molecule shown in a sequence 10;
the primer pair 6 consists of a single-stranded DNA molecule shown in a sequence 11 and a single-stranded DNA molecule shown in a sequence 12;
the primer pair 7 consists of a single-stranded DNA molecule shown in a sequence 13 and a single-stranded DNA molecule shown in a sequence 14;
the primer pair 8 consists of a single-stranded DNA molecule shown in a sequence 15 and a single-stranded DNA molecule shown in a sequence 16;
the primer pair 9 consists of a single-stranded DNA molecule shown in a sequence 17 and a single-stranded DNA molecule shown in a sequence 18;
the primer pair 10 consists of a single-stranded DNA molecule shown in a sequence 19 and a single-stranded DNA molecule shown in a sequence 20;
the primer pair 11 consists of a single-stranded DNA molecule shown in a sequence 21 and a single-stranded DNA molecule shown in a sequence 22;
the primer pair 12 consists of a single-stranded DNA molecule shown in a sequence 23 and a single-stranded DNA molecule shown in a sequence 24;
the primer pair 13 consists of a single-stranded DNA molecule shown in a sequence 25 and a single-stranded DNA molecule shown in a sequence 26;
the primer pair 14 consists of a single-stranded DNA molecule shown in a sequence 27 and a single-stranded DNA molecule shown in a sequence 28;
the primer pair 15 consists of a single-stranded DNA molecule shown in a sequence 29 and a single-stranded DNA molecule shown in a sequence 30.
The primer group 2 is a single-base extension primer (UEP) which is composed of a series of single-base primers with gradually increased molecular weights and gradually increased base numbers, and comprises a single-base extension primer 1, a single-base extension primer 2, a single-base extension primer 3, a single-base extension primer 4, a single-base extension primer 5, a single-base extension primer 6, a single-base extension primer 7, a single-base extension primer 8, a single-base extension primer 9, a single-base extension primer 10, a single-base extension primer 11, a single-base extension primer 12, a single-base extension primer 13, a single-base extension primer 14 and a single-base extension primer 15; each primer of the series only extends one base, the molecular weight of 15 extension products is increased in a gradient manner, and the number of bases is also increased in a gradient manner.
The single-base extension primer 1 is a single-stranded DNA molecule shown in a sequence 31;
the single-base extension primer 2 is a single-stranded DNA molecule shown in a sequence 32;
the single-base extension primer 3 is a single-stranded DNA molecule shown in a sequence 33;
the single-base extension primer 4 is a single-stranded DNA molecule shown in a sequence 34;
the single-base extension primer 5 is a single-stranded DNA molecule shown in a sequence 35;
the single-base extension primer 6 is a single-stranded DNA molecule shown in a sequence 36;
the single-base extension primer 7 is a single-stranded DNA molecule shown in a sequence 37;
the single-base extension primer 8 is a single-stranded DNA molecule shown as a sequence 38;
the single-base extension primer 9 is a single-stranded DNA molecule shown in a sequence 39;
the single-base extension primer 10 is a single-stranded DNA molecule shown in a sequence 40;
the single-base extension primer 11 is a single-stranded DNA molecule shown in a sequence 41;
the single-base extension primer 12 is a single-stranded DNA molecule shown in a sequence 42;
the single-base extension primer 13 is a single-stranded DNA molecule shown in sequence 43;
the single-base extension primer 14 is a single-stranded DNA molecule shown as a sequence 44;
the single-base extension primer 15 is a single-stranded DNA molecule shown in sequence 45.
The primers (single-stranded DNA molecules represented by SEQ ID Nos. 1 to 30) in the primer set 1 to the primer set 15 were mixed at an equimolar ratio to constitute an amplification primer Mix.
The single-base extension primers 1 to 15 (single-stranded DNA molecules represented by SEQ ID Nos. 31 to 45) in the primer set 2 were mixed at an equimolar ratio to constitute a single-base extension primer Mix.
Wherein the SNP sites are rs730012 (corresponding to primer pair 1 and single-stranded extension primer 1), rs4986893 (corresponding to primer pair 2 and single-stranded extension primer 2), rs4148738 (corresponding to primer pair 3 and single-stranded extension primer 3), rs6065 (corresponding to primer pair 4 and single-stranded extension primer 4), rs1045642 (corresponding to primer pair 5 and single-stranded extension primer 5), rs113681054 (corresponding to primer pair 6 and single-stranded extension primer 6), rs1330344 (corresponding to primer pair 7 and single-stranded extension primer 7), rs4149056 (corresponding to primer pair 8 and single-stranded extension primer 8), rs4244285 (corresponding to primer pair 9 and single-stranded extension primer 9), rs28399504 (corresponding to primer pair 10 and extension primer 10), rs1057910 (corresponding to primer pair 11 and single-stranded extension primer 11), rs2244613 (corresponding to primer pair 12 and single-stranded extension primer 12), 2032582 (corresponding to primer pair 13 and single-stranded extension primer 13), rs 9931 (corresponding primer pair 14 and single-stranded extension primer 14), rs8192935 (corresponding to primer pair 15 and single-stranded extension primer 15).
