CN112852958A - Kit for detecting anti-platelet effect of ticagrelor - Google Patents

Kit for detecting anti-platelet effect of ticagrelor Download PDF

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CN112852958A
CN112852958A CN202110364677.3A CN202110364677A CN112852958A CN 112852958 A CN112852958 A CN 112852958A CN 202110364677 A CN202110364677 A CN 202110364677A CN 112852958 A CN112852958 A CN 112852958A
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kit
ticagrelor
detecting
detection
genotype
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王珺楠
颜游游
王贺
黎子微
李龙波
齐超
高大松
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Jilin University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Abstract

The invention relates to a kit for detecting an antiplatelet effect of ticagrelor, and belongs to the field of molecular biology detection. Through researching rs773902G & gtA polymorphism in F2RL3 gene and analyzing correlation between each genotype and the antiplatelet efficacy of ticagrelor, a convenient and feasible detection kit is designed, and the kit has the advantages that: the designed specific qPCR detection kit can be used for detecting the SNP site genotype of a single nucleotide polymorphism, the detection result is completely coincident with the sequencing result, the detection is very quick and convenient, and simultaneously, the kit can be used as a feasible means for clinical detection within the economic bearing range of a patient.

Description

Kit for detecting anti-platelet effect of ticagrelor
Technical Field
The invention belongs to the field of molecular biological detection, and particularly relates to a kit for detecting an antiplatelet effect of ticagrelor.
Background
The 2018 report of cardiovascular diseases in China indicates that the number of patients with coronary heart disease in China is about 1100 ten thousand at present. Acute coronary syndrome is a serious type of coronary heart disease and is the main cause of death and disability. With the progress of society and the gradual extension of life expectancy, acute coronary syndrome has become a problem threatening the health of human beings and is a main cause of illness and death in developing countries. The acute coronary syndrome refers to acute ischemic syndrome of heart caused by fresh thrombosis secondary to rupture or erosion of unstable atherosclerotic plaque in coronary artery, and covers ST-elevation myocardial infarction, non-ST-elevation myocardial infarction and unstable angina pectoris. Platelet activation and its aggregation play a dominant role in coronary thrombosis, and antiplatelet therapy is therefore key to the treatment of acute coronary syndromes. When acute coronary syndrome is diagnosed, anti-platelet therapy should be performed as early as possible to reduce the risk of occurrence of cardiovascular events.
However, with the widespread clinical use of antiplatelet drugs, some patients begin to experience drug resistance, insufficient therapeutic effect, high risk of bleeding or thrombosis, and the like during treatment, and such phenomena are referred to as clinical resistance or laboratory resistance. Due to the phenomenon, personalized medicine is also gradually paid attention to, including genetic factors, and various factors, such as environmental factors, genetic factors, patient compliance and the like, need to be considered for antiplatelet personalized treatment of patients with acute coronary syndrome. Therefore, genetic polymorphisms, i.e., genetic factors, are of little importance in antiplatelet personalized therapy. To date, single nucleotide polymorphisms are the most common of all the types of polymorphisms found in the human genome, and are considered to be the primary cause behind genetic variation among the vast majority of individuals.
The research shows that thrombin can activate platelets by cutting and connecting F2R-like thrombin/trypsin receptor 3(F2RL3) to enable the platelets to release agonists such as adenosine diphosphate, ADP and platelet P2Y12 receptor are combined to play an important role in platelet activation and aggregation, and P2Y12 receptor is combined by ADP to activate a series of downstream proteins to cause the change of the structure of glycoprotein on the surface membrane of the platelets and enable the glycoprotein to be combined with adhesive protein, so that platelet aggregation is started. Thus, F2RL3 plays a key role in platelet activation, whereas F2RL3 is encoded by the F2RL3 gene, which is located on human chromosome 19 and on p 12.
Platelets can be inhibited by three drugs, each with a different mechanism of action. Aspirin can target cyclooxygenase, inhibit the formation of thromboxane a2, and induce permanent inhibition of platelet function, however, to ensure effective treatment and prevention of coronary thrombosis, other platelet aggregation pathways must be inhibited. Whereas P2Y12 receptor antagonists are the primary therapeutic agents for acute coronary syndrome. Thienopyridines like clopidogrel or prasugrel can be bioconverted into molecules that irreversibly bind to the P2RY12 receptor, while novel drugs like ticagrelor can reversibly bind to the P2RY12 receptor without biotransformation, thereby antagonizing ADP signaling and platelet activation.
Ticagrelor, formerly known as AZD6140, is a newly developed reversible P2Y12 receptor antagonist with a plasma half-life of 12 hours that can competitively bind to the P2Y12 receptor with ADP to inhibit ADP-induced platelet aggregation and thereby exert antiplatelet effects, whereas its level of inhibition of the P2Y12 receptor is dependent on the plasma level of ticagrelor and to a lesser extent is an active metabolite. Research shows that compared with clopidogrel, ticagrelor not only can reduce the incidence rate of cardiovascular events without increasing the risk of fatal bleeding, but also has the advantages of higher speed, better effect and smaller variability in platelet aggregation inhibition.
