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

A human cytochrome CYP2C19 and PAR1 gene polymorphism site detection kit and its use

Technical Field

The present invention relates to the field of biomedicine, and in particular to a human cytochrome P450CYP2C19 and PAR1 gene polymorphism site detection kit and its use.

Background Art

Ischemic heart disease and ischemic stroke are the top two causes of death worldwide announced by the World Health Organization (WHO). Atherosclerosis, plaque rupture, and thrombosis are the direct causes of cardiovascular and cerebrovascular events, and thrombotic diseases have become the number one killer of human health. Antiplatelet therapy is the main means of preventing thrombotic diseases.

Plaque rupture and thrombosis are the basic pathological changes that lead to acute cardiovascular events. Antithrombotic therapy, especially antiplatelet therapy, is the most important intervention measure for acute coronary syndrome. Platelets have no nucleus and are fragments produced in the cytoplasm of megakaryocytes in the bone marrow. Their maximum survival period in the circulation is about 10 days. Under normal circumstances, platelets are in an inactive state in the blood circulation. However, when the vascular endothelium is damaged or the atherosclerotic plaque ruptures, the matrix under the endothelium is exposed, and activation factors appear, platelets can be activated and play an important role in the physiological hemostasis process.

Antiplatelet drugs are developed to target various links and related targets of platelet activation, and can act on various stages of platelet activation, adhesion, and aggregation.

The main drugs currently used are:

Cyclooxygenase (COX) inhibitors: Aspirin can promote the acetylation of serine 529 at the active site of COX-1, and irreversibly inhibit the activity of COX-1. COX-1 plays a key role in the initial step of prostaglandin biosynthesis. It can catalyze the conversion of arachidonic acid into prostaglandin H2 (PGH2), which is the direct precursor of TXA2. The result of aspirin inhibiting COX-1 is a decrease in the production of TXA2, which is a strong platelet aggregate. The decrease in TXA2 production ultimately affects the aggregation and release reaction of platelets.

ADP receptor inhibitor: clopidogrel. After oral administration, the drug is absorbed in the small intestine and is regulated by the proton pump P-glycoprotein encoded by the ABCB1 gene. After absorption, 85%-95% is hydrolyzed by carboxylase to inactive carboxylic acid metabolites SR26334, and only 10%-15% is metabolized to active products by the liver cytochrome P450 (CYP450) enzyme system. Clopidogrel's active metabolite R-130964 irreversibly binds to the ADP receptor (P2Y12) on the platelet surface through a covalent bond when circulating in the liver, blocking the inhibitory effect of ADP on adenylate cyclase, promoting cAMP-dependent VASP phosphorylation, inhibiting the binding of fibrinogen to platelet glycoprotein GPⅡb/Ⅲa receptors, and thus inhibiting platelet aggregation. In addition, clopidogrel can also block the secondary ADP-mediated activation of the glycoprotein GPⅡb/Ⅲa complex, which causes platelet activation and expansion, thereby inhibiting platelet aggregation induced by other agonists.

Clopidogrel must be bioconverted into active products to inhibit platelet aggregation. This bioconversion process requires cytochrome P450 encoded by the CYP2C19 gene to be metabolized in the liver, and the active product can selectively and irreversibly block adenosine diphosphate-dependent platelet aggregation.

Different individuals have different reactivity to clopidogrel. About 40% of Asian patients still show residual platelet aggregation after treatment, which is the so-called "clopidogrel resistance". The main reason is that CYP2C19*2, CYP2C19*3CYP2C19 gene mutations lead to a reduction in the active metabolites of clopidogrel. The latest clinical research results have confirmed that individuals with poor metabolism caused by CYP2C19 gene mutations have an increased risk of embolism re-formation, an increased risk of cardiovascular and cerebrovascular events, and an increased mortality rate. For individuals with CYP2C19 gene mutations, the dose of clopidogrel needs to be increased (loading dose of 300mg or 600mg) to achieve the effect of blocking platelet aggregation.

Protease activated receptor-1 (PAR-1) is a G protein-coupled receptor on the cell surface and a thrombin receptor. Thrombin is the strongest platelet activator and mediates cell responses through PAR1. Human platelets express PAR1 receptors, which can couple with G12/13 and Gq families and indirectly activate the Gi signaling pathway by secreting ADP, causing a series of changes in platelet activation and inducing coagulation reactions. It plays a key role in thrombosis and hemostasis.

Therefore, SNP detection of the above-mentioned multiple genes has important clinical significance for achieving personalized use of antiplatelet drugs and reducing the mortality rate of cardiovascular diseases.

Summary of the invention

The purpose of the present invention is to provide a human cytochrome CYP2C19 and PAR1 gene polymorphism site detection kit and use.

In a first aspect, the present invention provides the use of the PAR-1:rs168753 gene locus and/or its detection reagent for preparing a reagent or a kit for detecting the sensitivity of a detected subject to an antiplatelet drug.

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

In another preferred embodiment, the detected object includes: humans or non-human animals (such as livestock, poultry, experimental animals, etc.).

In another preferred embodiment, the antiplatelet drug is selected from the following group:

Cyclooxygenase (COX) inhibitors and ADP receptor inhibitors.

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

In another preferred embodiment, the ADP receptor inhibitors include: clopidogrel and ticlopidine.

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

In another preferred embodiment, if the PAR-1:rs168753 gene locus of the detected subject mutates from A to T, it indicates that the subject is sensitive to clopidogrel.

In another preferred embodiment, if the PAR-1:rs168753 gene locus of the detected subject mutates from A to T, it means that 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 detected subject is TT, it means that the subject is more sensitive to the combined use of clopidogrel-aspirin.

In another preferred embodiment, if the subject undergoes the following mutations at one or more gene loci selected from the following group, the subject is not recommended to use clopidogrel (is insensitive to clopidogrel):

CYP2C19*2:rs4244285G→A;

CYP2C19*3:rs4986893G→A;

CYP2C19*17: rs12248560C→T.

In another preferred embodiment, the reagents include primers, probes, chips, or antibodies.

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

(A) Specific primers used for gene detection;

(B) Specific probes for gene detection;

(C) Chips used for gene detection;

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

Wherein, the specific primers, the specific probes, and the chip are directed to gene loci selected from the following group: CYP2C19*2: rs4244285 gene loci, CYP2C19*3: rs4986893 gene loci, CYP2C19*17: rs12248560 gene loci, and PAR1: rs168753 gene loci;

The mutated gene contains a mutated gene site selected from the following group: CYP2C19*2: rs4244285 gene site, CYP2C19*3: rs4986893 gene site, CYP2C19*17: rs12248560 gene site PAR1: rs168753 gene site.

