CN109423513B - Human cytochrome CYP2C19 and PAR1 gene polymorphic site detection kit and application thereof - Google Patents

Abstract

The invention provides a detection method for genetic correlation of a stroke medicine, which adopts a method based on cytochrome P450CYP2C19 x 2: rs4244285; CYP2C19 x 3: rs4986893; CYP2C19 × 17: rs12248560 and PAR-1 rs168753, and 4 polymorphic sites of 2 genes, and performing real-time fluorescent quantitative allele probe detection to determine whether the target belongs to drug-induced susceptible population. The method can be used for guiding and adjusting clinical medication schemes, providing basis for clinical personalized treatment and preventing adverse reactions of the drugs. The method can simultaneously detect 4 polymorphic sites of 2 genes, and has the advantages of simplicity, convenience, accuracy, rapidness, high throughput and the like.

Description

Human cytochrome CYP2C19 and PAR1 gene polymorphic site detection kit and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a human cytochrome P450CYP2C19 and PAR1 gene polymorphic site detection kit and application thereof.

Background

Ischemic heart disease and ischemic stroke are the first two of global causes of death published by the World Health Organization (WHO), atherosclerosis, plaque rupture and thrombosis are direct causes of cardiovascular and cerebrovascular events, thrombotic diseases become the first killers of human health, and antiplatelet therapy is a main means for preventing thrombotic diseases.

Plaque rupture and thrombosis are the fundamental pathological changes leading to acute cardiovascular events, and antithrombotic, especially antiplatelet, therapy is the most important intervention for acute coronary syndromes. Platelets are anucleate, fragments produced from the cytoplasm of bone marrow megakaryocytes. Its maximum life cycle in the cycle is about 10d. Under normal conditions, platelets are in an inactive state in the blood circulation, and when vascular endothelial injury or atherosclerotic plaque rupture, exposure of the subendothelial matrix, presence of activating factors, the platelets are activated and play an important role in physiological hemostasis.

The antiplatelet drug is developed aiming at each link and related target points of platelet activation, and can act on each stage of platelet activation, adhesion and aggregation in mechanism.

The drugs mainly used at present are:

cyclooxygenase (COX) inhibitors: aspirin can promote acetylation of serine at 529 th site of COX-1 active site, and irreversibly inhibit COX-1 activity. COX-1 plays a key role in the initial steps of prostanoid biosynthesis, catalyzing the conversion of arachidonic acid to prostaglandin H2 (PGH 2), where PGH2 is the direct precursor of TXA 2. Inhibition of COX-1 by aspirin results in a decrease in the production of TXA2, which is a strong platelet multimer, and the decrease in the production of TXA2 ultimately affects the platelet aggregation and release response.

ADP receptor inhibitors: clopidogrel. The medicine is absorbed in small intestine after oral administration, is regulated and controlled by proton pump P-glycoprotein encoded by ABCB1 gene, 85% -95% is hydrolyzed into inactive carboxylic acid metabolite SR26334 by carboxylase after absorption, and only 10% -15% is metabolized into active product by liver cytochrome P450 (CYP 450) enzyme system. The clopidogrel active metabolite R-130964 is irreversibly combined with an ADP receptor (P2Y 12) on the surface of a platelet through a covalent bond when the clopidogrel active metabolite passes through liver circulation, blocks the inhibition effect of ADP on adenylate cyclase, promotes cAMP-dependent VASP phosphorylation, inhibits the combination of fibrinogen and platelet glycoprotein GP IIb/IIIa receptors, and further inhibits the aggregation of the platelet. In addition, clopidogrel can also block platelet activation and amplification caused by secondary ADP mediated activation of glycoprotein GPIIb/IIIa compound, thereby inhibiting platelet aggregation induced by other agonists.

Since clopidogrel must be biologically converted to an active product to inhibit platelet aggregation. The biotransformation process needs cytochrome P450 encoded by CYP2C19 gene to be metabolized in liver, and the active product can selectively and irreversibly block the platelet aggregation dependent on adenosine diphosphate.

Different individuals have different reactivities towards clopidogrel, with about 40% of asian patients still showing residual platelet aggregation after treatment, the so-called "clopidogrel resistance". The main reason is that CYP2C19 x 2, CYP2C19 x 3CYP2C19 gene mutation causes the reduction of active metabolites of clopidogrel. The results of recent clinical studies confirm that the risk of embolism reformation is increased, the risk of cardiovascular and cerebrovascular events is increased and the fatality rate of the weakly metabolized individuals caused by CYP2C19 gene mutation is increased. For individuals with CYP2C19 gene mutation, the dose of clopidogrel (loading dose is 300mg or 600 mg) needs to be increased to achieve the effect of blocking platelet aggregation.

Protease activated receptor-1 (PAR-1) is a G protein coupled receptor on the cell surface, a thrombin receptor. Thrombin is the strongest platelet activator and mediates cellular responses through PAR 1. Human platelets express the PAR1 receptor, PAR1 is able to couple to the G12/13 and Gq families and indirectly activate the Gi signaling pathway by secreting ADP, causing a series of alterations in platelet activation, inducing a coagulation response. Plays a key role in the process of thrombosis and hemostasis.

Therefore, SNP detection by the multiple genes has important clinical significance for realizing personalized medication of antiplatelet drugs and reducing the fatality rate of cardiovascular diseases.

Disclosure of Invention

The invention aims to provide a human cytochrome CYP2C19 and PAR1 gene polymorphic site detection kit and application thereof.

In a first aspect of the invention, the PAR-1.

In another preferred embodiment, the reagent or kit further comprises a detection reagent for detecting one or more genetic loci selected from the group consisting of: CYP2C19 x 2: rs4244285 gene site, CYP2C19 x 3: rs4986893 gene site, and CYP2C19 x 17: rs12248560 gene locus.

In another preferred example, the detected object includes: human or non-human animals (e.g., livestock, poultry, laboratory animals, etc.).

In another preferred embodiment, the antiplatelet agent is selected from the group consisting of:

cyclooxygenase (COX) inhibitors and ADP receptor inhibitors.

In another preferred embodiment, the Cyclooxygenase (COX) inhibitor comprises: aspirin, diclofenac, meloxicam, nabumetone, or a combination thereof.

In another preferred embodiment, the ADP receptor inhibitor comprises: clopidogrel, and ticlopidine.

In another preferred embodiment, the antiplatelet agent is selected from the group consisting of: clopidogrel and aspirin.

In another preferred example, if the PAR-1.

In another preferred example, if the PAR-1 rs168753 gene locus of the detected subject is mutated from A to T, the subject is more sensitive to the combination of clopidogrel and aspirin.

In another preferred embodiment, if the genotype of the PAR-1 rs168753 gene locus of the subject is TT, the subject is more sensitive to the combination of clopidogrel and aspirin.

In another preferred example, if the subject is subjected to the following mutations at one or more gene sites selected from the group consisting of clopidogrel (clopidogrel insensitive):

CYP2C19*2：rs4244285G→A；

CYP2C19*3：rs4986893G→A；

CYP2C19*17：rs12248560C→T。

in another preferred embodiment, the reagent comprises a primer, a probe, a chip, or an antibody.

In another preferred embodiment, the kit contains one or more reagents selected from the group consisting of:

(A) Specific primers for gene detection;

(B) Specific probes for gene detection;

(C) A chip for gene detection;

(D) A specific antibody for detecting an amino acid mutation corresponding to the mutated gene;

wherein the specific primer, the specific probe and the chip are directed to gene loci selected from the group consisting of: CYP2C19 × 2: rs4244285 gene site, CYP2C19 x 3: rs4986893 gene site, CYP2C19 x 17: rs12248560 Gene site and PAR1: rs168753 Gene site;

the mutated gene comprises a mutated genetic locus selected from the group consisting of: CYP2C19 x 2: rs4244285 gene locus, CYP2C19 x 3: rs4986893 gene site, CYP2C19 x 17: the rs12248560 gene site PAR1: rs168753 gene site.

In another preferred embodiment, the primer comprises:

primer pair 1, directed against CYP2C19 x 2: rs4244285 gene locus:

the 5'-3' sequence of the upstream primer is as follows: AGATGCAATAATTTTCCCACTATC (Seq ID No. 1),

the 5'-3' sequence of the downstream primer is as follows: ATAAAGTCCCGAGGGGTTGTTGATG (Seq ID No. 2);

primer pair 2, directed against CYP2C19 x 3: rs4986893 gene site:

the 5'-3' sequence of the upstream primer is as follows: GATGGAAAAATTGAATGAAAACATCA (Seq ID No. 3),

the 5'-3' sequence of the downstream primer is as follows: CTGGGAAATCCAAAATTCTATATTG (Seq ID No. 4);

primer pair 3, directed against CYP2C19 x 17: rs12248560 gene site:

the 5'-3' sequence of the upstream primer is as follows: TCTTCTGATGCCCACGTGGCGCGCATT (Seq ID No. 5),

the 5'-3' sequence of the downstream primer is as follows: TAGTTATTCTGAATATACCACATT (Seq ID No. 6).