The special primer provided by the first aspect of the invention ensures the specificity and correctness of the primer from the following two aspects:
1. the single base extension primer is specifically combined with the product of the first PCR amplification, so that the specificity of the first PCR primer and the product is ensured;
2. the molecular weight of the product after the single base extension reaction is automatically generated by the primer design result, and if the molecular weight of the PCR primer or the single base extension primer or the reaction product does not accord with the correct molecular weight, the subsequent experiment fails, so the reliability and the correctness of the result are further ensured, and the occurrence of false positive is prevented.
In a second aspect of the present invention, there is provided an agent for detecting SNP sites of genes associated with drug resistance to anticoagulation and/or antiplatelet drugs, the agent comprising a first agent and a second agent; wherein:
the first reagent comprises the primer pair 1-the primer pair 15 in the primer set 1 according to the first aspect of the invention, and PCR buffer solution, MgCl 2 dNTPs and DNA polymerase.
The second reagent comprises the single base extension primer 1-the single base extension primer 15 in the primer set 2 according to the first aspect of the invention, and a single base extension buffer, a single base extension enzyme, and a single base extension termination Mix.
In the reagent I, the concentration of 30 primers in the primer pairs 1-15 in the reagent I is 0.5. mu.M. The PCR buffer is commercially available, PThe components in the CR buffer are commonly used PCR buffer components. Said MgCl 2 The concentration in reagent one was 2 mM. The concentration of the dNTP in the first reagent is 0.5 mM. The DNA polymerase can be Hotstar Taq DNA polymerase, and the concentration in reagent one is 0.2U/. mu.L.
In the second reagent, the concentrations of the single-base extended primer 1 to the single-base extended primer 15 in the second reagent are all 0.5. mu.M. The single base extension buffer, single base extension enzyme, and single base extension termination Mix are all commercially available, for example, from Agena BIOSCIENCE.
In the preparation for detecting SNP sites of genes related to anticoagulation and/or antiplatelet drug resistance provided by the second aspect of the invention, the first reagent and the second reagent are used in combination, namely, a multiple nested PCR reaction system is formed.
The third aspect of the invention provides a special primer for detecting SNP sites of genes related to anticoagulation and/or antiplatelet drug resistance provided by the first aspect of the invention, or an application of the preparation for detecting SNP sites of genes related to anticoagulation and/or antiplatelet drug resistance provided by the second aspect of the invention in preparation of products for detecting anticoagulation and/or antiplatelet drug resistance.
Wherein the SNP site is as described above.
In a fourth aspect of the present invention, there is provided a method for detecting SNP sites of genes associated with drug resistance to anticoagulation and/or antiplatelet drugs, the method comprising the steps of:
1) carrying out PCR amplification by using the primer pair 1-the primer pair 15 in the primer group 1 and using the genome DNA of a sample to be detected as a template to obtain a PCR amplification product;
2) carrying out alkaline phosphatase digestion on the PCR amplification product to obtain a digestion product;
3) carrying out single base extension reaction on the digestion product by using a single base extension primer 1-a single base extension primer 15 in a primer group 2 to obtain a single base extension reaction product;
4) and purifying the single base extension reaction product, and then carrying out matrix-assisted laser desorption ionization time-of-flight mass spectrometry to obtain the SNP locus information of the gene related to the drug resistance of the anticoagulant and/or antiplatelet drug in the sample to be detected.
Wherein, in the step 1), the PCR amplification procedure is as follows:
pre-denaturation at 94 ℃ for 2 min; then carrying out 45 cycles at 94 ℃ for 20s, 56 ℃ for 30s and 72 ℃ for 60 s; then 3min at 72 ℃; finally, the mixture is stored at 4 ℃.
In step 2), the alkaline phosphatase digestion conditions are as follows: storing at 37 deg.C, 40min, 85 deg.C, 5min, and 4 deg.C.
In step 3), the procedure of the single base extension reaction is as follows: 30s at 94 ℃; firstly, carrying out 40 cycles of 94 ℃ for 5s, then carrying out 5s at 52 ℃ and 5s at 80 ℃; then 3min at 72 ℃; finally, the mixture is stored at 4 ℃.
In step 4), the purification may be resin purification.
Wherein the SNP site is as defined above.
The special primer, the related preparation and the method for detecting the SNP locus of the related gene of the drug resistance of the anticoagulant and/or antiplatelet drug can efficiently and simultaneously complete the detection of the accurate medication of 6 clinical commonly used anticoagulant and/or antiplatelet therapeutic drugs at one time, have very high application value for guiding the accurate medication of patients with cerebrovascular diseases and toxic and side effects caused by overdose, and are suitable for popularization and application.
Various reagents, materials and the like used in the following examples are commercially available products unless otherwise specified; unless otherwise specified, all the tests and detection methods used in the following examples are conventional in the art and can be obtained from textbooks, tool books or academic journals.
Example 1
This example illustrates the specific primers provided by the present invention for detecting SNP sites of anticoagulant and/or antiplatelet drug resistance related genes.
6 drugs are commonly used according to anticoagulation and/or antiplatelet: SNP sites related to precise medication of warfarin, aspirin, clopidogrel, dabigatran etexilate, rivaroxaban, ticagrelor: rs730012, rs6065, rs4986893, rs4148738, rs1045642, rs113681054, rs4149056, rs4244285, rs28399504, rs1330344, rs1057910, rs2244613, rs2032582, rs9923231 and rs8192935, and specific PCR primers and single-base extension primers are designed to form a multi-nested PCR reaction system, so that accurate medication early warning detection of the 6 medicines for anticoagulation and/or antiplatelet treatment is realized by adopting matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) technology (MASSARRAY platform).