Currently, there are few studies on whether the antiplatelet activity of ticagrelor is affected by F2RL3 gene polymorphism, and the results are inconsistent. Therefore, the research on the influence of the genetic polymorphism on the antiplatelet effect of ticagrelor has obvious clinical significance. The research on domestic and foreign documents shows that the F2RL3 gene polymorphism is closely related to the antiplatelet effect of ticagrelor and plays a key role in the antiplatelet process. Whitley et al, an American scholar et al found that SNP rs773902 in F2RL3 gene can change in vitro thrombin-induced platelet reactivity through platelet function assessment and genotyping of healthy people, and found that ticagrelor can eliminate the characteristic of platelet hyperreactivity brought by rs773902A allele. However, this conclusion is limited to in vitro tests and still needs to be confirmed in vivo studies and in populations of other ethnic, sexual, and disease states.
At present, genotypes are mainly obtained by gene sequencing, the accuracy of the gene sequencing is high, but the price is high, the sequencing time is long, so that the large-scale application of the gene sequencing to clinic is still unrealistic at present, extra cost and heavy economic burden can be brought to patients and society, and an economical and practical detection kit is still needed to replace the gene sequencing.
Disclosure of Invention
The invention provides a kit for detecting an antiplatelet effect of ticagrelor, and aims to solve the problems of high price, long sequencing time and unsuitability for clinical use when a genotype is detected by gene sequencing.
The technical scheme adopted by the invention is as follows: comprises the following reagents:
rs773902 tube one:
dyeing agent:
Figure BDA0003006778820000021
Top/Tip Green qPCR SuperMIX 100ul
forward primer 1: 5ul
Reverse primer 1: 5ul
ddH2O：80ul
rs773902 tube two:
dyeing agent:
Figure BDA0003006778820000031
Top/Tip Green qPCR SuperMIX 100ul
forward primer 2: 5ul
Reverse primer 2: 5ul
ddH2O：80ul
Standard 1 tube three:
rs773902AA genotype DNA: 10ul of
Standard 2 tube four:
rs773902AG genotype DNA: 10ul of
Standard 3 tube five:
rs773902AG genotype DNA: 10 ul.
The nucleotide sequence of the forward primer 1 is shown as SEQ ID No. 1:
5'-ACCTCGTCTGCCCTCCACCAT-3'；
the nucleotide sequence of the reverse primer 1 is shown as SEQ ID No. 2:
5'-CCAGGAGCAGCAGGAGGTCAGCATC-3'；
the unique tail sequence at the 3' end of the reverse primer 1 is the last base C;
the nucleotide sequence of the forward primer 2 is shown as SEQ ID No. 3:
5'-TGCTGCTGATGAACCTCGCGA-3'；
the unique tail sequence at the 3' end of the forward primer 2 is the last base A;
the nucleotide sequence of the reverse primer 2 is shown as SEQ ID No. 4:
5'-AGTGCCAGCAGGAGGTCAGCAGT-3'；
the unique tail sequence at the 3' end of the reverse primer 2 is the last base T.
The invention relates to a real-time fluorescence quantification (qPCR) detection method aiming at the antiplatelet effect evaluation and medication guidance of ticagrelor of an acute coronary syndrome patient, which designs a convenient and feasible detection kit by researching rs773902G & gtA polymorphism in F2RL3 gene and analyzing the correlation between each genotype and the antiplatelet effect of ticagrelor, and has the advantages that: the designed specific qPCR detection kit can be used for detecting the genotype of a Single Nucleotide Polymorphism (SNP) site, the detection result is completely coincident with the sequencing result, the detection is very quick and convenient, and simultaneously, the kit can be used as a feasible means for clinical detection within the economic bearing range of a patient.
Drawings
FIG. 1 is a graph of rs773902G > A, dissolution of AA genotype;
FIG. 2 is a graph of rs773902G > A, AG genotype solubilization;
FIG. 3 is a graph of rs773902G > A, GG genotype lysis;
FIG. 4 is a diagram of alleles and primers;
FIG. 5 is a diagram showing the positions of SNPs of interest in a sample DNA;
FIG. 6 is a graph of allele-1 binding to the sample DNA in a correct match and extension;
FIG. 7 is an extension diagram of the reverse primer.
Detailed Description
Comprises the following reagents:
rs773902 tube one:
dyeing agent:
Figure BDA0003006778820000041
Top/Tip Green qPCR SuperMIX 100ul
forward primer 1: 5ul
Reverse primer 1: 5ul
ddH2O：80ul
rs773902 tube two:
dyeing agent:
Figure BDA0003006778820000042
Top/Tip Green qPCR SuperMIX 100ul
forward primer 2: 5ul
Reverse primer 2: 5ul
ddH2O：80ul
Standard 1 tube three:
rs773902AA genotype DNA: 10ul of
Standard 2 tube four:
rs773902AG genotype DNA: 10ul of
Standard 3 tube five:
rs773902AG genotype DNA: 10 ul.
The nucleotide sequence of the forward primer 1 is shown as SEQ ID No. 1:
5'-ACCTCGTCTGCCCTCCACCAT-3'；
the nucleotide sequence of the reverse primer 1 is shown as SEQ ID No. 2:
5'-CCAGGAGCAGCAGGAGGTCAGCATC-3'；
the unique tail sequence at the 3' end of the reverse primer 1 is the last base C;
the nucleotide sequence of the forward primer 2 is shown as SEQ ID No. 3:
5'-TGCTGCTGATGAACCTCGCGA-3'；
the unique tail sequence at the 3' end of the forward primer 2 is the last base A;
the nucleotide sequence of the reverse primer 2 is shown as SEQ ID No. 4:
5'-AGTGCCAGCAGGAGGTCAGCAGT-3'；
the unique tail sequence at the 3' end of the reverse primer 2 is the last base T.
The detection method comprises the following steps:
(1) 2 pairs of allele-specific forward primers and reverse primers with unique tail sequences at the 3' end are designed aiming at rs773902G & gtA in the F2RL3 gene;