Primer pair 4, for PAR-1

The 5'-3' sequence of the upstream primer is as follows: CTTTTGCCTTGTTGATGCGTTCAC (Seq ID No. 7),

the 5'-3' sequence of the downstream primer is as follows: CAACAATGCCACCTTAGATC (Seq ID No. 8).

In another preferred embodiment, the primers further comprise an extension primer comprising the primer sequence Seq ID No. 1-14.

In another preferred embodiment, the reagent or kit is used for real-time fluorescent quantitative PCR detection.

In another preferred example, in the real-time fluorescent quantitative PCR, the annealing temperature is between 60 and 67 ℃, and the length of the PCR amplified fragment is between 80 and 300bp.

In another preferred example, in the real-time fluorescent quantitative PCR, the annealing temperature of the fluorescent probe is between 60 and 70 ℃.

In another preferred example, the two ends of the probe are modified by chemical groups, the 5' end is modified by a fluorescence exciting group, and the 3 end is modified by a fluorescence group 23826.

In another preferred embodiment, the reagent or kit further comprises a probe sequence selected from the group consisting of:

in another preferred embodiment, the kit further comprises a probe sequence selected from the group consisting of:

probe 1, for CYP2C19 x 2: rs4244285 gene locus:

P1：FAM-TATGGGTTCCCCGGGAAATAA-BHQ1(SEQ ID NO.9)

P2:ROX-TATGGGTTCCCTGGGAAATAA-BHQ2(SEQ ID NO.10)；

probe 2, for CYP2C19 x 3: rs4986893 gene site:

P3：VIC-TTACCTGGATTCAGGGGGT-BHQ1(SEQ ID NO.11)

P4：TAMRA-TTACCTGGATCCAGGGGGT-BHQ2(SEQ ID NO.12)；

probe 3, directed against CYP2C19 x 17rs12248560 gene locus:

P5：FAM-TTCTCAAAGCATCTCTGATGT-BHQ1(SEQ ID NO.13)

P6：ROX-TTCTCAAAGTATCTCTGATGT-BHQ2(SEQ ID NO.14)。

probe 4, at PAR1: rs168753 gene site:

P13：FAM-CTGAAAAATAAAATTAAAAAAATTTT-BHQ1(SEQ ID NO.15)

P14：ROX-CTGAAAAATAAAATAAAAAAAATTTT-BHQ2(SEQ ID NO.16)。

in another preferred embodiment, the probe further comprises an extension primer set of probe sequences P1-P14.

In a second aspect of the invention, a kit is provided, which comprises a PAR1: rs168753 gene locus detection reagent.

In another preferred embodiment, the kit further comprises a detection reagent for detecting one or more genetic loci selected from the group consisting of: CYP2C19 x 2: rs4244285 gene site, CYP2C19 x 3: rs4986893 gene site, and CYP2C19 x 17: rs12248560 gene locus.

In another preferred embodiment, the kit contains one or more reagents selected from the group consisting of:

(A) Specific primers for gene detection;

(B) Specific probes for gene detection;

(C) A chip for gene detection;

(D) A specific antibody for detecting an amino acid mutation corresponding to the mutated gene;

wherein the specific primer, the specific probe and the chip are directed to gene loci selected from the group consisting of: CYP2C19 x 2: rs4244285 gene site, CYP2C19 x 3: rs4986893 gene site, CYP2C19 x 17: rs12248560 gene site and PAR-1 rs168753 gene site;

the mutated gene comprises a mutated genetic locus selected from the group consisting of:

CYP2C19 x 2: rs4244285 gene site, CYP2C19 x 3: rs4986893 gene site, CYP2C19 x 17: rs12248560 Gene site and PAR1: rs168753 Gene site.

In another preferred embodiment, the primer comprises:

primer pair 1, primer pair directed against CYP2C19 x 2: rs4244285 gene locus:

the upstream primer 5'-3' sequence is: AGATGCAATAATTTTCCCACTATC (Seq ID No. 1),

the downstream primer 5'-3' sequence is: ATAAGTCCCGAGGGGTTGTTGATG (Seq ID No. 2);

primer pair 2, directed against CYP2C19 x 3: rs4986893 gene site:

the 5'-3' sequence of the upstream primer is as follows: GATGGAAAAATTGAATGAAAACATCA (Seq ID No. 3),

the 5'-3' sequence of the downstream primer is as follows: CTGGGAAATCCAAAATTCTATATTG (Seq ID No. 4);

primer pair 3, directed against CYP2C19 x 17: rs12248560 gene site:

the upstream primer 5'-3' sequence is: TCTTCTGATGCCCACGTGGCGCGCATT (Seq ID No. 5),

the 5'-3' sequence of the downstream primer is as follows: TAGTTATTCTGAATATACCACATT (Seq ID No. 6).

Primer pair 4, for PAR-1, rs168753 gene site

The 5'-3' sequence of the upstream primer is as follows: CTTTTGCCTTGTTGATGCGTTCAC (Seq ID No. 7),

the 5'-3' sequence of the downstream primer is as follows: CAACAATGCCACCTTAGATC (Seq ID No. 8).

In another preferred embodiment, the primers further comprise an extension primer comprising the primer sequence Seq ID No. 1-8.

In another preferred embodiment, the kit is used for real-time fluorescent quantitative PCR detection.

In another preferred example, in the real-time fluorescent quantitative PCR, the annealing temperature is between 60 and 67 ℃, and the length of the PCR amplified fragment is between 80 and 300bp.

In another preferred example, in the real-time fluorescent quantitative PCR, the annealing temperature of the fluorescent probe is between 60 and 70 ℃.

In another preferred example, the two ends of the probe are modified by chemical groups, the 5' end is modified by a fluorescence exciting group, and the 3 end is modified by a fluorescence group 23826.

In another preferred embodiment, the kit further comprises a probe sequence selected from the group consisting of:

in another preferred embodiment, the kit further comprises a probe sequence selected from the group consisting of:

probe 1, for CYP2C19 x 2: rs4244285 gene locus:

P1：FAM-TATGGGTTCCCCGGGAAATAA-BHQ1(SEQ ID NO.9)

P2:ROX-TATGGGTTCCCTGGGAAATAA-BHQ2(SEQ ID NO.10)；

probe 2, directed against CYP2C19 x 3: rs4986893 gene site:

P3：VIC-TTACCTGGATTCAGGGGGT-BHQ1(SEQ ID NO.11)

P4：TAMRA-TTACCTGGATCCAGGGGGT-BHQ2(SEQ ID NO.12)；

probe 3, directed against CYP2C19 x 17rs12248560 gene locus:

P5：FAM-TTCTCAAAGCATCTCTGATGT-BHQ1(SEQ ID NO.13)

P6：ROX-TTCTCAAAGTATCTCTGATGT-BHQ2(SEQ ID NO.14)。

probe 4, at PAR1: rs168753 gene site:

P13：FAM-CTGAAAAATAAAATTAAAAAAATTTT-BHQ1(SEQ ID NO.15)

P14：ROX-CTGAAAAATAAAATAAAAAAAATTTT-BHQ2(SEQ ID NO.16)。

in another preferred embodiment, the probe further comprises an extension primer series of probe sequences P1-P14.

In a third aspect of the present invention, there is provided a method for in vitro detecting the presence or absence of a single nucleotide variation in a sample, comprising the steps of:

(a) Amplifying the polynucleotide of the sample by using a specific primer to obtain an amplification product; and

(b) Detecting the presence or absence of the following single nucleotide variations in the amplification product:

CYP2C19*2：rs4244285G→A；

CYP2C19*3：rs4986893G→A；

CYP2C19 × 17: rs12248560C → T; and

PAR-1:rs168753A→T。

in another preferred embodiment, the amplification product has a length of 80-2000bp and contains one or more single nucleotide variations selected from the group consisting of:

CYP2C19*2：rs4244285G→A；

CYP2C19*3：rs4986893G→A；

CYP2C19*17：rs12248560C→T。

PAR-1:rs168753A→T。

in a fourth aspect of the present invention, there is provided a method of detecting sensitivity of an individual to an antiplatelet agent, comprising the steps of:

(i) Detecting the individual's genetic locus selected from the group consisting of:

CYP2C19*2：rs4244285；

CYP2C19*3：rs4986893；

CYP2C19*17：rs12248560；

PAR-1:rs168753。

in another preferred embodiment, the genetic mutation is a single nucleotide variation selected from the group consisting of:

CYP2C19*2：rs4244285G→A；

CYP2C19*3：rs4986893G→A；

CYP2C19*17：rs12248560C→T。

PAR-1:rs168753A→T。

in another preferred embodiment, the individual is a human.

In another preferred embodiment, if PAR-1

If CYP2C19 x 2: rs4244285; and/or CYP2C19 x 3: rs4986893; and/or CYP2C19 x 17: the absence of the mutation in rs12248560 indicates clopidogrel sensitivity.

Within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., examples) can be combined with each other to constitute a new or preferred technical solution. Not to be reiterated herein, but to the extent of space.