In another preferred embodiment, the primers include:

Primer pair 1, targeting CYP2C19*2: rs4244285 gene locus:

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

The 5′-3′ sequence of the downstream primer was: ATAAAGTCCCGAGGGTTGTTGATG (Seq ID No. 2);

Primer pair 2, targeting CYP2C19*3: rs4986893 gene locus:

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

The 5′-3′ sequence of the downstream primer was: CTGGGAAATCCAAAATTCTATATTG (Seq ID No. 4);

Primer pair 3, targeting CYP2C19*17:rs12248560 gene locus:

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

The 5’-3’ sequence of the downstream primer is: TAGTTATTCTGAATATATACCACATT (Seq ID No. 6).

Primer pair 4, targeting PAR-1:rs168753 gene locus

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

The 5’-3’ sequence of the downstream primer is: CAACAAATGCCACCTTAGATC (Seq ID No. 8).

In another preferred embodiment, the primers further include extension primers containing primer sequences Seq ID No.1-14.

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

In another preferred embodiment, in the real-time fluorescence quantitative PCR, the annealing temperature is between 60-67° C., and the length of the PCR amplified fragment is 80-300 bp.

In another preferred embodiment, in the real-time fluorescence quantitative PCR, the annealing temperature of the fluorescent probe is between 60-70°C.

In another preferred embodiment, both ends of the probe are modified with chemical groups, the 5' end is modified with a fluorescent excitation group, and the 3' end is modified with a fluorescent quenching group.

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

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

Probe 1, targeting CYP2C19*2: rs4244285 gene locus:

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

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

Probe 2, targeting CYP2C19*3: rs4986893 gene locus:

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

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

Probe 3, targeting the CYP2C19*17rs12248560 gene locus:

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

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

Probe 4, targeting PAR1:rs168753 gene locus:

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

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

In another preferred embodiment, the probe further comprises a series of extension primers for the probe sequences P1-P14.

The second aspect of the present invention provides a kit, which includes a PAR1:rs168753 gene site detection reagent.

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

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

(A) Specific primers used for gene detection;

(B) Specific probes for gene detection;

(C) Chips used for gene detection;

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

Wherein, the specific primers, the specific probes, and the chip are directed to gene loci selected from the following group: CYP2C19*2: rs4244285 gene loci, CYP2C19*3: rs4986893 gene loci, CYP2C19*17: rs12248560 gene loci, and PAR-1: rs168753 gene loci;

The mutated gene contains a mutated gene site selected from the group consisting of:

CYP2C19*2: rs4244285 gene locus, CYP2C19*3: rs4986893 gene locus, CYP2C19*17: rs12248560 gene locus and PAR1: rs168753 gene locus.

In another preferred embodiment, the primers include:

Primer pair 1, targeting CYP2C19*2: rs4244285 gene locus:

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

The 5′-3′ sequence of the downstream primer was: ATAAAGTCCCGAGGGTTGTTGATG (Seq ID No. 2);

Primer pair 2, targeting CYP2C19*3: rs4986893 gene locus:

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

The 5′-3′ sequence of the downstream primer was: CTGGGAAATCCAAAATTCTATATTG (Seq ID No. 4);

Primer pair 3, targeting CYP2C19*17:rs12248560 gene locus:

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

The 5’-3’ sequence of the downstream primer is: TAGTTATTCTGAATATATACCACATT (Seq ID No. 6).

Primer pair 4, targeting PAR-1:rs168753 gene locus

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

The 5’-3’ sequence of the downstream primer is: CAACAAATGCCACCTTAGATC (Seq ID No. 8).

In another preferred embodiment, the primers further include extension primers containing primer sequences Seq ID No.1-8.

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

In another preferred embodiment, in the real-time fluorescence quantitative PCR, the annealing temperature is between 60-67° C., and the length of the PCR amplified fragment is 80-300 bp.

In another preferred embodiment, in the real-time fluorescence quantitative PCR, the annealing temperature of the fluorescent probe is between 60-70°C.

In another preferred embodiment, both ends of the probe are modified with chemical groups, the 5' end is modified with a fluorescent excitation group, and the 3' end is modified with a fluorescent quenching group.

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

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

Probe 1, targeting CYP2C19*2: rs4244285 gene locus:

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

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

Probe 2, targeting CYP2C19*3: rs4986893 gene locus:

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

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

Probe 3, targeting the CYP2C19*17rs12248560 gene locus:

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

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

Probe 4, targeting PAR1:rs168753 gene locus:

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

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

In another preferred embodiment, the probe further comprises a series of extension primers for the probe sequences P1-P14.

The third aspect of the present invention provides a method for detecting whether a sample has a single nucleotide variation in vitro, comprising the steps of:

(a) amplifying a polynucleotide of a sample using specific primers to obtain an amplification product; and

(b) Detect whether the following single nucleotide variations exist in the amplified product:

CYP2C19*2:rs4244285G→A;

CYP2C19*3:rs4986893G→A;

CYP2C19*17:rs12248560C→T; and

PAR-1:rs168753A→T.

In another preferred embodiment, the length of the amplified product is 80-2000 bp and contains one or more single nucleotide variations selected from the following group:

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 for detecting the sensitivity of an individual to an antiplatelet drug, comprising the steps of:

(i) detecting a gene locus selected from the following group of loci of the individual and comparing it with a corresponding normal gene locus to determine whether a gene mutation exists:

CYP2C19*2:rs4244285;

CYP2C19*3:rs4986893;

CYP2C19*17:rs12248560;

PAR-1:rs168753.

In another preferred embodiment, the gene mutation is a single nucleotide variation selected from the following group:

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:rs168753 has a mutation, and/or

If there is no mutation in CYP2C19*2:rs4244285; and/or CYP2C19*3:rs4986893; and/or CYP2C19*17:rs12248560, it indicates sensitivity to clopidogrel.

Within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

DETAILED DESCRIPTION

The inventors have obtained a gene polymorphism site related to the sensitivity of antiplatelet drugs through extensive and in-depth research. The experimental results show that the sensitivity of an individual to antiplatelet drugs can be assessed by detecting the gene polymorphism site of the present invention. On this basis, the present invention was completed.

The present invention relates to a method for identifying four polymorphic sites of two genes, namely, cytochrome P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753, by using a fluorescent hydrolysis probe (Taqman) method. A kit formed by the method is used to determine whether the target to be tested belongs to a susceptible population for medication, thereby guiding and adjusting the clinical medication regimen, providing a basis for clinical personalized treatment, and preventing adverse drug reactions. Therefore, the kit and detection method provided by the present invention can be used to guide personalized medication for stroke.

Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terminology used herein is intended only to describe specific embodiments, and is not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. As used herein, the term "about" when used in reference to a specific 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 therebetween (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 described.

Cytochrome P450

Cytochrome P450 (CYP) is a superfamily oxidase that is widely distributed in various life forms, from lower bacteria to higher animals and plants. It is an important component of multifunctional oxidases and exists in the microsomes of multiple tissue cells. In the human body, this enzyme is mainly present in the liver tissue. The activity of P450 determines the metabolic rate and clearance of drugs in the body, so it is also called a drug-metabolizing enzyme. The genes of the P450 enzyme system mainly include three families: CYP1, CYP2, and CYP3. The corresponding important enzymes are: CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5.