(2) respectively adding human DNA samples into the premix (SuperMIX, forward primer, reverse primer, ddH 2O);
(3) loading the DNA chip on a machine, setting amplification conditions, and carrying out DNA product amplification;
(4) and analyzing the genotype according to the melting curve, evaluating the antiplatelet efficacy of ticagrelor and guiding medication.
The invention is further illustrated below by means of experimental examples.
PCR primer sequences:
Figure BDA0003006778820000051
2. the reagent and the DNA template are placed on ice, and the PCR condition reaction system is as follows:
Figure BDA0003006778820000052
Figure BDA0003006778820000061
Figure BDA0003006778820000062
Figure BDA0003006778820000063
Figure BDA0003006778820000064
Figure BDA0003006778820000071
Figure BDA0003006778820000072
Figure BDA0003006778820000073
3. and (3) placing the 96-hole PCR reaction plate into a centrifuge 3000r at room temperature, centrifuging for 2min, and then placing the plate into a PCR instrument for detection.
PCR cycling program the following:
Figure BDA0003006778820000074
5. results read, genotyping:
as can be seen from FIG. 1, the occurrence of a specific lysis curve having a peak value of about 3.2 at Tm82 ℃ and a peak value of about 0.9 at Tm85 ℃ indicates that the genotype of the sample is AA;
as can be seen from FIG. 2, the occurrence of a specific lysis curve having a peak value of about 2.8 at Tm82 ℃ and a peak value of about 1.2 at Tm85 ℃ indicates that the genotype of the sample is AG;
FIG. 3 shows that the genotype of the sample is GG, as shown by a specific melting curve having a peak value of about 2.3 at Tm82 ℃ and a specific melting curve having a peak value of about 1.9 at Tm85 ℃;
the QPCR detection is carried out on the standard products 1, 2 and 3, namely the tubes three, four and five, simultaneously when the gene sequencing is carried out on the sample DNA, because the genotypes corresponding to the standard products 1, 2 and 3 are AA type, AG type and GG type respectively, when the dissolution curve obtained by the sample DNA detection is similar to the dissolution curve shape of the standard product and the peak value is similar, the genotype of the sample DNA is the genotype of the corresponding standard product. Wherein, the allele detected by the forward primer 1 and the reverse primer 1 is a G allele, and if the peak value of the sample DNA dissolution curve is the same as that of the standard 1 dissolution curve, the peak value is the lowest, which indicates that one allele of the sample DNA is an A allele; similarly, the allele detected by the forward primer 2 and the reverse primer 2 is the A allele, and if the peak value of the sample DNA dissolution curve is the same as that of the standard 3 dissolution curve, the peak value is the lowest, the result shows that one allele of the sample DNA is the G allele.
The gene sequencing has high accuracy, but the price is high, the sequencing time is long, the specific qPCR detection kit established by the invention is used for detecting the SNP genotype, and the designed primer is optimized and modified for multiple times, so that the specificity is high, and the occurrence of primer dimer is avoided.
The principle is as follows: in order to distinguish alleles, two pairs of allele-specific forward primers and reverse primers with unique tail sequences at the 3' ends are respectively designed (see figure 4), if the target genotype is homozygote, the peak value of the dissolution curve formed by one pair of primers is the highest, and the peak value of the dissolution curve formed by the other pair of primers is the lowest; if the primer is heterozygote, the two pairs of primers respectively form two melting curves with moderate specific peak values. In order to distinguish alleles, two pairs of primers were designed with different size segments, so that the temperature (Tm) at which the melting curve peaks appeared was different. FIG. 5 shows the target SNP, in the first round of PCR, the allele-1 primer binds and extends with the target SNP G allele in the sample DNA correctly matched (FIG. 6), and the target fragment is amplified by the reverse primer, then the 2 nd round PCR is carried out, the sequence is continuously amplified, the fluorescence signal is continuously enhanced, and finally the reaction end point is reached, and a specific peak can appear in the dissolution curve. If the sample is homozygote, two kinds of high-low specific peaks are generated, and if the sample is heterozygote, the two groups of primers are respectively combined for amplification, and 2 medium-high specific peaks appear at different temperatures (Tm), so that the genotype of the sample is detected.
6. Evaluating the antiplatelet effect of ticagrelor and guiding the medication.
rs773902GG ultrafast metabolism type, has good curative effect, and is recommended to be taken continuously or in reduced dose;
the rs773902AA and rs773902AG are of ultra-slow metabolism type, and the drugs have poor curative effect, and other types of antiplatelet drugs are recommended.
Sequence listing
<110> Jilin university
<120> kit for detecting anti-platelet effect of ticagrelor
<130> JLUWangJn
<141> 2021-04-03
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Synthesis (Artificial Synthesis)
<400> 1
acctcgtctg ccctccacca t 21
<210> 2
<211> 25
<212> DNA
<213> Artificial Synthesis (Artificial Synthesis)
<400> 2
ccaggagcag caggaggtca gcatc 25
<210> 3
<211> 21
<212> DNA
<213> Artificial Synthesis (Artificial Synthesis)
<400> 3
tgctgctgat gaacctcgcg a 21
<210> 4
<211> 23
<212> DNA
<213> Artificial Synthesis (Artificial Synthesis)
<400> 4
agtgccagca ggaggtcagc agt 23