The specific PCR primer and the single-base extension primer comprise:
Sequence 14, rs1330344 PCR primer 2: 5'-ACGTTGGATGAAGAAACACTTGTGTGGCCC-3' are provided.
Sequence 16, rs4149056 PCR primer 2: 5'-ACGTTGGATGAATCTGGGTCATACATGTGG-3' are provided.
Sequence 17, rs4244285PCR primer 1: 5'-ACGTTGGATGCACTTTCCATAAAAGCAAGG-3' are provided.
Sequence 19, rs28399504 PCR primer 1: 5'-ACGTTGGATGAGTGCAAGCTCACGGTTGTC-3' are provided.
Sequence 21, rs1057910 PCR primer 1: 5'-ACGTTGGATGTGTCACAGGTCACTGCATGG-3' are provided.
Sequence 22, rs1057910 PCR primer 2: 5'-ACGTTGGATGCTACACAGATGCTGTGGTGC-3' is added.
Sequence 23, rs2244613 PCR primer 1: 5'-ACGTTGGATGATTGAGGCAGGACTTTCTGG-3' are provided.
Sequence 24, rs2244613 PCR primer 2: 5'-ACGTTGGATGTCAGTTCCTTAGCAATGCAC-3' is added.
Sequence 27, rs9923231 PCR primer 1: 5'-ACGTTGGATGGCTAGGATTATAGGCGTGAG-3' are provided.
Sequence 28, rs9923231 PCR primer 2: 5'-ACGTTGGATGTCTGGGAAGTCAAGCAAGAG-3' are provided.
Sequence 29, rs8192935 PCR primer 1: 5'-ACGTTGGATGTCAACAGGCAAAAAAACTGG-3' are provided.
Sequence 32, rs4986893 single base extension primer: 5'-CTTGGCCTTACCTGGAT-3' is added.
Sequence 33, rs4148738 single base extension primer: 5'-GGGGAGGAACTAAAACC-3' are provided.
Sequence 34, rs6065 single base extension primer: 5'-TGCCCCCAGGGCTCCTGA-3' are provided.
Sequence 36, rs113681054 single base extension primer: 5'-CTCGGCTCACTGCAAGCTC-3' are provided.
Sequence 37, rs1330344 single base extension primer: 5'-TGAAGGCTCTTCCCAT-3' are provided.
Sequence 38, rs4149056 single base extension primer: 5'-GAAGCATATTACCCATGAAC-3' are provided.
Sequence 39, rs4244285 single base extension primer: 5'-AAGTAATTTGTTATGGGTTCC-3' are provided.
Sequence 40, rs28399504 single base extension primer: 5'-gTAACAAGAGGAGAAGGCTTCA-3' are provided.
Sequence 41, i.e. rs1057910 single base extension primer: 5'-CAGGCTGGTGGGGAGAAGGTCAA-3' are provided.
Sequence 44, rs9923231 single base extension primer: 5'-GGATTATAGGCGTGAGCCACCGCACC-3' are provided.
Sequence 45, rs8192935 single base extension primer: 5'-GAATACAAACAATATATTACATCATAAT-3' is added.
Example 2
This example illustrates the method for detecting SNP sites of genes associated with drug resistance of anticoagulant and/or antiplatelet drugs provided by the present invention.
1. Template whole genome DNA extraction
Genomic DNA of 384 exceptional week blood is extracted and uniformly diluted to 20-30 ng/. mu.L.
2. PCR reaction
The reaction was prepared in 5 μ L PCR 96-well plates: mu.L of template DNA, 1. mu.L of amplification primer Mix, and PCR buffer (containing 15mM Mg) 2+ )0.5μL，MgCl 2 mu.L (25mM), 0.4. mu.L dNTP (25mM), 0.1. mu.L Hotstar Taq (5U/. mu.L), and 5. mu.L of the resulting solution was supplemented with MBG (molecular Biology grade) water. The 384 well plates were sealed with a sealing membrane.
The PCR amplification primers Mix consist of primers shown in sequences 1-30 and water, wherein the concentration of each primer is 0.5. mu.M.
The PCR reaction program was set up as follows:
3. alkaline phosphatase treatment (SAP digestion reaction)
Adding the following components into the PCR reaction product obtained in the step 2 to obtain an SAP digestion reaction system:
SAP Buffer 0.17. mu.L, SAP Enzyme 0.30. mu.L, MBG water 1.53. mu.L.
Performing SAP digestion reaction on the SAP digestion reaction system according to the following digestion reaction program:
37℃，40min；85℃，5min；4℃，∞。
4. single base extension reaction
Adding the following components into the digestion reaction product obtained in the step 3 to obtain a single base extension reaction system:
0.2. mu.L of single-base extension Buffer iPLEX Buffer plus (Agena BIOSCIENCE, Ref:01431, lot:0000021844), 0.041. mu.L of single-base extension Enzyme iPLEX Enzyme (Agena BIOSCIENCE, Ref:01432, lot:0000021845), 0.2. mu.L of single-base extension termination Mix iPLEX Terminator (Agena BIOSCIENCE, Ref:01430, lot:0000021435), 0.619. mu.L of MBG water, and 0.940. mu.L of single-base extension primer.