Drugs metabolized by the P450 family account for more than 80% of drugs sold on the market. Once the function of the enzyme changes, it will affect the metabolism and efficacy of the drug. In the study of adverse drug reactions, about 48% are closely related to the polymorphism of the P450 enzyme gene.

P450 genetic polymorphism is the main cause of P450 enzyme deficiency, decreased or increased expression, and changes in substrate specificity, which ultimately lead to changes in drug metabolomics and cause different individuals to have different drug responses to the same drug.

CYP2C19 is an important member of the second subfamily. The CYP2C19 gene is located on chromosome region 10q24.2 and consists of 9 exons. It has 92% sequence homology with CYP2C9. The protein composed of 490 amino acids is mainly expressed in liver tissue and also in the duodenum. It is one of the important drug metabolizing enzymes in the human body. Among the drugs metabolized by P450 enzymes, 12% are metabolized by CYP2C19.

CYP2C19 has many SNP sites, and its genetic polymorphism plays an important role in the metabolism of cardiovascular and cerebrovascular drugs.

At present, among the 25 mutant alleles of CYP2C19 that have been discovered, at least 10 have caused changes in enzyme activity, the most common of which are CYP2C19*2, CYP2C19*3 and CYP2C19*17 (rs12248560). Among them, the slow metabolizers are mainly CYP2C19*2: and CYP2C19*3, and the fast metabolizers are mainly CYP2C19*17.

The splicing of proteins during the translation of CYP2C19*2 mRNA will lead to mutation inactivation, while CYP2C19*3 can form a terminator, destroying the activity of the transcribed protein. According to statistics, the two mutation sites of CYP2C19*2 and CYP2C19*3 can explain almost 100% of the related poor metabolic genetic defects in East Asians and 85% of Caucasians. A large amount of evidence confirms that different races have great differences in their ability to metabolize CYP2C19 substrates; 2–5% of Caucasians are poor metabolizers, while 13–23% of Asians are poor metabolizers. This is due to the high frequency of CYP2C19*2 and CYP2C19*3 alleles in the Asian population.

Protease-activated receptor-1 (PAR-1)

PAR-1 is a G protein-coupled receptor on the cell surface and a thrombin receptor. Thrombin is the strongest platelet activator and mediates cell reactions through PAR1. Human platelets express PAR1 receptors, which can couple with G12/13 and Gq families and indirectly activate the Gi signaling pathway by secreting ADP, causing a series of changes in platelet activation and inducing coagulation reactions. It plays a key role in thrombosis and hemostasis.

The present invention proposes a detection method based on the correlation between stroke medication and genetics to determine whether the target to be tested belongs to the drug-susceptible population. Primers and probes are synthesized based on the nucleotide sequences of the four polymorphic sites of the two genes cytochrome P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753, and real-time fluorescence quantitative hydrolysis probe PCR is performed on the sample to be tested. The genotype of the PCR amplification product is determined based on the fluorescence value generated by the reaction. It is determined whether the sample belongs to the drug-sensitive population based on the difference in fluorescence.

In a preferred embodiment of the present invention, the primers used include:

CYP2C19*2: rs4244285 primer set

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

The 5′-3′ sequence of the downstream primer (Seq ID No. 2) was: ATAAAGTCCCGAGGGTTGTTGATG;

CYP2C19*3: rs4986893 primer set

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

The 5′-3′ sequence of the downstream primer (Seq ID No. 4) was: CTGGGAAATCCAAAATTCTATATTG;

CYP2C19*17: rs12248560 primer set

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

The 5′-3′ sequence of the downstream primer (Seq ID No. 6) was: TAGTTATTCTGAATATATACCACATT;

PAR1:rs168753 primer set

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

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

The primers of the present invention may also be an extension primer series containing the above primer sequences (Seq ID No.1-Seq ID No.14).

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

Preferably, in real-time fluorescence quantitative PCR, the annealing temperature of the fluorescent probe is between 60-70° C. Wherein, in the real-time fluorescence quantitative allele-specific PCR, the annealing temperature is between 60-67° C., the length of the PCR amplification fragment is 820-300 bp; and the concentration range of the genomic DNA to be tested is between 1-20 ng.

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

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

Probe 1, targeting CYP2C19*2: rs4244285 gene locus:

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

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

Probe 2, targeting CYP2C19*3: rs4986893 gene locus:

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

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

Probe 3, targeting the CYP2C19*17rs12248560 gene locus:

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

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

Probe 4, targeting PAR1:rs168753 gene locus:

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

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

The probe of the present invention comprises: chemical group modification at both ends, a fluorescence excitation group at the 5' end and a fluorescence quenching group at the 3' end, and an extension primer series containing the probe sequence (P1-P14).

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

The present invention also provides a diagnostic reagent for four polymorphic sites based on two genes of cytochrome P450CYP2C19*2:rs4244285;CYP2C19*3:rs4986893;CYP2C19*17:rs12248560; and PAR1:rs168753. It is used to implement the detection method described in the present invention, including an optimized real-time fluorescent quantitative hydrolysis probe multiplex PCR reaction system; the primers; a plasmid positive control containing human P450CYP2C19*2:rs4244285;CYP2C19*3:rs4986893;CYP2C19*17:rs12248560;PAR1:rs168753 gene genomic DNA fragments; Taq DNA polymerase; DMSO and other PCR enhancers.

The present invention also provides a kit for implementing the detection method of the present invention, comprising the above-mentioned cytochrome P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753 two genes for diagnosing the four polymorphic sites.

The present invention provides a diagnostic reagent for drug-sensitive population based on the technical solution of identifying the genotypes of four polymorphic sites of cytochrome P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753. The four sets of primers described in the present invention are used in the real-time fluorescent quantitative hydrolysis probe PCR.

The present invention identifies four polymorphic sites of P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753 and is used in preparing a diagnostic reagent for drug-sensitive populations. The diagnostic reagent is a kit comprising the primers of the present invention, a reaction buffer, a plasmid containing human P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753 genomic DNA fragments, Taq DNA polymerase, and a fluorescent probe.

In the study of genetic polymorphism of people sensitive to stroke medication, the present invention establishes a simple, accurate, rapid and high-throughput real-time fluorescent quantitative probe hydrolysis PCR method for screening people sensitive to stroke medication, which can also be applied to the study of the correlation between SNP and other diseases.

The invention synthesizes corresponding primers based on the nucleic acid sequences on both sides of human P450CYP2C19*2:rs4244285;CYP2C19*3:rs4986893;CYP2C19*17:rs12248560; and PAR1:rs168753SNP, designs corresponding nucleotide probes based on SNP sites, designs the SNP sites in the middle of the probes, and labels the 5' end of the nucleotides of the probes with fluorescent dyes, while labeling the 3' end with fluorescent quenching dyes. Utilizes an optimized PCR reaction program, amplifies genomic DNA extracted from human blood samples through real-time fluorescent probe hydrolysis quantitative PCR reaction, and performs gene polymorphism analysis based on the fluorescence reading data of different wavelengths labeled by each probe.