Detailed Description

The inventor obtains a gene polymorphism site related to the sensitivity of the antiplatelet drugs through extensive and intensive research, and an experimental result shows that the detection of the gene polymorphism site can evaluate the sensitivity of an individual to the antiplatelet drugs. On the basis of this, the present invention has been completed.

The invention relates to a method for screening cytochrome P450CYP2C19 x 2 by using a fluorescence hydrolysis probe (Taqman) method: rs4244285; CYP2C19 x 3: rs4986893; CYP2C19 × 17: rs12248560; and 4 polymorphic sites of PAR1: rs168753 2 genes. The kit formed by the method is used for judging whether the target to be detected belongs to the drug-taking susceptible population or not, so as to guide and adjust the clinical drug-taking scheme, provide basis for clinical personalized treatment and prevent the adverse reaction of the drug. Therefore, the kit and the detection method provided by the invention can be used for guiding the individual medication of the cerebral apoplexy.

Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.

Cytochrome P450

Cytochrome P450 (CYP) is a superfamily oxidase, widely distributed in various life forms, from low-grade bacteria to higher animals and plants, and is an important component of multifunctional oxidase, and exists in microsomes of multiple tissue cells, and the enzyme mainly exists in liver tissues in human bodies. P450 activity determines the rate of metabolism and clearance of the drug in vivo and is therefore also referred to as a drug metabolizing enzyme. The genes of the P450 enzyme system mainly comprise three families of CYP1, CYP2 and CYP3, and corresponding several important enzymes comprise: CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5.

The P450 family of drugs involved in metabolism accounts for more than 80% of the drugs sold on the market, and once the function of the enzyme is changed, the metabolism and the curative effect of the drugs are influenced. In the study of adverse reactions to drugs, approximately 48% were closely related to polymorphisms of the P450 enzyme gene.

The P450 genetic polymorphism is the main reason for causing deletion, reduction or increase of expression quantity and change of substrate specificity of P450 enzyme, and finally causes change of pharmacokinetics, so that different individuals generate different drug responses to the same drug.

CYP2C19 is an important member in the second subfamily, the CYP2C19 gene is located in the chromosome region 10q24.2, is composed of 9 exons, has 92% sequence homology with CYP2C9, is mainly expressed in liver tissues, is also expressed in duodenum, and is one of important drug metabolism enzymes in human bodies. Of the drugs responsible for metabolism by the P450 enzyme, 12% are metabolized by CYP2C 19.

CYP2C19 has a plurality of SNP sites, and the genetic polymorphism thereof plays an important role in the metabolic process of cardiovascular and cerebrovascular medicines.

Currently, at least 10 of the 25 mutant alleles of CYP2C19 that have been found result in altered enzyme activity, most commonly CYP2C19 x 2 and CYP2C19 x 3 and CYP2C19 x 17 (rs 12248560). Wherein the slow metabolic form is represented by CYP2C19 x 2: and CYP2C19 × 3, and CYP2C19 × 17 is the main fast metabolic form.

Protein cleavage during CYP2C19 x 2mRNA translation results in mutational inactivation, whereas CYP2C19 x 3 can constitute a terminator and disrupt the activity of the transcribed protein. Statistically, the two mutation sites CYP2C19 x 2 and CYP2C19 x 3 explain the associated weak metabolic genetic defect in almost 100% of east asian and 85% of caucasian species. There is a large body of evidence that there are large differences in the metabolic capacity of CYP2C19 substrates among different human species; 2-5% of caucasians are hypometabolizers, while 13-23% of Asians are hypometabolizers. This is due to the high frequency of CYP2C19 x 2 and CYP2C19 x 3 alleles in asian population.

Protease activated receptor-1 (PAR-1)

PAR-1 is a G protein-coupled receptor on the cell surface, a thrombin receptor. Thrombin is the strongest platelet activator and mediates cellular responses through PAR 1. Human platelets express the PAR1 receptor, PAR1 is able to couple to the G12/13 and Gq families and indirectly activate the Gi signaling pathway by secreting ADP, causing a series of alterations in platelet activation, inducing a coagulation response. Plays a key role in the process of thrombosis and hemostasis.

The invention provides a detection method for judging whether a target to be detected belongs to a drug-taking susceptible population or not based on the correlation between a stroke drug and heredity. According to cytochrome P450CYP2C19 x 2: rs4244285; CYP2C19 x 3: rs4986893; CYP2C19 × 17: rs12248560; and nucleotide sequences of 4 polymorphic sites of PAR1: rs168753 2 genes to synthesize primers and probes, carrying out real-time fluorescent quantitative hydrolysis probe PCR on a sample to be detected, and judging the genotype of a PCR amplification product according to a fluorescence value generated by reaction. And judging whether the sample belongs to the drug sensitive population according to the difference of the fluorescence.

In a preferred embodiment of the invention, the primers used comprise:

CYP2C19 x 2: rs4244285 primer set

The 5'-3' sequence of the upstream primer (Seq ID No. 1) is as follows: agattatgcaatatttcccactatc,

the 5'-3' sequence of the downstream primer (Seq ID No. 2) is as follows: ATAAGTCCCGAGGGGTTGTTGATG;

CYP2C19 x 3: rs4986893 set primer

The upstream primer 5'-3' sequence (Seq ID No. 3) is: GATGGAAAAATTGAATGAAAACATCA,

the 5'-3' sequence of the downstream primer (Seq ID No. 4) is as follows: CTGGGAAATCCAAAATTCTATATTG;

CYP2C19 × 17: rs12248560 set of primers

The upstream primer 5'-3' sequence (Seq ID No. 5) is: TCTTCTGATGCCCACGTGGCGCGCATT,

the 5'-3' sequence of the downstream primer (Seq ID No. 6) is as follows: TAGTTATTCTGAATATACCATATT;

PAR1: rs168753 set primer

The 5'-3' sequence of the upstream primer (Seq ID No. 7) is: CTTTTGCCTTGTTGATGTCGTTCAC,

the 5'-3' sequence of the downstream primer (Seq ID No. 8) is as follows: CAACAAATGGCCACCTTAGATC.

The primer of the present invention may be an extension primer set comprising the above-mentioned primer sequence (Seq ID No.1-Seq ID No. 14).

Preferably, the annealing temperature is between 60 and 67 ℃, and the length of the PCR amplified fragment is between 80 and 300bp.

Preferably, in real-time fluorescent quantitative PCR, the annealing temperature of the fluorescent probe is between 60 and 70 ℃. In the real-time fluorescence quantitative allele specificity PCR, the annealing temperature is 60-67 ℃, and the length of a PCR amplification fragment is 820-300bp; the concentration range of the genomic DNA to be detected is between 1 ng and 20 ng.

Preferably, in real-time fluorescent quantitative PCR, the probes used include:

in another preferred embodiment, the kit further comprises a probe sequence selected from the group consisting of:

probe 1, for CYP2C19 x 2: rs4244285 gene locus:

P1：FAM-TATGGGTTCCCCGGGAAATAA-BHQ1(SEQ ID NO.9)

P2:ROX-TATGGGTTCCCTGGGAAATAA-BHQ2(SEQ ID NO.10)；

probe 2, for CYP2C19 x 3: rs4986893 gene site:

P3：VIC-TTACCTGGATTCAGGGGGT-BHQ1(SEQ ID NO.11)

P4：TAMRA-TTACCTGGATCCAGGGGGT-BHQ2(SEQ ID NO.12)；

probe 3, directed against CYP2C19 x 17rs12248560 gene locus:

P5：FAM-TTCTCAAAGCATCTCTGATGT-BHQ1(SEQ ID NO.13)

P6：ROX-TTCTCAAAGTATCTCTGATGT-BHQ2(SEQ ID NO.14)。

probe 4, at PAR1: rs168753 gene site:

P13：FAM-CTGAAAAATAAAATTAAAAAAATTTT-BHQ1(SEQ ID NO.15)

P14：ROX-CTGAAAAATAAAATAAAAAAAATTTT-BHQ2(SEQ ID NO.16)。

the probe of the invention comprises: the double ends are modified by chemical groups, and the fluorescent exciting group at the 5' end and the fluorescent quenching group at the 3 end. And an extension primer set containing a probe sequence (P1-P14).

In the present invention, the stroke may be developed from cardiovascular diseases.

The invention also provides a cytochrome P450CYP2C19 x 2-based food product comprising: rs4244285; CYP2C19 × 3: rs4986893; CYP2C19 × 17: rs12248560; and 4 polymorphic sites of PAR1: rs168753 2 genes. The detection method is used for implementing the detection method, and comprises an optimized real-time fluorescent quantitative hydrolysis probe multiplex PCR reaction system; the primer is used; human P450-containing CYP2C19 x 2: rs4244285; CYP2C19 x 3: rs4986893; CYP2C19 × 17: rs12248560; plasmid positive control of PAR1: rs168753 gene genome DNA fragment; taq DNA polymerase; PCR enhancers such as DMSO.

The present invention also provides a kit for carrying out the detection method according to the present invention, comprising the cytochrome P450CYP2C19 x 2: rs4244285; CYP2C19 x 3: rs4986893; CYP2C19 × 17: rs12248560; and 4 polymorphic sites of PAR1: rs168753 2 genes.