The above single-base extension primer Mix consisted of the above 15 specific primers (SEQ ID Nos. 31 to 45) and water, wherein the concentration of each primer was 0.5. mu.M.
The single base extension reaction was carried out using the above single base extension reaction system according to the following reaction procedure to obtain a single base extension reaction product.
Single base extension reaction procedure:
5. resin purification
And (3) performing resin purification on the single base extension reaction product obtained in the step (4), wherein the resin purification comprises the following steps:
1) and (3) centrifuging the 384 reaction plates containing the single-base extension products obtained in the step (4) at 3000rpm for 2min, adding 16 mu L of MBG water into each hole, sealing the reaction plates by using a sealing film, and performing instant centrifugation at 3000 rpm.
2) A clean A4 paper is taken, a resin plate (specification is 6mg) is placed on the paper, a proper amount of resin (Agena BIOSCIENCE, product ref:08040 lot:0000022403) is taken by a spoon and placed on the resin plate, a plastic cover plate is used for repeatedly pushing the resin from side to side, and the resin is compacted to ensure that the resin content in each hole is uniform.
3) The centrifuged 384 reaction plate was inverted over the resin plate with the resin laid, with the wells of the two plates in one-to-one correspondence. The plates were inverted with the resin plate on top and the 384 reaction plate on the bottom. The back of the resin plate was gently tapped evenly to drop the resin into a 384 reaction plate containing the single base extension product.
4) The 384 reaction plate containing the single base extension product and the resin is sealed with a sealing film, and the shaker is rotated vertically at a low speed for 30min to bring the resin into full contact with the reactants.
5) And centrifuging the 384 reaction plates after the reaction is finished at 3000rpm for 5min to enable the resin to sink to the bottom of the tube, wherein the supernatant is the purified product.
6. And (3) matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) detection:
the purified product obtained in the step 5 is transferred to a chip (Agena BIOSCIENCE, product ref:01509lot:0000022421) through a sample applicator, and is put into a mass spectrometer for mass spectrometry (mass spectrometry detection molecular weight range: 4500-.
As shown in fig. 1-15, SNP locus information results of rs730012, rs6065, rs4986893, rs4148738, rs1045642, rs113681054, rs4149056, rs4244285, rs28399504, rs1330344, rs1057910, rs2244613, rs2032582, rs9923231 and rs8192935 are obtained. As can be seen from these figures, the detection rate of each site was 99% or more, indicating that the primer set and method of the present invention can be used for information detection of these SNP sites.
7. And (4) interpretation of results: by mass spectrum detection results, referring to the following table 1, accurate prediction and drug-forbidden early warning are performed for risks of 6 clinical commonly used anticoagulant and/or antiplatelet therapeutic drugs.
TABLE 1 SNP site information guided phenotypic analysis
Note that: in the above table, if the clinical significance interpretation of the detection results of different detection sites of the same drug by the same patient is inconsistent, the clinician refers to the contribution degree of the different detection sites to the metabolism and resistance of the drug, and further adjusts the targeted drug administration scheme for the patient.
Example 3
This example illustrates the method for detecting SNP sites of 6 genes associated with drug resistance of anticoagulant and/or antiplatelet drugs and the application of the primers.
1. Template DNA (Whole genome) extraction
Peripheral blood of 8 cases of examinees is taken as samples 1-8 to be examined (sequentially corresponding to the examinees 1-8), and the examinees 1-8 are all diagnosed with hypertension, coronary heart disease (stable angina, unstable angina or old myocardial infarction) and peripheral atherosclerotic plaques.
2. And (3) PCR reaction: see example 2, step 2.
3. Alkaline phosphatase treatment (SAP digestion reaction): see example 2, step 3.
4. Single base extension reaction: see example 2, step 4.
5. Resin purification: see example 2, step 5.
6. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection: see example 2, step 6.
According to the detection result, in 8 samples tested in the embodiment, the detection rate of each site is 100%. The results are shown in Table 2.
TABLE 28 test results of samples tested
| Detected | Sample | 1 | |
|
|
|
|
|
|
| rs730012 | CC | AC | AA | CC | CC | AA | AA | AC | |
| Rs6065 | CC | TC | CC | CC | CC | CC | TT | CC | |
| rs1330344 | CC | TC | TC | TC | CC | CC | TT | CC | |
| rs4986893 | GA | AA | GG | GA | GG | GG | GG | AA | |
| rs4244285 | GA | GA | GG | GA | GA | GG | GG | AA | |
| rs28399504 | GA | GA | AA | GA | AA | AA | AA | GG | |
| rs4148738 | TT | CC | CC | TT | CC | CC | TG | CC | |
| rs2244613 | GG | TG | TT | TG | TT | TG | GG | TT | |
| rs2032582 | CC | TT | AA | CC | AT | AA | CC | AC | |
| rs8192935 | GG | AA | AA | GG | AA | AA | GG | AG | |
| rs1045642 | GG | AG | AA | GG | AA | AG | GG | AA | |
| rs113681054 | CC | CC | CT | TT | TT | CC | CT | CC | |
| rs4149056 | CC | CT | CC | TT | TT | CC | CT | CT | |
| rs1057910 | AA | AC | AC | AC | AC | AA | CC | AC | |
| rs9923231 | GG | GA | GA | GA | GA | GG | AA | GA |
From the results of table 2 it can be seen that:
the samples 1 and 6 can be used for normally taking warfarin, and the samples 2, 3, 4, 5, 7 and 8 can reduce the warfarin dosage, so that the clinical treatment effect can be achieved, and the side effect of gastrorrhagia can be reduced.