In the present invention, the optimization process of the real-time fluorescent probe hydrolysis quantitative PCR method adopted includes the use of TaqDNA polymerase, reasonable primer and probe design and modification, and optimization of the multiplex PCR reaction system.

Due to the presence of non-specific amplification and primer dimers in PCR amplification, the present invention uses hot-start TaqDNA polymerase. The biggest advantage of this polymerase is that it is different from ordinary Taq DNA polymerase. Through chemical modification, when the temperature is lower than 50°C, its polymerase activity is completely inhibited, and after 10 minutes at 95°C, its polymerase activity is released. Therefore, when performing DNA synthesis, it greatly improves the specificity of PCR amplification and reduces the generation of primer dimers.

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

In the present invention, the fluorescent probe hydrolysis method (Taqman PCR) in the real-time fluorescent quantitative PCR technology is used to synthesize primers and probes based on the nucleotide sequences of four polymorphic sites of two genes, cytochrome P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753, and real-time fluorescent quantitative hydrolysis probe PCR is performed on the sample to be tested, and the genetic polymorphism of the PCR amplification product gene is judged based on the fluorescence value generated by the reaction. Comparison of the fluorescence differences of the genomic amplification products among the four primer sets with those of the standard reference samples (cloned plasmids containing human cytochrome P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753 genomic DNA fragments) improved the reliability and accuracy of the data analysis results.

The present invention uses a probe PCR amplification to add a specific fluorescent probe at the same time as adding a pair of primers. The probe is an oligonucleotide, and a reporter fluorescent group and a quencher fluorescent group are marked at both ends. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the 5'-3' exonuclease activity of the Taq enzyme will enzymatically degrade the probe, so that the reporter fluorescent group and the quencher fluorescent group are separated, so that the fluorescent monitoring system can receive the fluorescent signal, that is, each time a DNA chain is amplified, a fluorescent molecule is formed, and the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product. A series of optimizations are performed on the reaction system, such as primer concentration, annealing temperature, fragment amplification length, template DNA concentration, reagent additives in the reaction system, etc., so as to accurately and quickly detect the P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753 SNP sites in the human genome. The principles and methods of the technical solution of the present invention are also applicable to the SNP detection of other genes.

The present invention provides 4 sets of specific primers for amplifying specific sequences on human P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753 genes; and uses the Taqman method of real-time fluorescence quantitative PCR to detect 4 SNP sites of human P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753. The present invention is the first to establish a method for simultaneously detecting the distribution of four polymorphisms of P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753 by real-time fluorescence quantitative PCR hydrolysis probe technology (Taqman technology). The detection scheme of the four SNP combinations of the present invention has the characteristics of simple operation, rapidity, high accuracy, high throughput, etc., and is suitable for large-scale P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753 gene SNP distribution detection and its correlation with diseases. At the same time, it determines whether the target to be tested belongs to the group susceptible to medication, and then guides and adjusts the clinical medication plan, provides a basis for clinical personalized treatment, prevents adverse drug reactions, and achieves precise medication.

The present invention uses real-time fluorescence quantitative PCR Taqman method to analyze four polymorphic sites of human P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753. The method is used to detect the distribution of three SNP site combinations of P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; in Chinese healthy population patients with relapse of stroke drug treatment, and determine the close correlation between specific 3SNPs and relapse of stroke drug treatment, thereby demonstrating the clinical applicability and feasibility of the present invention.

The present invention evaluates the distribution of the CYP2C19*2; CYP2C19*3; and CYP2C19*17 3 SNP combination in Chinese healthy population patients with recurrent stroke drug treatment, and further finds that these 3 SNP sites may serve as high-risk factors for drug resistance in patients with stroke treated with clopidogrel/aspirin, and can detect susceptible populations from patients with recurrent stroke drug treatment, thereby guiding and adjusting clinical medication regimens. In addition, as a new clopidogrel/aspirin resistance biomarker combination, it can greatly improve the accuracy of medication and prevent adverse drug reactions. In summary, the combined detection of the CYP2C19*2; CYP2C19*3; and CYP2C19*17 3 SNP sites can not only be used to prevent adverse drug reactions, thereby guiding and adjusting clinical medication regimens, but also provide a basis for clinical personalized treatment and achieve precision medication.

The detection of the combination of 4 SNP sites of P450CYP2C19*2:rs4244285;CYP2C19*3:rs4986893;CYP2C19*17:rs12248560; and PAR1:rs168753 plays an important role and clinical significance in preventing adverse drug reactions, guiding and adjusting clinical medication regimens, and realizing accurate medication. Therefore, the present invention not only provides an experimental basis for clopidogrel/aspirin treatment, but also establishes a high-throughput real-time fluorescence quantitative PCR Taqman method for identification, laying a theoretical and experimental foundation for realizing early accurate medication. Thereby greatly reducing the incidence of adverse drug reactions, it has immeasurable significance not only for individual patients but also for the development and improvement of the economic and medical level of the entire society.

The main advantages of the present invention are:

A simple, accurate, rapid and high-throughput method for screening people sensitive to stroke medications has been established.

The present invention will be further described in detail below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where detailed conditions are not specified are usually carried out according to conventional conditions such as the conditions described in "Molecular Cloning Laboratory Guide" by Sambrook.J et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

Example 1 Establishment of Real-time Fluorescence Quantitative Allele-specific PCR and Its Clinical Evaluation and Application

1. Establishment of real-time fluorescence quantitative allele-specific PCR method

Real-time fluorescence quantitative PCR technology has the characteristics of real-time monitoring, quantification and high throughput, and it is easy to operate and highly sensitive. Fluorescence quantitative PCR is divided into probe quantitative PCR (Taqman method) and fluorescent dye quantitative PCR. Probe PCR (Taqman method) refers to the addition of a specific fluorescent probe when adding a pair of primers during amplification. The probe is an oligonucleotide with a reporter fluorescent group and a quencher fluorescent group labeled at both ends. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the 5'-3' exonuclease activity of the Taq enzyme will enzymatically degrade the probe, separating the reporter fluorescent group and the quencher fluorescent group, so that the fluorescence monitoring system can receive the fluorescent signal, that is, for each DNA chain amplified, a fluorescent molecule is formed, achieving complete synchronization between the accumulation of the fluorescent signal and the formation of the PCR product. The product is judged based on the corresponding fluorescence detected by the reaction. Compared with fluorescent dyes, Taqman probes have sequence specificity and only bind to complementary regions. In addition, the fluorescent signal has a one-to-one correspondence with the number of amplified copies. Therefore, it has strong specificity and high sensitivity, and the conditions are easy to optimize. Compared with hybridization probes, Taqman probes only require the design of one probe, so the probe design is cheaper and more convenient, and it can also meet the basic requirements of quantitative PCR.