Claims (8)

1. A kit for detecting an antiplatelet effect of ticagrelor, comprising the following reagents:
rs773902 tube one:
dyeing agent: 2 is good
Figure FDA0003006778810000011
Top/Tip Green qPCR SuperMIX 100ul；
Forward primer 1: 5 ul;
reverse primer 1: 5 ul;
ddH2O：80ul；
rs773902 tube two:
dyeing agent: 2 is good
Figure FDA0003006778810000012
Top/Tip Green qPCR SuperMIX 100ul；
Forward primer 2: 5 ul;
reverse primer 2: 5 ul;
ddH2O：80ul
standard 1 tube three:
rs773902AA genotype DNA: 10 ul;
standard 2 tube four:
rs773902AG genotype DNA: 10 ul;
standard 3 tube five:
rs773902AG genotype DNA: 10 ul.
2. The kit for detecting an antiplatelet effect of ticagrelor according to claim 1, wherein: the nucleotide sequence of the forward primer 1 is shown as SEQ ID No. 1.
3. The kit for detecting an antiplatelet effect of ticagrelor according to claim 1, wherein: the nucleotide sequence of the reverse primer 1 is shown as SEQ ID No. 2.
4. The kit for detecting an antiplatelet effect of ticagrelor according to claim 3, wherein: the unique tail sequence at the 3' end of the reverse primer 1 is the last base C.
5. The kit for detecting an antiplatelet effect of ticagrelor according to claim 1, wherein: the nucleotide sequence of the forward primer 2 is shown as SEQ ID No. 3.
6. The kit for detecting an antiplatelet effect of ticagrelor according to claim 5, wherein: the unique tail sequence at the 3' end of the forward primer 2 is the last base A;
7. the kit for detecting an antiplatelet effect of ticagrelor according to claim 1, wherein: the nucleotide sequence of the reverse primer 2 is shown as SEQ ID No. 4.
8. The kit for detecting an antiplatelet effect of ticagrelor according to claim 7, wherein: the unique tail sequence at the 3' end of the reverse primer 2 is the last base T.
CN202110364677.3A 2021-04-04 2021-04-04 Kit for detecting anti-platelet effect of ticagrelor Pending CN112852958A (en)

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Cited By (1)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117004705A (en) * 2023-07-06 2023-11-07 重庆京因生物科技有限责任公司 Kit for rapidly detecting ticagrelor drug genes

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