The invention relates to a method for screening cytochrome P450CYP2C19 x 2: rs4244285; CYP2C19 x 3: rs4986893; CYP2C19 × 17: rs12248560; and PAR1: rs168753 on the basis of 4 polymorphism site genotypes of the technical scheme, provides a drug sensitive population diagnostic reagent, and the 4 sets of primers are adopted in the real-time fluorescent quantitative hydrolysis probe PCR.

The present invention relates to screening of P450CYP2C19 x 2: rs4244285; CYP2C19 x 3: rs4986893; CYP2C19 × 17: rs12248560; and PAR1 rs168753 in the preparation of medicament sensitive people diagnostic reagent, the diagnostic reagent comprises the primer, reaction buffer solution, human P450CYP2C19 x 2: rs4244285; CYP2C19 × 3: rs4986893; CYP2C19 × 17: rs12248560; and PAR1: rs168753 genome DNA fragment, taq DNA polymerase and fluorescent probe kit.

The invention establishes a simple, convenient, accurate, rapid and high-flux real-time fluorescent quantitative probe hydrolysis PCR method for detecting the stroke drug sensitive population screening in the research of the stroke drug sensitive population genetic polymorphism, and can also be applied to the research of the correlation of SNP and other diseases.

The present invention is based on human P450CYP2C19 x 2: rs4244285; CYP2C19 x 3: rs4986893; CYP2C19 × 17: rs12248560; and synthesizing corresponding primers with nucleic acid sequences at two sides of PAR1: rs168753SNP, designing corresponding nucleotide probes according to the SNP sites, designing the SNP sites in the middle of the probes, labeling fluorescent dye at the 5 'end of the nucleotides of the probes, and labeling fluorescent quenching dye at the 3' end. And (3) utilizing an optimized PCR reaction program, carrying out real-time fluorescent probe hydrolysis quantitative PCR reaction, amplifying the genomic DNA extracted from the human blood sample, and carrying out gene polymorphism analysis according to the fluorescent reading data with different wavelengths marked by each probe.

In the invention, the optimization process of the adopted real-time fluorescent probe hydrolysis quantitative PCR method comprises the application of TaqDNA polymerase, reasonable primer and probe design and modification and the optimization of a multiple PCR reaction system.

Due to non-specific amplification and the existence of primer dimer in PCR amplification, the invention uses the hot-start Taq DNA polymerase, the polymerase has the greatest advantage that the polymerase is different from the common Taq DNA polymerase, the polymerase activity is completely inhibited when the temperature is lower than 50 ℃ through chemical modification, and the polymerase activity is released after 10 minutes at 95 ℃. Therefore, the specificity of PCR amplification is greatly improved when the DNA synthesis is carried out, and the generation of primer dimer is reduced.

At the same time, by selecting more optimal PCR reaction conditions, such as primer concentration range (0.5-5 mM); the primer length range (20-30 bp); the probe length range (15-21 bp); the annealing temperature (57-67 ℃) during the reaction; the length of the amplified fragment (80-300 bp); the concentration range (0.5-10 ng) of the genome DNA to be detected and the like further improve the specificity and the amplification efficiency of PCR amplification.

In the invention, a fluorescent probe hydrolysis method (Taqman PCR) in a real-time fluorescent quantitative PCR technology is applied, and the method is carried out according to the following formula that cytochrome P450CYP2C19 x 2: rs4244285; CYP2C19 × 3: rs4986893; CYP2C19 × 17: rs12248560; and nucleotide sequence synthesis primers and probes of 4 polymorphic sites of PAR1: rs168753 2 genes, carrying out real-time fluorescent quantitative hydrolysis probe PCR on a sample to be detected, and judging the gene polymorphism of a PCR amplification product gene according to a fluorescent value generated by reaction. The differences in fluorescence of the genomic amplification products between the 4 sets of primers compared to standard reference samples (cloned plasmid containing human cytochrome P450CYP2C19 x 2.

The invention utilizes a specific fluorescent probe added while adding a pair of primers during probe PCR amplification, wherein the probe is an oligonucleotide, and two ends of the probe are respectively marked with a reporter fluorescent group and a quenching fluorescent group. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme cuts and degrades the probe, so that the report fluorescent group and the quenching fluorescent group are separated, a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescent molecule is formed when each DNA chain is amplified, and the accumulation of the fluorescence signal and the formation of a PCR product are completely synchronous. A series of optimization is carried out on a reaction system such as primer concentration, annealing temperature, fragment amplification length, template DNA concentration, reagent additives of the reaction system and the like, so that the method can accurately and rapidly detect the concentration of P450CYP2C19 x 2 in a human genome: rs4244285; CYP2C19 x 3: rs4986893; CYP2C19 × 17: rs12248560; and PAR1: rs168753SNP locus, the principle and the method of the technical scheme of the invention are also suitable for SNP detection of other genes.

The invention provides 4 sets of specific primers for amplifying the expression of human P450CYP2C19 x 2: rs4244285; CYP2C19 x 3: rs4986893; CYP2C19 × 17: rs12248560; and a specific sequence on the PAR1: rs168753 gene; detecting human P450CYP2C19 x 2 by using a Taqman method of real-time fluorescent quantitative PCR: rs4244285; CYP2C19 × 3: rs4986893; CYP2C19 × 17: rs12248560; and PAR1: rs168753 4SNP loci. The invention establishes the method for simultaneously detecting P450CYP2C19 x 2 by a real-time fluorescence quantitative PCR hydrolysis probe technology (Taqman technology) for the first time: rs4244285; CYP2C19 x 3: rs4986893; CYP2C19 × 17: rs12248560; and PAR1: rs168753 4 polymorphism distribution. The detection scheme of the 4SNP combinations has the characteristics of simple and convenient operation, rapidness, high accuracy, high flux and the like, and is suitable for carrying out large-scale P450CYP2C19 x 2: rs4244285; CYP2C19 × 3: rs4986893; CYP2C19 × 17: rs12248560; and the distribution detection of PAR1: rs168753 gene SNP and the research of the correlation of the gene and the disease. Meanwhile, whether the target to be detected belongs to the medicine-taking susceptible population or not is judged, so that the clinical medicine taking scheme is guided and adjusted, a basis is provided for clinical personalized treatment, adverse reaction of the medicine is prevented, and accurate medicine taking is realized.

The invention analyzes human P450CYP2C19 x 2 by a real-time fluorescence quantitative PCR Taqman method: rs4244285; CYP2C19 x 3: rs4986893; CYP2C19 × 17: rs12248560; and PAR1: rs168753 4 polymorphic sites. And applying the method to detect the expression of P450CYP2C19 x 2: rs4244285; CYP2C19 x 3: rs4986893; CYP2C19 × 17: rs12248560; the distribution of the 3SNP site combinations in the patients with relapse of stroke drug therapy of Chinese healthy people determines the close correlation between the specific 3SNP and the drug therapy relapse of stroke, thereby demonstrating the clinical applicability and feasibility of the invention.

The present invention is directed to CYP2C19 x 2; CYP2C19 x 3; and CYP2C19 x 17 SNP in the evaluation of the distribution of the combination in the treatment of the relapse patients with the stroke drugs in Chinese healthy people, further finds that the 3SNP loci can be used as high-risk factors for the drug resistance of the event drugs of the clopidogrel/aspirin treatment stroke patients, can detect susceptible people from the treatment of the relapse patients with the stroke drugs, and further realizes the guidance and adjustment of clinical medication schemes. In addition, as a brand-new clopidogrel/aspirin drug resistance biomarker combination, the administration accuracy can be greatly improved, and adverse reactions of the drugs can be prevented. As described above, for CYP2C19 x 2; CYP2C19 x 3; and CYP2C19 x 17, the 3SNP loci can be used for combined detection, so that the adverse reaction of the medicament can be prevented, the clinical medication scheme can be further guided and adjusted, a basis can be provided for clinical personalized treatment, and accurate medication can be realized.

P450CYP2C19 × 2: rs4244285; CYP2C19 x 3: rs4986893; CYP2C19 × 17: rs12248560; and PAR1: rs168753 4SNP loci have important functions and clinical significance for preventing adverse reaction of medicaments, guiding and adjusting clinical medication schemes and realizing accurate medication, so the invention not only provides experimental basis for clopidogrel/aspirin treatment, but also establishes a high-flux real-time fluorescence quantitative PCR Taqman method for screening and lays theoretical and experimental foundation for realizing early accurate medication. Thereby greatly reducing the incidence of the adverse reaction of the medicine, and having immeasurable significance for the development and improvement of the economic and medical level of the whole society as well as the individual patients.