The basic principle of Taqman method for detecting SNP is that specific fluorescent probes can only bind to the corresponding completely complementary nucleic acid templates, but the probes cannot bind to the templates if there are non-specific nucleic acid sequences in the nucleic acid templates. Therefore, a pair of double-labeled Taqman probes are used to target different genotypes of biallelic SNPs, and only the completely matched probes can amplify the corresponding genotypes; labeling these two probes with two fluorescent dyes of different wavelengths can complete the genotype determination of a single SNP site in a single PCR reaction.

The present invention adopts the above-mentioned Taqman method based on improved real-time fluorescence quantitative PCR: a double PCR reaction is carried out in one reaction tube to detect quadruple fluorescence. By simultaneously carrying out reactions in two reaction wells, the detection of 6 fluorescence channels of the combination of CYP2C19*2, CYP2C19*3 and CYP2C19*17 three SNP sites can be completed at one time. Clinical experiments were conducted on 1068 aspirin-treated patients and 1081 clopidogrel/aspirin-treated patients to evaluate their possibility as a drug metabolism detection combination. First, CYP2C19*2; CYP2C19*3; CYP2C19*17 were combined: those carrying at least one *2 or *3 variation were called loss-of-function allele carriers. Those carrying at least one *17 variation were called gain-of-function allele carriers. Their distribution is shown in Table 1.

Table 1

In carriers of the CYP2C19 loss-of-function allele, clopidogrel-aspirin combination did not provide a clear benefit compared with aspirin alone. However, in carriers of the normal-function allele, clopidogrel-aspirin combination reduced stroke recurrence by 50% compared with aspirin alone.

The invention is based on the nucleotide sequences between 100 and 200 nucleotides on both sides of the SNP sites in the human genome (GenBank: NCBI Reference Sequence: P450 CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR-1: rs168753), designs corresponding primer pairs to amplify the target DNA fragment; at the same time, designs 8 Taqman probes labeled with different fluorescent groups specific to the nucleic acid sequences for the four SNP sites, and uses the real-time fluorescence quantitative PCR Taqman method to detect the eight SNP sites on the P450 CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753 genes.

The invention synthesizes corresponding primers based on the sequences on both sides of the SNPs (GenBank: NCBI Reference Sequence: rs4244285; rs4986893; rs12248560; and rs168753) of the human P450CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753), designs the SNP site in the middle of the Taqman probe, amplifies the DNA extracted from the human blood sample through the real-time fluorescence quantitative PCR Taqman reaction, and performs SNP analysis of the target gene according to the fluorescence value displayed by the amplification.

2. Experimental methods:

1. Obtain the sample to be tested: Extraction of human genomic DNA from blood: Take 500 μL of human blood, extract it using a blood DNA extraction kit, measure the absorbance at 260 nm to calculate the DNA concentration, and dilute it to a concentration of 10 ng/uL.

2. Based on the nucleotide sequences between 100-200 on both sides of the SNP site in the human genome (GenBank: NCBI Reference Sequence: P450 CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560; and PAR1: rs168753), the corresponding primer pairs were designed to amplify the DNA target fragments; at the same time, 8 Taqman probes labeled with different fluorescent groups specific to the nucleic acid sequences were designed for these 4 SNP sites, and the real-time fluorescence quantitative PCR Taqman method was used to detect the 8 SNP sites on the P450CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR1 genes.

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

CYP2C19*2: rs4244285(SEQ ID NO.17)

AGATATGCAATAATTTTCCCACTATCATTGA TTATTTCCC(G/A)GGAACCCATA ACAAATTACTTAAAAACCTTGCTTTTATGGAAAGTGATATTTTGGAGAAAGTAAAAGAACACCAAGAATCGATGGACATCAACAACCCTCGGGACTTTAT

CYP2C19*3: rs4986893(SEQ ID NO.18)

GATGGAAAAATTGAATGAAAACATCAGGATTGTAAGC ACCCCCTG(A/G)ATCCAGGTAA GGCCAAGTTTTTTGCTTCCTGAGAAACCACTTACAGTCTTTTTTTCTGGGAAATCCAAAATTCTATATTG

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

AATGTGGTATATATTCAGAATAACTAATGTTTGGAAGTTGTTTTGTTTTGCTAAAACAAAGTTTTAGCAAACGATTTTTTTTTTCAAATTTGTGTCTTCTG TTCTCAAAG(C/T)ATCTCT GATGTAAGAGATAATGCGCCACGATGGGCATCAG AAGA

PAR1:rs1687537 (SEQ ID NO.20)

ATAAACAAAAGTAAAATATGCTCTCTGCTTGTCGCTTTTGCCTTGTTGATGCGTTCACTTTTTACATTT AAAATTTTTTT(A/T)ATTTTATTTTTCAG AATCAAAAGCAACAAATGCCACCTTAGATCCCCGGTCATTTCTTCT

Table 2 Real-time fluorescence quantitative PCR primers and Taqman probes

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

Table 3 shows the predicted fluorescence detection results of two wells of real-time fluorescence quantitative PCR Taqman proposed by the present invention (the table is the abbreviation of fluorescence)

Table 3

3. Cloning and sequencing of human CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR1 gene DNA fragments. The sequencing results are consistent with the gene library sequence.

4. Implementation of real-time fluorescence quantitative allele-specific PCR: Real-time fluorescence quantitative PCR Taqman reaction was performed on human genomic DNA (gDNA, 10 ng/μL). The positive control was a plasmid containing human CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR1 genomic DNA fragments cloned and sequenced by the present invention, and the negative control was water. The reaction system and conditions were as follows:

The reaction conditions were 95°C, 5 min; 95°C for 30 sec; 62°C for 60 sec; and 40 cycles.

5. Evaluation of the Real-time Fluorescence Quantitative Allelic PCR Method

5.1. Test on positive control plasmid:

The eight pBluescript II (SK (+) plasmids prepared by the present invention, each containing genomic DNA fragments of four SNP sites, namely, CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR1, were used as positive controls for the PCR reaction. The real-time fluorescence quantitative PCR system was evaluated, such as adjusting the primer concentration, probe concentration, and Mg ion concentration, and the reaction conditions were further optimized, and the feasibility of the method for clinical application was discussed.

Taking CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR1 gene plasmids as templates, the reaction conditions were: 95°C for 10 minutes, 95°C for 15 seconds, 64°C for 60 seconds; 40 cycles. SNP analysis was performed based on the fluorescence values obtained after amplification.