Clinical sample verification shows that:
the samples 1 and 6 are normally taken with warfarin, and the samples 2, 3, 4, 5, 7 and 8 can reduce the warfarin dosage, so that the clinical treatment effect can be achieved, and the side effect of gastrorrhagia is avoided.
Therefore, the detection result of the special primer and the method for detecting the SNP locus of the gene related to the drug resistance of the anticoagulant and/or antiplatelet drug is completely consistent with the clinical phenotype, and the special primer and the method for detecting the SNP locus of the gene related to the drug resistance of the anticoagulant and/or antiplatelet drug can well predict the risk of the drug resistance of the anticoagulant and/or antiplatelet drug.
Comparative example 1
The design of the primer is the key of the invention, the combination rate of the primer and the template directly influences the PCR amplification and single base extension efficiency, thereby influencing the sensitivity and the detection rate of the detection result, in particular, the design requirement of the single base extension primer is very high, except for the requirement that the sequence is completely matched with the sequence in front of the detection site in a base complementary manner, the length design of the 15 single base extension primers needs to lead the product to be increased in a gradient manner on the molecular weight, so that the product generates the molecular weight difference, and the mass spectrum detection is realized.
For each SNP site, the inventors designed 3 sets of PCR primers and single-base extension primers in parallel (primer set 3 in examples 1 to 3, and primer sets 1 to 2 in tables 3 to 4), and tested 384 samples with the primer sets 1 to 2, respectively, according to the method of example 2 (i.e., the operating parameters in this comparative example were the same as those in example 2 except that the primer set 3 used in example 2 was replaced with the primer set 1 in Table 3 or the primer set 2 in Table 4).
TABLE 3 primer set 1
TABLE 4 primer set 2
The detection result shows that the conditions of low site detection rate and poor clustering effect are found by using the primer group 1 or the primer group 2. The specific detection rates were as follows:
in the 384 samples tested, the detection rate of each site when using primer set 1 was as follows: the detection rate of rs730012 is 58.2%, the detection rate of rs6065 is 39.5%, the detection rate of rs4986893 is 82.6%, the detection rate of rs4148738 is 59.2%, the detection rate of rs1045642 is 91.3%, the detection rate of rs113681054 is 88.2%, the detection rate of rs4149056 is 23.6%, the detection rate of rs4244285 is 56.3%, the detection rate of rs28399504 is 59.3%, the detection rate of rs1330344 is 93.5%, the detection rate of rs1057910 is 59.2%, the detection rate of rs2244613 is 88.6%, the detection rate of rs2032582 is 62.3%, the detection rate of rs9923231 is 56.9%, and the detection rate of rs8192935 is 85.6%.
In the 384 samples tested, the detection rate of each site was as follows, using primer set 2: the detection rate of rs730012 is 59.3%, the detection rate of rs6065 is 85.2%, the detection rate of rs4986893 is 92.3%, the detection rate of rs4148738 is 88.3%, the detection rate of rs1045642 is 56.3%, the detection rate of rs113681054 is 33.7%, the detection rate of rs4149056 is 59.7%, the detection rate of rs4244285 is 51.9%, the detection rate of rs28399504 is 92.4%, the detection rate of rs1330344 is 53.0%, the detection rate of rs1057910 is 91.4%, the detection rate of rs2244613 is 20.6%, the detection rate of rs2032582 is 56.3%, the detection rate of rs9923231 is 71.6%, and the detection rate of rs 81935 is 56.3%.
Therefore, compared with the comparative example, the primer group 3 in the embodiment 2 has the advantages of high detection rate, high sensitivity, good clustering effect, high specificity and high reliability.
Comparative example 2
The same procedure as in example 2 was repeated, except that a primer set against the relevant site rs671 was additionally added to all primers used in example 2.
Wherein, the primer sequence of the nitroglycerin resistance related site rs671 is as follows:
rs671-PCR primer 1: 5'-ACGTTGGATGTTGGTGGCTACAAGATGTCG-3', respectively;
rs671-PCR primer 2: 5'-ACGTTGGATGAGGTCCCACACTCACAGTTT-3', respectively;
rs671-UEP primer: 5'-CCACACTCACAGTTTTCACTT-3' are provided.
As a result, it was found that, while maintaining the various operating parameters of example 2, even if 1 primer set of nitroglycerin other than the 15 sites against the rs671 site was added, the compatibility of the reaction system and the reaction conditions of example 2 was deteriorated, resulting in a decrease in the detection rate of the 15 SNP sites and a deterioration in the clustering effect.