The main advantages of the invention are:

a simple, accurate, rapid and high-flux method for screening drug-sensitive people for detecting stroke is established.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Example 1 establishment of real-time fluorescent quantitation of allele-specific PCR and clinical evaluation and applications thereof

Establishment of real-time fluorescence quantitative allele specificity PCR method

The real-time fluorescence quantitative PCR technology has the characteristics of real-time monitoring, quantification, high flux and the like, and is simple and convenient to operate and high in sensitivity. The fluorescent quantitative PCR is classified into probe quantitative PCR (Taqman method) and fluorescent dye quantitative PCR. The probe PCR (Taqman method) is that a pair of primers is added and a specific fluorescent probe is added at the same time during amplification, the probe is an oligonucleotide, and two ends of the probe are respectively marked with a reporter fluorescent group and a quenching fluorescent group. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme cuts and degrades the probe, so that the reporter fluorescent group and the quenching fluorescent group are separated, a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescent molecule is formed when one DNA chain is amplified, and the accumulation of the fluorescence signal and the formation of a PCR product are completely synchronous. And judging the product according to the corresponding fluorescence detected by the reaction. Compared with fluorescent dye, the Taqman probe has sequence specificity and is only combined to a complementary region, and fluorescent signals and amplified copy numbers have one-to-one correspondence, so that the Taqman probe has strong specificity and high sensitivity, and the conditions are easy to optimize; compared with a hybridization probe, the Taqman probe only needs to be designed into one probe, so the probe design is cheaper and convenient, and the basic quantitative PCR requirement can be fulfilled.

The basic principle of the Taqman method for detecting SNP is that a specific fluorescent probe can only be combined with a corresponding complete complementary nucleic acid template, but the probe cannot be combined with the template when the nucleic acid template has non-specific nucleic acid sequence. Therefore, a pair of double-labeled Taqman probes are applied to amplify the corresponding genotypes of the double-allelic SNP by only the completely matched probes respectively aiming at the different genotypes of the double-allelic SNP; the two probes are respectively marked by two fluorescent dyes with different wavelengths, so that the genotype judgment of a single SNP site can be completed in one PCR reaction.

The invention adopts the improved real-time fluorescence quantitative PCR Taqman method: double PCR reactions were performed in one reaction tube and quadruple fluorescence was detected. The reaction to CYP2C19 x 2 can be completed at one time by simultaneously carrying out two reaction holes; and 6 fluorescence channels combined by CYP2C 19X 3 and CYP2C 19X 17 3SNP sites. Clinical trials were conducted on 1068 aspirin-treated groups and 1081 clopidogrel/aspirin-treated patients, and the possibility of using them as a drug metabolism test combination was evaluated. First, CYP2C19 x 2; CYP2C19 x 3; CYP2C19 × 17 in combination: carriers of loss-of-function alleles are called carrying at least one x 2 or 3 variation. Carriers of function-gain alleles are called carrying at least one x 17 variation. The distribution is shown in Table 1.

TABLE 1

Compared with the single aspirin, the clopidogrel-aspirin combination has no obvious benefit for CYP2C19 function loss and other gene carriers. In the normal allele carriers, compared with the single use of aspirin, the clopidogrel-aspirin combination reduces the recurrence of stroke by 50%.

The invention designs a corresponding primer pair to amplify DNA target fragments based on the nucleotide Sequence between 100 and 200 on both sides of the SNP locus in human whole genome (GenBank: NCBI Reference Sequence: P450CYP2C19 x 2; simultaneously, 8 Taqman probes with different specific fluorescent group markers of nucleic acid sequences are designed aiming at the 4SNP sites, and a real-time fluorescence quantitative PCRT Taqman method is used for detecting P450CYP2C19 x 2: rs4244285; CYP2C19 × 3: rs4986893; CYP2C19 × 17: rs12248560; and 8 SNP sites on the PAR1: rs168753 gene.

The present invention is based on human P450CYP2C19 x 2: rs4244285; CYP2C19 x 3: rs4986893; CYP2C19 × 17: rs12248560; and SNP (GenBank: NCBI Reference Sequence: rs4244285; rs4986893; rs12248560; and rs 168753) of PAR1: rs168753 gene, corresponding primers were synthesized, and SNP sites were designed in the middle of the Taqman probe. Amplifying DNA extracted from a human blood sample by real-time fluorescence quantitative PCR Taqman reaction, and carrying out SNP analysis on a target gene according to a fluorescence value displayed by amplification.

The second experimental method comprises the following steps:

1. obtaining a sample to be tested: extracting human genome DNA in blood, taking 500 mu L of human blood, extracting by using a blood DNA extraction kit, measuring the light absorption value of 260nm to calculate the DNA concentration, and diluting the DNA concentration to 10 ng/uL.

2. Designing a corresponding primer pair to amplify the DNA target fragment based on the nucleotide Sequence between 100 and 200 on both sides of the SNP site in the human genome (GenBank: NCBI Reference Sequence: P450CYP2C 19X 2; simultaneously, 8 Taqman probes marked by specific different fluorophores of nucleic acid sequences are designed aiming at the 4SNP loci, and a real-time fluorescence quantitative PCR Taqman method is used for detecting P450CYP2C19 x 2; CYP2C19 x 3; CYP2C19 x 17; and 8 SNP sites on the PAR1 gene.

The specific design is as follows (middle underline marks as probe; SNP site in parentheses):

CYP2C19*2：rs4244285(SEQ ID NO.17)

AGATATGCAATAATTTTCCCACTATCATTGATTATTTCCC(G/A)GGAACCCATAACAAATTACTTAAAAACCTTGCTTTTATGGAAAGTGATATTTTGGAGAAAGTAAAAGAACACCAAGAATCGATGGACATCAACAACCCTCGGGACTTTAT

CYP2C19*3：rs4986893(SEQ ID NO.18)

GATGGAAAAATTGAATGAAAACATCAGGATTGTAAGCACCCCCTG(A/G)ATCCAGGTAAGGCCAAGTTTTTTGCTTCCTGAGAAACCACTTACAGTCTTTTTTTCTGGGAAATCCAAAATTCTATATTG

CYP2C19*17：rs12248560(SEQ ID NO.19)

AATGTGGTATATATTCAGAATAACTAATGTTTGGAAGTTGTTTTGTTTTGCTAAAACAAAGTTTTAGCAAACGATTTTTTTTTTCAAATTTGTGTCTTCTGTTCTCAAAG(C/T)ATCTCTGATGTAAGAGATAATGCGCCACGATGGGCATCAG AAGA

PAR1:rs1687537(SEQ ID NO.20)

ATAAACAAAAGTAAAATATGCTCTCTGCTTGTCGCTTTTGCCTTGTTGATGCGTTCACTTTTTACATTTAAAATTTTTTT(A/T)ATTTTATTTTTCAGAATCAAAAGCAACAAATGCCACCTTAGATCCCCGGTCATTTCTTCT

TABLE 2 real-time fluorescent quantitative PCR primers and Taqman probes

The correspondence between the fluorescence value of the primer-probe constructed by the present invention and the SNP type of the gene is shown in Table 3.

Table 3 shows the two-hole prediction fluorescence detection results of the real-time fluorescence quantitative PCR Taqman (the interior of the table is abbreviated as fluorescence)

TABLE 3

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3. Human CYP2C19 x 2; CYP2C19 x 3; CYP2C19 x 17; and the DNA fragment of the PAR1 gene is cloned and sequenced, and the sequencing result is consistent with the sequence comparison of the gene library

4. Implementation of real-time fluorescent quantitative allele-specific PCR: real-time fluorescent quantitative PCR Taqman reaction was performed on human genomic DNA (gDNA, 10 ng/. Mu.L). The positive control is the human CYP2C19 x 2 which is cloned, sequenced and identified by the invention; CYP2C19 x 3; CYP2C19 x 17; and PAR1 genomic DNA fragment, negative control water. The reaction system and conditions were as follows:

the reaction conditions are 95 ℃ and 5min; 30 seconds at 95 ℃; 60 seconds at 62 ℃;40 cycles.

5. Evaluation of real-time fluorescent quantitative allelic PCR method

5.1. Testing on positive control plasmid:

8 prepared according to the invention each contained CYP2C19 x 2; CYP2C19 x 3; CYP2C19 x 17; and pBluescript II (SK (+) plasmid of PAR 14 SNP locus genome DNA fragment is used as positive control of PCR reaction, and the real-time fluorescence quantitative PCR system is evaluated, such as primer concentration, probe concentration, mg ion concentration and the like are adjusted, and further reaction conditions are optimized, and the clinical application feasibility of the method is discussed.

CYP2C19 x 2; CYP2C19 x 3; CYP2C19 x 17; and PAR1 gene plasmid as template, reaction conditions: 10 minutes at 95 ℃, 15 seconds at 95 ℃ and 60 seconds at 64 ℃; and 40 cycles. SNP analysis was performed based on the fluorescence value obtained after amplification.

In this example, 4 sets of primers and 8 probes were used for the fluorescent quantitative PCR amplification reaction.