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

5.2. Test on human genomic DNA samples and obtain test results on genomic DNA samples.

6. Optimization of Real-time Fluorescence Quantitative PCR Taqman Method

6.1 Due to the non-specific amplification and the presence of primer dimers in PCR amplification, the present invention uses a chemically modified hot-start DNA polymerase. Due to the non-specific amplification and the presence of primer dimers in PCR amplification, the present invention uses a hot-start Taq DNA polymerase. The biggest advantage of this polymerase is that it is different from ordinary Taq DNA polymerase. Through chemical modification, when the temperature is lower than 50°C, its polymerase activity is completely inhibited, and its polymerase activity is released after 10 minutes at 95°C. Therefore, it greatly improves the specificity of PCR amplification when performing DNA synthesis, while reducing the generation of primer dimers.

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

2. Clinical evaluation and clinical application:

The detection method of the four polymorphic sites of CYP2C19*2, CYP2C19*3, CYP2C19*17 and PAR-1 genes established by the present invention is used to screen the aspirin-treated population and the clopidogrel/aspirin-treated population, and it can be determined whether the target to be tested belongs to the medication-susceptible population, and the clinical medication regimen can be guided and adjusted, providing a basis for clinical personalized treatment and preventing adverse drug reactions.

The distribution of each SNP type in the population is shown in Table 4. It can be seen that the four SNP sites are highly correlated with clopidogrel/aspirin resistance. The detection of this SNP combination can determine whether the target belongs to the drug-susceptible population, guide and adjust the clinical medication plan, provide a basis for clinical personalized treatment, and prevent adverse drug reactions.

Table 4 Screening results of 4SNP combination in aspirin-treated population and clopidogrel/aspirin-treated population

Abbreviation: NE, not detected.

In the table above, the incidence rate is the stroke recurrence rate after medication. In the clopidogrel/aspirin combination group, the stroke recurrence rate of PAR-1 variant carriers (AT, or TT) was significantly lower than that of wild-type patients (among which, the recurrence rate of AT type was reduced by 20.3% compared with AA type, and the recurrence rate of TT type was reduced by 46.2% compared with AA type). Therefore, it is recommended that PAR-1 variant carriers (AT, or TT) add clopidogrel. In patients with CYP2C19*2, CYP2C19*3, and CYP2C19*17 without mutations, the incidence rate of aspirin alone was significantly higher than that of clopidogrel-aspirin combination, so clopidogrel-aspirin combination is recommended.

Example 2: Detection of CYP2C19*2, CYP2C19*3, CYP2C19*17 and PAR-1 genotypes by sequencing

At present, the gold standard for gene polymorphism sites is sequencing. This example uses sequencing to study the distribution of CYP2C19*2, CYP2C19*3, CYP2C19*17 and PAR-1 genes in healthy people. The reliability and accuracy of the method of the present invention are evaluated by comparing the results obtained with real-time fluorescence quantitative PCR Taqman.

Sequencing refers to the use of conventional PCR technology to amplify the target gene fragment of the sample to be tested, and then use the traditional dideoxy method to determine the sequence and compare it with the original sequence to analyze the polymorphic sites. It is currently the most commonly used method for SNP research. This method can be used to determine the differences in species at different levels within and between populations, and can be used for genotyping and biodiversity research.

This example takes the screening distribution of CYP2C19*2, CYP2C19*3, CYP2C19*17 and PAR-1 genes in a healthy population as an example, and uses sequencing to detect the genotype distribution in a healthy population.

The sequencing detection method in this example studies the distribution of CYP2C19*2; CYP2C19*3; CYP2C19*17 and PAR-1 gene SNPs in healthy people. By comparing with other ethnic groups, the differences in the sensitivity of this group of SNP sites in the Chinese population to clopidogrel/aspirin were analyzed. At the same time, the comparison with the above-mentioned Taqman method was carried out to prove the consistency of the detection results of the two methods.

Four sets of corresponding primers were synthesized based on the nucleotide sequences on both sides of the polymorphic sites of human CYP2C19*2; CYP2C19*3; CYP2C19*17 and PAR-1 genes. Each set of primers targets a specific polymorphic site, which is located in the middle of the PCR amplification product, thus facilitating sequencing and analysis.

Preferably, the above PCR reaction procedure includes:

(a) Due to the presence of nonspecific amplification and primer dimers in PCR amplification, the present invention uses pfu DNA polymerase, which has 5'-3' DNA synthase activity and 3'-5' DNA exonuclease activity. Therefore, it can not only synthesize DNA but also instantly identify and remove mismatched nucleotides, greatly improving the specificity of PCR amplification and reducing the generation of primer dimers.

(b) By modifying the 3’ end of the primer with thiophosphorylation, the primer end is prevented from being degraded by the 3’-5’ exonuclease activity of pfu DNA polymerase, thereby further improving the specificity of amplification.

(c) The amplification efficiency and specificity of PCR were further improved by more precise optimization and selection of primer concentration range (0.5-5mM), primer length range (20-30bp), annealing temperature during reaction (60℃-67℃), amplified fragment length (200-400bp), genomic DNA concentration range to be tested (5-15ng), reaction system, etc.

This embodiment utilizes the characteristic that the primer cannot be extended when there is a mismatch at the 3' end of the PCR reaction, designs the allele SNP site at the 3' end of the fluorescent quantitative PCR primer, and appropriately modifies it, and uses pfu DNA polymerase and other means. In addition, a series of conditions such as primer concentration, annealing temperature, fragment amplification length, template DNA concentration, etc. are optimized. Through DNA agarose gel electrophoresis detection, reliable and accurate results are obtained according to the presence or absence of amplification products and the length of BanI enzyme-cut fragments. The present invention can not only accurately detect CYP2C19*2; CYP2C19*3; CYP2C19*17 and PAR-1 genes in the human genome, but is also suitable for other gene mutations and SNP detection.

1. Experimental methods:

1. Obtain the sample to be tested: Extraction of human genomic DNA from blood: Take 500 μL of human blood, extract it using a blood DNA extraction kit, measure the absorbance at 260 nm to calculate the DNA concentration, and dilute it to a concentration of 10 ng/μL.

2. Primer design: Based on the nucleotide sequence between 200-300 bp on both sides of the SNP site in the human genome (GenBank: NCBI Reference Sequence: CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; CYP2C19*17: rs12248560 and PAR1: rs168753), the corresponding primer pairs were designed for DNA target fragment amplification, see Table 5:

Table 5 Design of primers for sequencing analysis

3. Simultaneously, the 3’ end of each primer is modified by thiophosphorylation.

4. Cloning and sequencing of human CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR-1 genomic DNA fragments:

4.1. Primer design: Based on the nucleotide sequences between 100-200 on both sides of the SNP site in the human genome (GenBank: NCBI Reference Sequence: CYP2C19*2: rs4244285; CYP2C19*3: rs4986893; and CYP2C19*17: rs12248560; and PAR-1: rs168753), the corresponding primer pairs were designed to amplify the target DNA fragment;

Primer sequences are shown in Table 5:

The obtained sequences are as follows:

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)

ATAAACAAAAGTAAAATATGCTCTCTGCTTGTCGCTTTTGCCTTGTTGATGCGTTCACTTTTTACATTT AAAATTTTTTT(A/T)ATTTTATTTTTCAG AATCAAAAGCAACAAATGCCACCTTAGATCCCCGGTCATTTCTTCT

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

Add the following reagents to a 200 μL micro PCR reaction tube:

Add deionized sterile water to a final volume of 50 uL.