Specifically, the detection rates of the respective sites were as follows: the detection rate of rs730012 is 85.3%, the detection rate of rs6065 is 56.1%, the detection rate of rs4986893 is 56.2%, the detection rate of rs4148738 is 85.0%, the detection rate of rs1045642 is 51.4%, the detection rate of rs113681054 is 90.3%, the detection rate of rs4149056 is 85.3%, the detection rate of rs4244285 is 47.6%, the detection rate of rs28399504 is 59.3%, the detection rate of rs1330344 is 88.6%, the detection rate of rs1057910 is 56.3%, the detection rate of rs2244613 is 90.2%, the detection rate of rs2032582 is 85.3%, the detection rate of rs9923231 is 74.9%, and the detection rate of rs8192935 is 85.3%.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Beijing university Hospital
<120> special primer for detecting anticoagulant and antiplatelet drug resistance related gene SNP locus and application
<160> 45
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
acgttggatg actcctccac ccaccttatc 30
<210> 2
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
acgttggatg ttctgaagcc aaaggcactg 30
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
acgttggatg gactgtaagt ggtttctcag 30
<210> 4
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
acgttggatg aacatcagga ttgtaagcac 30
<210> 5
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
acgttggatg gtaaagacta gggttgaggg 30
<210> 6
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
acgttggatg gagaggactg ttggatctac 30
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
acgttggatg ttagccagac tgagcttctc 30
<210> 8
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
acgttggatg acctgaaagg caatgagctg 30
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
acgttggatg ttgcctatgg agacaacagc 30
<210> 10
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
acgttggatg aaggcatgta tgttggcctc 30
<210> 11
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
acgttggatg agtgcagtgg tgggatctc 29
<210> 12
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
acgttggatg cctgtagtcc cagctacttg 30
<210> 13
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
acgttggatg aggtcacgct atggaagaag 30
<210> 14
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
acgttggatg aagaaacact tgtgtggccc 30
<210> 15
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
acgttggatg ccaatggtac tatgggagtc 30
<210> 16
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
acgttggatg aatctgggtc atacatgtgg 30
<210> 17
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
acgttggatg cactttccat aaaagcaagg 30
<210> 18
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
acgttggatg gcaataattt tcccactatc 30
<210> 19
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
acgttggatg agtgcaagct cacggttgtc 30
<210> 20
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
acgttggatg catgagagac agagcacaag 30
<210> 21
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
acgttggatg tgtcacaggt cactgcatgg 30
<210> 22
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
acgttggatg ctacacagat gctgtggtgc 30
<210> 23
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
acgttggatg attgaggcag gactttctgg 30
<210> 24
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
acgttggatg tcagttcctt agcaatgcac 30
<210> 25
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
acgttggatg gtctggacaa gcactgaaag 30
<210> 26
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
acgttggatg agtaagcagt agggagtaac 30
<210> 27
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
acgttggatg gctaggatta taggcgtgag 30
<210> 28
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
acgttggatg tctgggaagt caagcaagag 30
<210> 29
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
acgttggatg tcaacaggca aaaaaactgg 30
<210> 30
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
acgttggatg atattccacc acccagagag 30
<210> 31
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ccaccttatc tgttccc 17
<210> 32
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
cttggcctta cctggat 17
<210> 33
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ggggaggaac taaaacc 17
<210> 34
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
<210> 35
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
<210> 36
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ctcggctcac tgcaagctc 19
<210> 37
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
tgaaggctct tcccat 16
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
<210> 39
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
aagtaatttg ttatgggttc c 21
<210> 40
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gtaacaagag gagaaggctt ca 22
<210> 41
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
caggctggtg gggagaaggt caa 23
<210> 42
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
ggctggagaa gctgcatcgc tcacc 25
<210> 43
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gaaagataag aaagaactag aaggt 25
<210> 44
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
ggattatagg cgtgagccac cgcacc 26
<210> 45
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
gaatacaaac aatatattac atcataat 28
Claims (6)
1. A special primer for detecting anticoagulant and/or antiplatelet drug resistance related gene SNP locus is characterized in that:
the special primer consists of a primer group 1 and a primer group 2, wherein:
the primer group 1 is a specific PCR primer and consists of the following primers: primer pair 1, primer pair 2, primer pair 3, primer pair 4, primer pair 5, primer pair 6, primer pair 7, primer pair 8, primer pair 9, primer pair 10, primer pair 11, primer pair 12, primer pair 13, primer pair 14, and primer pair 15;
the primer pair 1 consists of a single-stranded DNA molecule shown in a sequence 1 and a single-stranded DNA molecule shown in a sequence 2;