5.2. And (4) testing on the human genome DNA sample to obtain a test result on the genome DNA sample.

6. Optimization of real-time fluorescent quantitative PCR Taqman method

6.1 due to the nonspecific amplification in PCR amplification and the existence of primer dimer, the invention uses the chemically modified hot-start DNA polymerase, and due to the nonspecific amplification in PCR amplification and the existence of primer dimer, the invention uses the hot-start Taq DNA polymerase, the polymerase has the greatest advantage that the polymerase is different from the common Taq DNA polymerase, through chemical modification, when the temperature is lower than 50 ℃, the polymerase activity is completely inhibited, and the polymerase activity is released after 10 minutes at 95 ℃. Therefore, the specificity of PCR amplification is greatly improved when the DNA synthesis is carried out, and the generation of primer dimer is reduced.

6.2 simultaneously, by selecting more optimized PCR reaction conditions, such as primer concentration range (0.5-5 mM); the primer length range (20-30 bp); the probe length range (15-21 bp); the annealing temperature (57-67 ℃) during the reaction; the length of the amplified fragment (80-300 bp); the concentration range (0.5-10 ng) of the genome DNA to be detected and the like further improve the specificity and the amplification efficiency of PCR amplification.

Clinical evaluation and clinical application:

CYP2C19 x 2 established by the invention is adopted; CYP2C19 x 3; a method for detecting 4 polymorphic sites of CYP2C19 x 17 and PAR-1 genes. The aspirin treatment population and the clopidogrel/aspirin treatment population are screened, whether the target to be detected belongs to the drug-taking susceptible population or not can be judged, the clinical medication scheme is guided and adjusted, the basis is provided for clinical personalized treatment, and the adverse reaction of the drug is prevented.

The distribution of each SNP type in the population is shown in Table 4, so that the 4SNP loci have high correlation with the drug resistance of clopidogrel/aspirin, and the detection of the SNP combination can judge whether the target to be detected belongs to the drug-taking susceptible population, so as to guide and adjust the clinical medication scheme, provide basis for clinical personalized treatment and prevent the adverse reaction of the drug.

TABLE 4 screening results of the SNP combination in aspirin-treated and clopidogrel/aspirin-treated populations

Abbreviation NE, not tested.

In the above table, the incidence rate is the rate of stroke recurrence after administration, and in the clopidogrel/aspirin combination group, the rate of stroke recurrence of PAR-1 variant carriers (AT, or TT) was significantly lower than that of wild-type patients (in which the rate of recurrence was reduced by 20.3% for AT type versus AA and 46.2% for TT type versus AA), so that clopidogrel addition to PAR-1 variant carriers (AT, or TT) was recommended. In patients with non-mutated CYP2C19 x 2, CYP2C19 x 3 and CYP2C19 x 17, the incidence of aspirin alone is significantly higher than that of clopidogrel-aspirin combination, and thus clopidogrel-aspirin combination is suggested.

Example 2: detecting CYP2C19 x 2 by a sequencing method; CYP2C19 x 3; CYP2C19 and PAR-1 genotype

The current gold standard of gene polymorphism sites is sequencing method, and the sequencing method is adopted in the embodiment to carry out sequencing on CYP2C19 x 2; CYP2C19 x 3; the distribution of CYP2C19 x 17 and PAR-1 genes in healthy populations was studied. The reliability and accuracy of the method of the invention were assessed by comparison with the results obtained from real-time fluorescent quantitative PCR Taqman.

The sequencing method is to amplify a target gene fragment of a sample to be detected by a conventional PCR technology, determine a sequence by a conventional dideoxy method, and compare the sequence with an original sequence to analyze polymorphic sites, and is the most commonly used method for SNP research at present. The method can be used for measuring the difference of species molecule levels in different levels in and among populations, and can be used for the research of genotyping and biodiversity.

The present example refers to CYP2C19 x 2; CYP2C19 x 3; CYP2C19 x 17 and PAR-1 genes are screened and distributed in healthy population as an example, and the genotype distribution in the healthy population is detected by a sequencing method.

In the sequencing detection method of this example, CYP2C19 × 2; CYP2C19 x 3; the distribution of SNPs in CYP2C19 x 17 and PAR-1 genes in healthy humans was studied. Through comparison with other races, differences of the group of SNP loci in clopidogrel/aspirin medication sensitivity in Chinese population are analyzed. Simultaneously, the method is compared with the Taqman method, and the consistency of the detection results of the two methods is proved

According to human CYP2C19 x 2; CYP2C19 x 3; 4 sets of corresponding primers are respectively synthesized by nucleotide sequences at two sides of polymorphic sites of CYP2C19 x 17 and PAR-1 genes, each set of primer is directed at a specific polymorphic site, and the polymorphic site is positioned in the middle of a PCR amplification product, so that sequencing and analysis are facilitated

Preferably the PCR reaction procedure described above comprises:

(a) Because of the non-specific amplification and the existence of primer dimer in PCR amplification, pfu DAN polymerase is used, and the polymerase has the activity of DNA synthetase at the 5 '-end to the 3' -end and the activity of DNA exonuclease at the 3'-5' -end, so that the invention can not only carry out DNA synthesis, but also immediately identify and excise mismatched nucleotides, greatly improve the specificity of PCR amplification and simultaneously reduce the generation of primer dimer.

(b) The end of the 3' end of the primer is modified by phosphorothioation, so that the end of the primer is prevented from being degraded by the activity of pfu DAN polymerase 3' -5' exonuclease, and the amplification specificity is further improved.

(c) The PCR amplification efficiency and the amplification specificity are further improved by more accurate optimization and selection of a primer concentration range (0.5-5 mM), a primer length range (20-30 bp), an annealing temperature (60-67 ℃) during reaction, an amplified fragment length (200-400 bp), a genome DNA concentration range (5-15 ng) to be detected, a reaction system and the like.

In the embodiment, by utilizing the characteristic that a primer cannot be extended when mismatch exists at the end of the 3 'end in the PCR reaction, the allele SNP locus is designed at the end of the 3' end of the fluorescent quantitative PCR primer, and is subjected to appropriate modification, means such as application of pfu DNA polymerase and the like are added, in addition, a series of conditions such as primer concentration, annealing temperature, fragment amplification length, template DNA concentration and the like of the PCR are optimized, and reliable and accurate results are obtained according to the existence of an amplification product and the length of a BanI enzyme digestion fragment through DNA agarose gel electrophoresis detection. The invention can not only accurately detect CYP2C19 x 2 in the human genome; CYP2C19 x 3; CYP2C19 x 17 and PAR-1 gene, and is also suitable for other gene mutation and SNP detection.

The experimental method comprises the following steps:

1. obtaining a sample to be tested: extracting human genome DNA in blood, taking 500 mu L of human blood, extracting by using a blood DNA extraction kit, measuring the light absorption value at 260nm to calculate the DNA concentration, and diluting the DNA concentration to 10 ng/mu L.

2. Designing a primer: based on the nucleotide Sequence in the human genome (GenBank: NCBI Reference Sequence: CYP2C 19X 2:

TABLE 5 design of sequencing analysis primers

3. And simultaneously carrying out phosphorothioate modification on the tail end of the 3' end of each primer.

4. Human CYP2C19 x 2; CYP2C19 x 3; CYP2C19 x 17; and cloning and sequencing of PAR-1 genomic DNA fragments:

4.1. designing a primer: based on the nucleotide sequences in the human genome (GenBank: NCBI Reference Sequence: CYP2C 19X 2;

primer sequences are shown in table 5:

the sequences obtained were the following:

CYP2C19*2：rs4244285(SEQ ID NO.17):

AGATATGCAATAATTTTCCCACTATCATTGATTATTTCCC(G/A)GGAACCCATAACAAATTACTTAAAAACCTTGCTTTTATGGAAAGTGATATTTTGGAGAAAGTAAAAGAACACCAAGAATCGATGGACATCAACAACCCTCGGGACTTTAT

CYP2C19*3：rs4986893(SEQ ID NO.18):

GATGGAAAAATTGAATGAAAACATCAGGATTGTAAGCACCCCCTG(A/G)ATCCAGGTAAGGCCAAGTTTTTTGCTTCCTGAGAAACCACTTACAGTCTTTTTTTCTGGGAAATCCAAAATTCTATATTG

CYP2C19*17rs12248560(SEQ ID NO.19):

AATGTGGTATATATTCAGAATAACTAATGTTTGGAAGTTGTTTTGTTTTGCTAAAACAAAGTTTTAGCAAACGATTTTTTTTTTCAAATTTGTGTCTTCTGTTCTCAAAG(C/T)ATCTCTGATGTAAGAGATAATGCGCCACGATGGGCATCAGAAGA

PAR1:rs1687537(SEQ ID NO.20)

ATAAACAAAAGTAAAATATGCTCTCTGCTTGTCGCTTTTGCCTTGTTGATGCGTTCACTTTTTACATTTAAAATTTTTTT(A/T)ATTTTATTTTTCAGAATCAAAAGCAACAAATGCCACCTTAGATCCCCGGTCATTTCTTCT

4.2.PCR amplification reaction: the following PCR reaction system was used.

The following reagents were added to a 200. Mu.L micro PCR reaction tube:

deionized sterile water was added to a final volume of 50uL.

PCR conditions were 94 ℃ for 5 minutes, 94 ℃ for 30 seconds, 54 ℃ for 1 minute, 72 ℃ for 1 minute, 40 cycles, and then 72 ℃ for 10 minutes for extension.