The PCR reaction conditions were 94°C for 5 min, 94°C for 30 s, 54°C for 1 min, and 72°C for 1 min for 40 cycles, followed by extension at 72°C for 10 min.

4.3. PCR product cloning: The amplified PCR fragment was recovered using a PCR product recovery kit, and then the ends were blunted using T4 DNA polymerase. The target fragment was then recovered and purified using a gel recovery kit by agarose gel electrophoresis, and then inserted into the EcoRV site on the pBluesecript II SK(+) vector. The method is described in the literature (Sambrooks, Molecular Cloning Manual). The ligation product was transformed into the DH5 strain and positive clones were screened by PCR.

4.4. DNA sequencing of positive clones was performed and the sequencing results were consistent with the gene library sequence.

5. Establishment of positive control:

A plasmid containing CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR-1 genomic DNA fragments.

2. Result analysis:

1. Distribution of CYP2C19*2, CYP2C19*3, CYP2C19*17 and PAR-1 in the Chinese Han population

The present invention detects CYP2C19*2; CYP2C19*3; and CYP2C19*17 and PAR-1 SNP combination in Chinese population as shown in Table 6:

Table 6

From the comparison of the distribution percentages of the two methods in the population (see Table 4), the results of the detection method proposed in the present invention and the sequencing detection method are highly consistent. Therefore, the real-time fluorescence quantitative PCR Taqman method detection proposed in the present invention has high reliability and accuracy, and can be used to identify whether the target to be tested belongs to the drug-susceptible population, guide and adjust the clinical drug regimen, and provide a basis for clinical personalized treatment.

Example 3

This embodiment provides a real-time fluorescence quantitative PCR Taqman detection kit for human cytochrome P450 CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR1:rs168753 genomic single nucleotide polymorphism (SNP).

This kit can identify and detect CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR1:rs168753 sites in the human genome, and is suitable for the typing of this combined SNP analysis. The kit adopts the principle of fluorescent probe hydrolysis method (Taqman method), including the primer concentration and probe concentration that are most suitable for real-time fluorescence quantitative PCR reaction, and uses hot-start Taq DNA polymerase and the buffer and primer combination that best matches the multiple real-time fluorescence quantitative reaction, which can effectively inhibit non-specific PCR amplification and achieve the purpose of high-sensitivity and high-throughput real-time fluorescence quantitative PCR amplification reaction. When conducting experiments, the preparation of PCR reaction solution is very convenient and simple. It can accurately analyze CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR1:rs 168753 in the human genome.

Table 7 Composition of the kit

Reagent A: 2×PCR Master with hot start Taq DNA polymerase

Reagent B: CYP2C19*2; CYP2C19*3 two sets of primers and corresponding 4 SNP probes

Reagent D: CYP2C19*17; and PAR1:rs168753 two sets of primers and the corresponding 4 SNP probes

Working standard 1: 4 DNA clones containing human CYP2C19*2; CYP2C19*3 SNP sites

Working standard 2: 4 DNA clones containing human CYP2C19*17; and PAR-1:rs168753 SNP loci

In this example, the kit is used to identify and detect CYP2C19*2, CYP2C19*3, CYP2C19*17, and PAR1:rs168753 SNPs in the human genome. The applicable Real Time PCR amplification instruments include:

Real Time SystemThermal Cycler

Real Time System

System/Smart

II System(Cepheid)Smart

System/Smart

II System (Cepheid)

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

(Roche Diagnostics)

(Roche Diagnostics)

Mx3000P ^TM (Stratagene)

The kit can be transported at 2-8°C and stored at -20°C.

Validity period: This kit is valid for 12 months and should be used within the validity period.

This kit uses hot-start Taq DNA polymerase for PCR amplification, and detects PCR products by monitoring specific fluorescence in the reaction solution. The purpose of PCR amplification of CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR1:rs168753 genes in human genomic DNA is to repeat the three steps of thermal denaturation of DNA chains, primer annealing, and primer extension under the action of DNA polymerase, and the genomic DNA fragments of CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR1:rs168753 in the human genome can be amplified in a short time.

The DNA polymerase in the kit uses hot-start Taq DNA polymerase, which inhibits primer dimers generated during primer annealing and the non-specific amplification caused by them, greatly improving the accuracy of PCR amplification.

This kit uses a fluorescent probe detection method to identify and detect the CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR1:rs168753 SNPs in the human genome. By detecting the fluorescence signal intensity in the PCR reaction solution, the SNP can be accurately detected.

This kit is suitable for Real-time PCR reaction and can quickly and accurately detect and analyze CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR1:rs168753SNP in the human genome.

The reaction solution is pre-mixed with primers. When preparing the PCR reaction solution, you only need to add DNA template and sterile distilled water to carry out Real Time PCR reaction. The operation is simple and convenient.

The DNA polymerase uses a hot-start DNA polymerase, which is combined with the company's independently developed multiplex PCR buffer system, and has the characteristics of high amplification efficiency, high amplification sensitivity, and high amplification specificity.

The use of this kit includes the following steps:

1. Dilute the extracted human genomic DNA to 0.5-15 ng/μL with nucleic acid dilution buffer.

2. Take 2 μL of diluted sample DNA and add it to two PCR reaction wells respectively.

3. Take 8 μL of each of reaction tubes B and C in the kit and add them to two reaction wells respectively.

4. Take 10 μL of reagent from reaction tube A and add it into the reaction wells respectively.

5. Treat the negative control, working standard 1, and working standard 2 in the same manner as above.

6. Mix the reaction solution prepared above and then centrifuge at 5000 rpm for 3 minutes.

7. Place the reaction tube into the real-time fluorescence PCR amplification instrument for amplification reaction.

8. Cycle parameter setting: 95℃10min; 95℃20sec; 62℃60sec; 40 cycles.

The kit was used to detect and compare CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR-1:rs168753 SNPs in healthy Chinese Han people (Table 6), and the test results are as described in the above examples. It can be seen that the kit of the present invention can not only be used for the study of the distribution and disease correlation of CYP2C19*2; CYP2C19*3; CYP2C19*17; and PAR-1:rs168753 SNPs in normal people, but also can be used to identify whether the target to be tested belongs to the drug-susceptible population, guide and adjust the clinical drug regimen, and provide a basis for clinical personalized treatment.