the primer pair 2 consists of a single-stranded DNA molecule shown in a sequence 3 and a single-stranded DNA molecule shown in a sequence 4;
the primer pair 3 consists of a single-stranded DNA molecule shown in a sequence 5 and a single-stranded DNA molecule shown in a sequence 6;
the primer pair 4 consists of a single-stranded DNA molecule shown in a sequence 7 and a single-stranded DNA molecule shown in a sequence 8;
the primer pair 5 consists of a single-stranded DNA molecule shown in a sequence 9 and a single-stranded DNA molecule shown in a sequence 10;
the primer pair 6 consists of a single-stranded DNA molecule shown in a sequence 11 and a single-stranded DNA molecule shown in a sequence 12;
the primer pair 7 consists of a single-stranded DNA molecule shown in a sequence 13 and a single-stranded DNA molecule shown in a sequence 14;
the primer pair 8 consists of a single-stranded DNA molecule shown in a sequence 15 and a single-stranded DNA molecule shown in a sequence 16;
the primer pair 9 consists of a single-stranded DNA molecule shown in a sequence 17 and a single-stranded DNA molecule shown in a sequence 18;
the primer pair 10 consists of a single-stranded DNA molecule shown in a sequence 19 and a single-stranded DNA molecule shown in a sequence 20;
the primer pair 11 consists of a single-stranded DNA molecule shown in a sequence 21 and a single-stranded DNA molecule shown in a sequence 22;
the primer pair 12 consists of a single-stranded DNA molecule shown in a sequence 23 and a single-stranded DNA molecule shown in a sequence 24;
the primer pair 13 consists of a single-stranded DNA molecule shown in a sequence 25 and a single-stranded DNA molecule shown in a sequence 26;
the primer pair 14 consists of a single-stranded DNA molecule shown in a sequence 27 and a single-stranded DNA molecule shown in a sequence 28;
the primer pair 15 consists of a single-stranded DNA molecule shown in a sequence 29 and a single-stranded DNA molecule shown in a sequence 30;
the primer group 2 is a single-base extension primer and consists of the following primers: a single-base extension primer 1, a single-base extension primer 2, a single-base extension primer 3, a single-base extension primer 4, a single-base extension primer 5, a single-base extension primer 6, a single-base extension primer 7, a single-base extension primer 8, a single-base extension primer 9, a single-base extension primer 10, a single-base extension primer 11, a single-base extension primer 12, a single-base extension primer 13, a single-base extension primer 14, and a single-base extension primer 15;
the single-base extension primer 1 is a single-stranded DNA molecule shown in a sequence 31;
the single-base extension primer 2 is a single-stranded DNA molecule shown in a sequence 32;
the single-base extension primer 3 is a single-stranded DNA molecule shown in a sequence 33;
the single-base extension primer 4 is a single-stranded DNA molecule shown in a sequence 34;
the single-base extension primer 5 is a single-stranded DNA molecule shown in a sequence 35;
the single-base extension primer 6 is a single-stranded DNA molecule shown in a sequence 36;
the single-base extension primer 7 is a single-stranded DNA molecule shown in a sequence 37;
the single-base extension primer 8 is a single-stranded DNA molecule shown as a sequence 38;
the single-base extension primer 9 is a single-stranded DNA molecule shown in a sequence 39;
the single-base extension primer 10 is a single-stranded DNA molecule shown in a sequence 40;
the single-base extension primer 11 is a single-stranded DNA molecule shown in a sequence 41;
the single-base extension primer 12 is a single-stranded DNA molecule shown as a sequence 42;
the single-base extension primer 13 is a single-stranded DNA molecule shown in a sequence 43;
the single-base extension primer 14 is a single-stranded DNA molecule shown as a sequence 44;
the single-base extension primer 15 is a single-stranded DNA molecule shown as a sequence 45;
wherein the anticoagulant drug is warfarin, dabigatran etexilate or rivaroxaban; the antiplatelet drugs are aspirin, clopidogrel and ticagrelor;
the SNP loci are rs730012, rs4986893, rs4148738, rs6065, rs1045642, rs113681054, rs1330344, rs4149056, rs4244285, rs28399504, rs1057910, rs2244613, rs2032582, rs9923231 and rs 8192935.
2. A preparation for detecting SNP sites of genes related to drug resistance of anticoagulation and/or antiplatelet drugs, which is characterized in that:
the primers in the preparation consist of the specific primers of claim 1.
3. The formulation of claim 2, wherein:
the preparation comprises a first reagent and a second reagent;
the reagent comprises the primer group 1 of claim 1, PCR buffer solution and MgCl 2 Dntps and DNA polymerase;
the reagent two comprises the primer group 2 of claim 1, a single base extension buffer, a single base extension enzyme, and a single base extension termination Mix.
4. The formulation of claim 3, wherein:
the primers in the primer pair 1-the primer pair 15 in the primer group 1 are mixed in an equimolar ratio to form an amplification primer Mix;
the single-base extension primers 1 to 15 in the primer set 2 were mixed at an equimolar ratio to constitute a single-base extension primer Mix.
5. The formulation of claim 4, wherein:
in the reagent I, the concentration of 30 primers in the primer pairs 1-15 in the reagent I is 0.5 mu M;
said MgCl 2 The concentration in the first reagent is 2 mM;
the concentration of the dNTP in the reagent I is 0.5 mM;
the concentration of the DNA polymerase in the first reagent is 0.2U/. mu.L;
in the second reagent, the concentrations of the single-base extended primer 1 to the single-base extended primer 15 in the second reagent are all 0.5. mu.M.