4.3. Cloning of PCR product: the amplified PCR fragment is recovered by a PCR product recovery kit, the tail end of the PCR fragment is filled with T4DNA polymerase (T4 DNA polymerase), agarose gel electrophoresis is carried out, the target fragment is recovered and purified by a gel recovery kit and then inserted into an EcoRV site on a pBluescript II SK (+) vector, the method is shown in documents (Sambrooks, molecular cloning manual), the ligation product is transformed into a DH5 strain, and positive clones are screened by a PCR method.

4.4. And (4) carrying out DNA sequence determination on the positive clone, wherein the sequencing result is consistent with the sequence comparison of the gene library.

5. Establishment of positive control:

to contain CYP2C19 x 2; CYP2C19 x 3; CYP2C19 x 17; and PAR-1 genomic DNA fragments.

Secondly, analyzing results:

cyp2c19 x 2; CYP2C19 x 3; distribution of CYP2C19 x 17 and PAR-1 in the Han population of China

The present invention detects CYP2C19 x 2; CYP2C19 x 3; and CYP2C19 x 17 and PAR-1SNP in combination in chinese population as shown in table 6:

TABLE 6

From a comparison of the percentage distribution of the population in both methods (see table 4), the results of both the detection method proposed by the present invention and the sequencing detection method show a high degree of consistency. Therefore, the real-time fluorescence quantitative PCR Taqman method has higher reliability and accuracy in detection, can be used for discriminating whether a target to be detected belongs to a drug-taking susceptible population or not, guides and adjusts a clinical drug-taking scheme, and provides a basis for clinical personalized treatment.

Example 3

This example provides a human cytochrome P450CYP2C19 x 2; CYP2C19 x 3; CYP2C19 x 17; and PAR1: rs168753 genome Single Nucleotide Polymorphism (SNP) real-time fluorescence quantitative PCR Taqman detection kit.

The kit can be used for treating CYP2C19 x 2 in the human genome; CYP2C19 x 3; CYP2C19 x 17; and PAR1: rs168753 locus, and is suitable for typing of the combination SNP analysis. The kit adopts the principle of a fluorescent probe hydrolysis method (Taqman method), comprises the primer concentration which is most suitable for real-time fluorescent quantitative PCR reaction and the probe concentration, and adopts hot start Taq DNA polymerase and buffer solution and primer combination which are most matched with the multiplex real-time fluorescent quantitative reaction, so that nonspecific PCR amplification can be effectively inhibited, and the aim of high-sensitivity and high-flux real-time fluorescent quantitative PCR amplification reaction is fulfilled. When the experiment is carried out, the preparation of the PCR reaction solution is very convenient and simple. Human genome CYP2C19 x 2; CYP2C19 x 3; CYP2C19 x 17; and PAR1: rs 168753.

TABLE 7 composition of the kit

Reaction solution reagent a: 2 XPCR Master containing Hot Start Taq DNA polymerase

Reaction solution reagent B: CYP2C19 x 2; CYP2C19 x 3 two sets of primers and corresponding 4SNP probes

Reaction solution reagent D: CYP2C19 x 17; and PAR1: rs168753 two sets of primers and corresponding 4SNP probes

Working standard 1: human-containing CYP2C19 x 2; 4DNA clones at the CYP2C19 x 3SNP site

Working standard 2: human-containing CYP2C19 x 17; and 4DNA clones at the PAR-1

This example utilizes the present kit for human genome CYP2C19 x 2; CYP2C19 x 3; CYP2C19 x 17; and PAR1: rs168753SNP, the Real Time PCR amplification instrument comprises:

Thermal Cycler

Real Time System

Smart

System/Smart/>

II System(Cepheid)

Applied Biosystems 7900HT/7300/7500Real-Time PCR System、7500 Fast Real-Time PCR System、StepOnePlus ^TM Real-Time PCR System(Applied Biosystems)

(Roche Diagnostics)

Mx3000P ^TM (Stratagene)

the transportation of the kit can be carried out at the temperature of 2-8 ℃. During storage, the product should be stored at-20 ℃.

The validity period is as follows: the effective period of the kit is 12 months, and the kit is used in the effective period.

The kit uses hot start Taq DNA polymerase to carry out PCR amplification, and achieves the purpose of detecting PCR products by monitoring specific fluorescence in reaction liquid. CYP2C19 x 2 in human genome DNA; CYP2C19 x 3; CYP2C19 x 17; and PAR1: rs168753 gene for PCR amplification, through the thermal denaturation of DNA chain, primer annealing, primer extension under the action of DNA polymerase three steps cycle to cycle, can amplify human genome CYP2C19 x 2 in a short time; CYP2C19 x 3; CYP2C19 x 17; and a PAR1: rs168753 genomic DNA fragment.

The DNA polymerase in the kit uses hot start Taq DNA polymerase, so that primer dimer generated during primer annealing and non-specific amplification caused by the primer dimer are inhibited, and the accuracy of PCR amplification is greatly improved.

The kit is used for detecting human genome CYP2C19 x 2 in the human genome by adopting a fluorescent probe; CYP2C19 x 3; CYP2C19 x 17; and PAR1: rs168753SNP, and can accurately detect the SNP by detecting the fluorescence signal intensity in the PCR reaction solution.

The kit is suitable for Real-time PCR reaction, and can quickly and accurately carry out the Real-time PCR reaction on human genome CYP2C19 x 2; CYP2C19 x 3; CYP2C19 x 17; and PAR1: rs168753 SNP.

The primers are pre-mixed in the reaction liquid, when the PCR reaction liquid is prepared, the Real Time PCR reaction can be carried out only by adding the DNA template and sterile distilled water, and the operation is simple and convenient.

The DNA polymerase uses hot start DNA polymerase, is combined with a multiple PCR buffer solution system independently developed by a company, and has the characteristics of high amplification efficiency, high amplification sensitivity and high amplification specificity.

The kit comprises the following steps:

1. the extracted human genomic DNA was diluted to 0.5-15 ng/. Mu.L with a nucleic acid dilution buffer.

2. mu.L of each diluted sample DNA was added to 2 PCR reaction wells.

3. Take 8. Mu.L of each of reaction tubes B and C in the kit, add to 2 reaction wells, respectively.

4. mu.L of the reagent in the reaction tube A was added to the reaction well.

5. Negative controls were treated separately according to the same method as above; 1, a working standard product; a working standard 2;

6. the prepared reaction solution was mixed well and then centrifuged at 5000rpm for 3 minutes.

7. And (3) putting the reaction tube into a real-time fluorescent PCR (polymerase chain reaction) amplification instrument for amplification reaction.

8. Setting circulation parameters at 95 ℃ for 10min; 20 seconds at 95 ℃; 60 seconds at 62 ℃;40 cycles.

CYP2C19 x 2 of a healthy person of a normal Chinese Han family population is treated by adopting the kit; CYP2C19 x 3; CYP2C19 x 17; and PAR-1, rs168753SNP (Table 6), and the results are described in the above examples. It follows that the kit of the invention is not only useful for CYP2C19 x 2 in the normal population; CYP2C19 x 3; CYP2C19 x 17; and research on the correlation between PAR-1.

SNP detection was performed on randomly selected samples of 500 patients using the kit of this example. Then, medication guidance is carried out based on the following principles:

(a) When the PAR-1;

(b) When detecting the mutation of PAR-1;

(c) When CYP2C19 x 2: when rs4244285 shows single mutation (genotype GA) or double mutation (genotype AA), clopidogrel is not recommended;

(d) When CYP2C19 × 3 is detected: when single mutation (genotype GA) or double mutation (genotype AA) occurs in rs4986893, clopidogrel is not recommended;

(e) When CYP2C19 × 17: clopidogrel should be used with cautions when there is a single mutation (genotype CT) or double mutation (genotype TT) in rs 12248560.

The partially detected cases of typical patients with SNPs are shown in the following table.

TABLE 8

Note: "-" indicates that there is no mutation at this site (i.e., wild type); "- +" indicates the presence of a single mutation (heterozygous) at that site; "+ +" indicates the presence of double mutations (homozygous) at this site.

For example, for PAR-1 rs16875, "-" denotes AA; "- +" denotes AT; "+ +" TT. CYP2C19 x 2; CYP2C19 x 3; CYP2C19 x 17 and so on.

For patients 3-1 (52 cases of similar patients), the treatment effect is not obvious when the conventional aspirin is used. The combination of aspirin and clopidogrel is replaced later, and the treatment effect is obvious.

Aspirin was administered to patients 3-2a (63 similar patients in total) with insignificant effect, and the therapeutic effect was improved after increasing the amount of drug (2 times the conventional amount).

Aspirin was administered to patient 3-2b (62 cases of similar patients) with insignificant effect; the aspirin and the clopidogrel are combined later, so that the treatment effect is obvious.

For patients 3-3 (21 similar patients), clopidogrel administration is not recommended.

For patients 3-4 (similar patients in 1 case), clopidogrel administration is not recommended.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Beijing Temple Hospital affiliated to capital medical university

<120> human cytochrome CYP2C19 and PAR1 gene polymorphism site detection kit and application thereof

<130> P2017-0172

<160> 23

<170> PatentIn version 3.5

<210> 1

<211> 26

<212> DNA

<213> Artificial sequence

<400> 1

agatatgcaa taattttccc actatc 26

<210> 2

<211> 24

<212> DNA

<213> Artificial sequence

<400> 2

ataaagtccc gagggttgtt gatg 24

<210> 3

<211> 26

<212> DNA

<213> Artificial sequence

<400> 3

gatggaaaaa ttgaatgaaa acatca 26

<210> 4

<211> 25

<212> DNA

<213> Artificial sequence

<400> 4

ctgggaaatc caaaattcta tattg 25

<210> 5

<211> 25

<212> DNA

<213> Artificial sequence

<400> 5

ctgggaaatc caaaattcta tattg 25

<210> 6

<211> 26

<212> DNA

<213> Artificial sequence

<400> 6

tagttattct gaatatatac cacatt 26

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence

<400> 7

cttttgcctt gttgatgcgt tcac 24

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<400> 8

caacaaatgc caccttagat c 21

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence

<400> 9

tatgggttcc ccgggaaata a 21

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence

<400> 10

tatgggttcc ctgggaaata a 21

<210> 11

<211> 19

<212> DNA

<213> Artificial sequence

<400> 11

ttacctggat tcagggggt 19

<210> 12

<211> 19

<212> DNA

<213> Artificial sequence

<400> 12

ttacctggat ccagggggt 19

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence

<400> 13

ttctcaaagc atctctgatg t 21

<210> 14

<211> 21

<212> DNA

<213> Artificial sequence

<400> 14

ttctcaaagt atctctgatg t 21

<210> 15

<211> 26

<212> DNA

<213> Artificial sequence

<400> 15

ctgaaaaata aaattaaaaa aatttt 26

<210> 16

<211> 26

<212> DNA

<213> Artificial sequence

<400> 16

ctgaaaaata aaataaaaaa aatttt 26

<210> 17

<211> 151

<212> DNA

<213> human

<220>

<221> misc_feature

<222> (41)..(41)

<223> n = g or a

<400> 17

agatatgcaa taattttccc actatcattg attatttccc nggaacccat aacaaattac 60

ttaaaaacct tgcttttatg gaaagtgata ttttggagaa agtaaaagaa caccaagaat 120

cgatggacat caacaaccct cgggacttta t 151

<210> 18

<211> 126

<212> DNA

<213> human

<220>

<221> misc_feature

<222> (46)..(46)

<223> n = a or/g

<400> 18

gatggaaaaa ttgaatgaaa acatcaggat tgtaagcacc ccctgnatcc aggtaaggcc 60

aagttttttg cttcctgaga aaccacttac agtctttttt tctgggaaat ccaaaattct 120

atattg 126

<210> 19

<211> 155

<212> DNA

<213> human

<220>

<221> misc_feature

<222> (111)..(111)

<223> n = c or t

<400> 19

aatgtggtat atattcagaa taactaatgt ttggaagttg ttttgttttg ctaaaacaaa 60

gttttagcaa acgatttttt ttttcaaatt tgtgtcttct gttctcaaag natctctgat 120

gtaagagata atgcgccacg atgggcatca gaaga 155

<210> 20

<211> 141

<212> DNA

<213> human

<220>

<221> misc_feature

<222> (81)..(81)

<223> n=a/t

<400> 20

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tttacattta aaattttttt nattttattt ttcagaatca aaagcaacaa atgccacctt 120

agatccccgg tcatttcttc t 141

<210> 21

<211> 22

<212> DNA

<213> Artificial sequence

<400> 21

ataagtggtt ctatttaatg tg 22

<210> 22

<211> 28

<212> DNA

<213> Artificial sequence

<400> 22

ccactttcat cctgggctgt gctccctg 28

<210> 23

<211> 23

<212> DNA

<213> Artificial sequence

<400> 23

tcttattttt tctcatgagc atc 23

Claims (10)

1. The application of the PAR-1: rs168753 gene locus and/or a detection reagent thereof is characterized in that the application is used for preparing a reagent or a kit, the reagent or the kit is used for detecting the sensitivity of a detected object to clopidogrel-aspirin combined medication, and the detected object is a cerebral apoplexy patient.

2. The use of claim 1, wherein the reagent or kit further comprises a detection reagent for detecting one or more genetic loci selected from the group consisting of: CYP2C19 x 2: rs4244285 gene site, CYP2C19 x 3: the rs4986893 gene site, and CYP2C19 x 17: rs12248560 gene locus.

3. The use of claim 1, wherein the subject is susceptible to the antiplatelet agent clopidogrel if said subject is mutated in:

PAR-1: rs168753 A→T。

4. the use of claim 1, wherein a mutation of the PAR-1: rs168753 gene locus from a to T indicates that the subject is sensitive to a clopidogrel-aspirin combination.

5. The use of claim 1, wherein if the genotype of the PAR-1: rs168753 locus of said subject is TT, said subject is sensitive to a combination of clopidogrel and aspirin.

6. The use of claim 2, wherein clopidogrel is not advised if the subject is mutated at one or more of the genetic loci selected from the group consisting of:

CYP2C19*2： rs4244285 G→A；

CYP2C19*3： rs4986893 G→A；

CYP2C19*17： rs12248560 C→T。

7. the use of claim 1, wherein said reagent comprises a primer, a probe, a chip, or an antibody.

8. The use of claim 2, wherein the kit comprises one or more reagents selected from the group consisting of:

(a) Specific primers for gene detection;

(b) Specific probes for gene detection;

(c) A chip for gene detection;

(d) A specific antibody for detecting an amino acid mutation corresponding to the mutated gene;

wherein, the specific primer, the specific probe and the chip are directed to the gene locus selected from the following group: CYP2C19 × 2: rs4244285 gene site, CYP2C19 x 3: rs4986893 gene site, CYP2C19 x 17: rs12248560 Gene locus and PAR-1: rs168753 Gene locus;

the mutated gene comprises a mutated genetic locus selected from the group consisting of:

CYP2C19 x 2: rs4244285 gene locus, CYP2C19 x 3: rs4986893 gene site, CYP2C19 x 17: rs12248560 Gene site and PAR1: rs168753 Gene site.

9. The use of claim 8, wherein the primers comprise:

primer pair 1, primer pair directed against CYP2C19 x 2: rs4244285 gene locus:

the 5'-3' sequence of the upstream primer is as follows: AGATGCAATAATTTTCCCACTATC (Seq ID No. 1),

the 5'-3' sequence of the downstream primer is as follows: ATAAAGTCCCGAGGGGTTGTTGATG (Seq ID No. 2);

primer pair 2, primer pair 2 for CYP2C19 x 3: rs4986893 gene site:

the upstream primer 5'-3' sequence is: GATGGAAAAATTGAATGAAAACATCA (Seq ID No. 3),

the 5'-3' sequence of the downstream primer is as follows: CTGGGAAATCCAAAATTCTATATTG (Seq ID No. 4);

primer pair 3, directed against CYP2C19 x 17: rs12248560 gene site:

the 5'-3' sequence of the upstream primer is as follows: TCTTCTGATGCCCACGTGGCGCGCATT (Seq ID No. 5),

the downstream primer 5'-3' sequence is: TAGTTATTCTGAATATACCACATT (Seq ID No. 6);

primer pair 4, aiming at PAR-1: rs168753 gene locus

The 5'-3' sequence of the upstream primer is as follows: CTTTTGCCTTGTTGATGCGTTCAC (Seq ID No. 7),

the downstream primer 5'-3' sequence is: CAACAATGCCACCTTAGATC (Seq ID No. 8).

10. The use of claim 9, wherein the kit further comprises a probe sequence selected from the group consisting of:

probe 1, for CYP2C19 x 2: rs4244285 gene locus:

P1：FAM- TATGGGTTCCCCGGGAAATAA-BHQ1(SEQ ID NO.9)

P2: ROX- TATGGGTTCCCTGGGAAATAA-BHQ2(SEQ ID NO.10)；

probe 2, for CYP2C19 x 3: rs4986893 gene site:

P3：VIC- TTACCTGGATTCAGGGGGT-BHQ1(SEQ ID NO.11)

P4：TAMRA- TTACCTGGATCCAGGGGGT-BHQ2(SEQ ID NO.12)；

probe 3, directed against CYP2C19 x 17rs12248560 gene locus:

P5：FAM-TTCTCAAAGCATCTCTGATGT-BHQ1(SEQ ID NO.13)

P6：ROX- TTCTCAAAGTATCTCTGATGT-BHQ2(SEQ ID NO.14)；

probe 4, at PAR1: rs168753 gene site:

P13：FAM-CTGAAAAATAAAATTAAAAAAATTTT -BHQ1(SEQ ID NO.15)

P14：ROX- CTGAAAAATAAAATAAAAAAAATTTT -BHQ2(SEQ ID NO.16)。