Using the kit in this example, SNP detection was performed on samples of 500 randomly selected patients. Medication guidance was then provided based on the following principles:

(a) When the genotype of PAR-1:rs168753 is detected to be AA, aspirin alone is recommended for treatment;

(b) When a mutation is detected at the PAR-1:rs 168753 site (genotype AT or TT), clopidogrel and aspirin are recommended for combination therapy;

(c) When a single mutation (genotype GA) or double mutation (genotype AA) of CYP2C19*2:rs4244285 is detected, the use of clopidogrel is not recommended;

(d) When a single mutation (genotype GA) or double mutation (genotype AA) of CYP2C19*3:rs4986893 is detected, clopidogrel is not recommended;

(e) When a single mutation (genotype: CT) or double mutation (genotype: TT) of CYP2C19*17:rs12248560 is detected, clopidogrel should be used with caution.

The following table shows some typical patients with detected SNPs.

Table 8

Note: “――” indicates that there is no mutation at the site (i.e., wild type); “-+” indicates that there is a single mutation at the site (heterozygous); “++” indicates that there is a double mutation at the site (homozygous).

For example, for PAR-1:rs16875, "――" represents AA; "-+" represents AT; "++" represents TT. The same applies to CYP2C19*2, CYP2C19*3, and CYP2C19*17.

For patient 3-1 (52 similar patients in total), the conventional aspirin administration did not produce a significant therapeutic effect. Later, the combination of aspirin and clopidogrel was used, and the therapeutic effect was significant.

Aspirin was administered to patient 3-2a (a total of 63 similar patients), but the effect was not significant. After increasing the dosage (twice the normal dosage), the treatment effect improved.

Aspirin was administered to patient 3-2b (a total of 62 similar patients), but the effect was not significant; later, aspirin and clopidogrel were used in combination, and the treatment effect was obvious.

For patient 3-3 (a total of 21 similar patients), clopidogrel is not recommended.

For patients 3-4 (a total of 1 similar patient), clopidogrel is not recommended.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Sequence Listing

<110> Beijing Tiantan Hospital Affiliated to Capital Medical University

<120> A human cytochrome CYP2C19 and PAR1 gene polymorphism site detection kit and its use

<130> P2017-0172

<160> 23

<170> PatentIn version 3.5

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gatggaaaaa ttgaatgaaa acatca 26

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ctgggaaatc caaaattcta tattg 25

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cttttgcctt gttgatgcgt tcac 24

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Claims (10)

1. Use of the PAR-1:rs168753 gene locus and/or its detection reagent, characterized in that it is used to prepare a reagent or a kit, wherein the reagent or the kit is used to detect the sensitivity of the detected object to the combination of clopidogrel-aspirin, and the detected object is a stroke patient. 2. The method of claim 1, wherein the reagent or test kit further comprises a detection reagent for detecting one or more gene loci selected from the group consisting of: CYP2C19*2: rs4244285 gene loci, CYP2C19*3: rs4986893 gene loci, and CYP2C19*17: rs12248560 gene loci. 3. The use according to claim 1, characterized in that if the subject being tested has a mutation in the following gene, it indicates that the subject is sensitive to the antiplatelet drug clopidogrel: PAR-1: rs168753 A→T. 4. The method according to claim 1, wherein if the PAR-1:rs168753 gene locus of the detected object mutates from A to T, it indicates that the object is sensitive to the combination of clopidogrel and aspirin. 5. The method according to claim 1, wherein if the genotype of the PAR-1:rs168753 gene locus of the detected object is TT, it indicates that the object is sensitive to the combination of clopidogrel and aspirin. 6. The use according to claim 2, characterized in that if the subject has the following mutations at one or more gene loci selected from the following group, the subject is not recommended to use clopidogrel: CYP2C19*2: rs4244285 G→A; CYP2C19*3: rs4986893 G→A; CYP2C19*17: rs12248560 C→T. 7. The use according to claim 1, characterized in that the reagent comprises a primer, a probe, a chip, or an antibody. 8. The use according to claim 2, characterized in that 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) Chips for genetic testing; (d) a specific antibody for detecting the amino acid mutation corresponding to the mutated gene; Wherein, the specific primers, the specific probes, and the chip are directed to gene loci selected from the following group: CYP2C19*2: rs4244285 gene loci, CYP2C19*3: rs4986893 gene loci, CYP2C19*17: rs12248560 gene loci, and PAR-1: rs168753 gene loci; The mutated gene contains a mutated gene site selected from the group consisting of: CYP2C19*2: rs4244285 gene locus, CYP2C19*3: rs4986893 gene locus, CYP2C19*17: rs12248560 gene locus and PAR1: rs168753 gene locus. 9. The method according to claim 8, wherein the primer comprises: Primer pair 1, targeting CYP2C19*2: rs4244285 gene locus: The 5'-3' sequence of the upstream primer is: AGATATGCAATAATTTTCCCACTATC (Seq ID No. 1), The 5′-3′ sequence of the downstream primer was: ATAAAGTCCCGAGGGTTGTTGATG (Seq ID No. 2); Primer pair 2, targeting CYP2C19*3: rs4986893 gene locus: The 5'-3' sequence of the upstream primer is: GATGGAAAAATTGAATGAAAACATCA (Seq ID No. 3), The 5′-3′ sequence of the downstream primer was: CTGGGAAATCCAAAATTCTATATTG (Seq ID No. 4); Primer pair 3, targeting CYP2C19*17: rs12248560 gene locus: The 5'-3' sequence of the upstream primer is: TCTTCTGATGCCCATCGTGGCGCATT (Seq ID No. 5), The 5′-3′ sequence of the downstream primer was: TAGTTATTCTGAATATATACCACATT (Seq ID No. 6); Primer pair 4, targeting PAR-1: rs168753 gene locus The 5'-3' sequence of the upstream primer is: CTTTTGCCTTGTTGATGCGTTCAC (Seq ID No. 7), The 5’-3’ sequence of the downstream primer was: CAACAAATGCCACCTTAGATC (Seq ID No. 8). 10. The use according to claim 9, characterized in that the kit further comprises a probe sequence selected from the group consisting of: Probe 1, targeting CYP2C19*2: rs4244285 gene locus: P1: FAM-TATGGGTTCCCCGGGAAATAA-BHQ1 (SEQ ID NO.9) P2: ROX-TATGGGTTCCCTGGGAAATAA-BHQ2 (SEQ ID NO. 10); Probe 2, targeting CYP2C19*3: rs4986893 gene locus: P3: VIC-TTACCTGGATTCAGGGGGT-BHQ1(SEQ ID NO.11) P4: TAMRA-TTACCTGGATCCAGGGGGT-BHQ2 (SEQ ID NO. 12); Probe 3, targeting the CYP2C19 *17 rs12248560 gene locus: P5: FAM-TTCTCAAAGCATCTCTGATGT-BHQ1 (SEQ ID NO.13) P6: ROX- TTCTCAAAGTATCTCTGATGT-BHQ2 (SEQ ID NO. 14); Probe 4, targeting PAR1: rs168753 gene locus: P13: FAM-CTGAAAAATAAAATTAAAAAAATTTT-BHQ1(SEQ ID NO.15) P14: ROX-CTGAAAAATAAAATAAAAAAAATTTT-BHQ2 (SEQ ID NO. 16).