6. Use of a primer specific for use according to claim 1, or a formulation according to any one of claims 2 to 5, for the preparation of a product for the detection of anticoagulant and/or antiplatelet drug resistance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011458539.3A CN112553319B (en) | 2020-12-10 | 2020-12-10 | Special primer for detecting anticoagulant and/or antiplatelet drug resistance related gene SNP locus and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011458539.3A CN112553319B (en) | 2020-12-10 | 2020-12-10 | Special primer for detecting anticoagulant and/or antiplatelet drug resistance related gene SNP locus and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112553319A CN112553319A (en) | 2021-03-26 |
| CN112553319B true CN112553319B (en) | 2022-09-09 |
Family
ID=75062237
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011458539.3A Active CN112553319B (en) | 2020-12-10 | 2020-12-10 | Special primer for detecting anticoagulant and/or antiplatelet drug resistance related gene SNP locus and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112553319B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215244A (en) * | 2021-05-21 | 2021-08-06 | 广州合一生物科技有限公司 | Gene detection kit for antiplatelet drug personalized medication and application thereof |
| CN117512093A (en) * | 2023-11-07 | 2024-02-06 | 瑞因生物科技(广州)有限公司 | Primer group, reagent and method for detecting gene locus of human anticoagulation related medicine |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106893783A (en) * | 2017-04-05 | 2017-06-27 | 李爱娟 | It is a kind of for the accurate early warning of cardiovascular and cerebrovascular disease risk and the detection method and primer special of accurate medication |
| CN107022611A (en) * | 2017-04-05 | 2017-08-08 | 李爱娟 | It is a kind of to be used for the method and primer special of 4 kinds of accurate medications of common clinical cardiovascular and cerebrovascular disease medicine of detection |
| CN108070646A (en) * | 2017-06-23 | 2018-05-25 | 安徽安龙基因医学检验所有限公司 | A kind of accurate medication genetic test Solid phase PCR kit of cardiovascular and cerebrovascular disease |
-
2020
- 2020-12-10 CN CN202011458539.3A patent/CN112553319B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106893783A (en) * | 2017-04-05 | 2017-06-27 | 李爱娟 | It is a kind of for the accurate early warning of cardiovascular and cerebrovascular disease risk and the detection method and primer special of accurate medication |
| CN107022611A (en) * | 2017-04-05 | 2017-08-08 | 李爱娟 | It is a kind of to be used for the method and primer special of 4 kinds of accurate medications of common clinical cardiovascular and cerebrovascular disease medicine of detection |
| CN108070646A (en) * | 2017-06-23 | 2018-05-25 | 安徽安龙基因医学检验所有限公司 | A kind of accurate medication genetic test Solid phase PCR kit of cardiovascular and cerebrovascular disease |
Non-Patent Citations (3)
| Title |
|---|
| Drug-response related genetic architecture of Bangladeshi population;Tamim Ahsan等;《Meta Gene》;20190509;https://doi.org/10.1016/j.mgene.2019.100585，第1-15页 * |
| 基因多态性与新型口服抗凝药个体化用药概述;张君 等;《中国药师》;20191231;第22卷(第12期);2262-2266 * |
| 替格瑞洛所致呼吸困难与血浆中替格瑞洛和腺苷浓度的相关性;宋娟;《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》;20190115(第12期);第E079-145篇，论文全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112553319A (en) | 2021-03-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106893783B (en) | A kind of detection method and primer special for the accurate early warning of cardiovascular and cerebrovascular disease risk and accurate medication | |
| CN112553319B (en) | Special primer for detecting anticoagulant and/or antiplatelet drug resistance related gene SNP locus and application | |
| CN107034273B (en) | CYP2C19 and ABCB1 gene detecting kits | |
| CN107022611B (en) | Method and special primer for detecting accurate medication of 4 common clinical cardiovascular and cerebrovascular disease medicines | |
| CN111808944A (en) | Gene detection method for children personalized medicine | |
| CN107904306B (en) | Nucleic acid mass spectrum detection kit for detecting clopidogrel personalized medication related gene polymorphism sites and application | |
| CN106947815B (en) | Method and special primer for detecting accurate medication of aspirin and nitroglycerin | |
| WO2014151940A1 (en) | Method and system to predict ssri response | |
| CN111187824A (en) | Nicholol medication guide gene detection kit for antihypertensive drug | |
| CN111235251A (en) | Kit for detecting gene of nitrendipine individualized medication guidance of antihypertensive drug | |
| CN111235252A (en) | Method for distinguishing individual medication of nitrendipine by mass spectrometry through detecting product | |
| CN106701923B (en) | SNP marker related to treatment of ischemic stroke by antiplatelet drug clopidogrel and application thereof | |
| TW202146660A (en) | Compositions and methods for assessing the efficacy of inhibitors of neurotransmitter transporters | |
| CN111187823A (en) | Primer composition for distinguishing Nichol individual medicine type | |
| CN111235254A (en) | Primer composition for distinguishing nitrendipine individualized medication type | |
| CN111187820A (en) | Method for distinguishing Nistrol personalized medicine by detecting product and performing mass spectrometry | |
| CN111187822A (en) | Detection product for distinguishing individual drug type of Nicholol | |
| CN111187821A (en) | Method for distinguishing Nicol lol personalized medicine by using primer composition to perform mass spectrometry | |
| CN111235255A (en) | Method for distinguishing individual medication of nitrendipine by using primer composition through mass spectrometry | |
| CN111235253A (en) | Detection product for distinguishing nitrendipine individualized medication type | |
| CN111206083A (en) | Detection kit for medication guidance gene of telmisartan medicament for reducing hypertension | |
| CN112980946B (en) | Gene polymorphism detection kit and detection method for guiding administration of rocuronium bromide serving as skeletal muscle relaxant | |
| Wang et al. | Comparison of the antiplatelet effect of clopidogrel benzene sulfonate and clopidogrel hydrogen sulfate in stable coronary heart disease | |
| Maza et al. | Development of a Clinical Focus Pharmacovigilance System | |
| CN117051101A (en) | Primer and kit for detecting personalized medicine genes for cardiovascular and cerebrovascular diseases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |