CN106636337B - Clopidogrel drug resistance gene detection method based on multiple HRM analysis - Google Patents
Clopidogrel drug resistance gene detection method based on multiple HRM analysis Download PDFInfo
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Abstract
The invention relates to a clopidogrel drug resistance gene detection method based on multiple HRM analysis, wherein primers of the method are shown as SEQ ID NO. 1-10. The invention also provides a primer combination and a kit related to HRM analysis. Its advantages are: the invention establishes a HRM typing method aiming at a plurality of SNP sites, can detect three sites of rs4244285, rs4986893 and rs12248560 and/or two sites of rs1045642 and rs662 in the same reaction system, reduces the reagent dosage and shortens the detection time. The method provided by the invention is used for detecting the sample of the patient in the percutaneous coronary intervention radiography operation and verifying the sample by a Sanger sequencing method, so that the accuracy is high.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a clopidogrel drug-resistant gene detection method based on multiple HRM analysis.
Background
Clopidogrel is used for preventing thrombotic events after Percutaneous Coronary Intervention (PCI) for the treatment of patients with coronary heart disease. In clinical application, 4-30% of patients cannot achieve expected antiplatelet effect in conventional dose treatment, serious patients can cause cardiovascular adverse events such as myocardial infarction, thrombus, death and the like, and the phenomenon is called Clopidogrel Resistance (CR). Clopidogrel is a prodrug, inactive in vitro, and derived into thiol metabolites through enzymatic reactions to exert clinical effects. Related genes affecting drug transformation mainly include ABCB1 affecting drug absorption and liver drug enzyme metabolizing enzyme genes affecting metabolic processes. CYP2C19 plays a crucial role in the metabolic process of clopidogrel, polymorphism exists in CYP2C19 gene, wherein slow metabolic type mainly comprises CYP2C19 x 2(rs4244285) and CYP2C19 x 3(rs4986893) variation, and enzyme loses catalytic activity and activity is reduced respectively; the CYP2C19 x 17(rs12248560) mutation significantly increased the transcriptional activity of 2C19, a gene phenotype of ultra-rapid metabolism. The PON1 enzyme is a key enzyme in the clopidogrel metabolic process, controls 2-O-clopidogrel to generate active products, and PON1Q192R (rs662) gene polymorphism can influence the aggregation of platelets. The bioavailability of clopidogrel is influenced by the polymorphism of the ABCB1 gene encoding the multi-drug-resistant P-glycoprotein of the small intestine, and ABCB13435C & gtT (rs1045642) is related to the high expression of the P-gp of duodenum and the reduction of the bioavailability of the P-gp substrate. Therefore, it is suggested to perform CYP2C19, PON1 and ABCB1 gene tests before using clopidogrel, and to determine a suitable administration schedule depending on the patient genotype.
High-resolution melting (HRM) analysis is a recent genetic analysis method for mutation scanning and genotyping that has emerged abroad in recent years. The method is an efficient and stable PCR technology, is not limited by mutation base sites and types, does not need a sequence specific probe, and can finish the analysis of the genotype of a sample by directly carrying out high-resolution melting after the PCR is finished.
Chinese patent document CN104450935A discloses a method for detecting related gene polymorphism of clopidogrel medication by HRM technology. Chinese patent document CN105018583A discloses a detection kit for polymorphism of genes related to individual administration of warfarin and clopidogrel and application thereof, which can be used for identifying polymorphic sites of genes related to drug resistance of clopidogrel. Chinese patent document CN105671165A discloses a kit for detecting the efficient typing of gene loci related to clopidogrel drug resistance reaction based on a Sequenom Mass ARRAY typing system. However, the accuracy of clinical medical detection is crucial, and high-throughput detection based on the HRM technology is low in detection accuracy due to the fact that a large number of primers are used in the same reaction system and other reaction components are added, so that the clopidogrel drug-resistant gene detection primer and method capable of detecting multiple sites simultaneously and high in accuracy are necessary.
Disclosure of Invention
The invention aims to provide a primer combination for detecting clopidogrel drug resistance gene by HRM analysis, aiming at the defects in the prior art.
The invention further aims to provide a clopidogrel drug resistance gene detection method based on multiple HRM analysis.
The invention also aims to provide application of the primer combination for detecting the clopidogrel drug-resistant gene.
In order to achieve the purpose, the invention adopts the technical scheme that: a primer combination for detecting clopidogrel drug resistance gene by HRM analysis method is used for detecting three sites of rs4244285, rs4986893 and rs12248560, wherein the upstream primer sequence of the site of rs4244285 is shown as SEQ ID NO. 1, and the downstream primer sequence of the site of rs4244285 is shown as SEQ ID NO. 2; the upstream primer sequence of the rs4986893 site is shown as SEQ ID NO. 3, and the downstream primer sequence of the rs4986893 site is shown as SEQ ID NO. 4; the upstream primer sequence of the rs12248560 site is shown as SEQ ID NO. 5, and the downstream primer sequence of the rs12248560 site is shown as SEQ ID NO. 6.
A primer combination for detecting clopidogrel drug resistance gene by HRM analysis method is used for detecting two sites of rs662 and rs1045642, wherein the sequence of the upstream primer of the site of rs662 is shown as SEQ ID NO. 7, and the sequence of the downstream primer of the site of rs662 is shown as SEQ ID NO. 8; the sequence of the upstream primer at the site rs1045642 is shown as SEQ ID NO. 9, and the sequence of the downstream primer at the site rs1045642 is shown as SEQ ID NO. 10.
A primer combination for detecting clopidogrel drug resistance gene by HRM analysis method is used for detecting rs4244285, rs4986893, rs12248560, rs662 and rs1045642 sites, the upstream primer sequence of the rs4244285 site is shown as SEQ ID NO. 1, and the downstream primer sequence of the rs4244285 site is shown as SEQ ID NO. 2; the upstream primer sequence of the rs4986893 site is shown as SEQ ID NO. 3, and the downstream primer sequence of the rs4986893 site is shown as SEQ ID NO. 4; the upstream primer sequence of the rs12248560 site is shown as SEQ ID NO. 5, and the downstream primer sequence of the rs12248560 site is shown as SEQ ID NO. 6; the sequence of the upstream primer at the rs662 site is shown as SEQ ID NO. 7, and the sequence of the downstream primer at the rs662 site is shown as SEQ ID NO. 8; the sequence of the upstream primer at the site rs1045642 is shown as SEQ ID NO. 9, and the sequence of the downstream primer at the site rs1045642 is shown as SEQ ID NO. 10.
In order to achieve the second object, the invention adopts the technical scheme that: a HRM analysis method for detecting clopidogrel drug resistance gene, which comprises the following steps: and carrying out PCR amplification on the DNA sample, slowly raising the temperature of the reaction system by a program after the PCR reaction is finished, and recording a fluorescence signal in the temperature raising process to obtain a high-resolution melting curve.
Preferably, the PCR amplification reaction is performed in a fluorescent quantitative PCR analyzer, and the conditions for PCR amplification are as follows: pre-denaturation at 95 ℃ for 3min, and then 35 reaction cycles, wherein each cycle comprises denaturation at 95 ℃ for 15s and annealing extension at 30s, the annealing extension temperature of three sites of rs4244285, rs4986893 and rs12248560 is detected to be 60 ℃, and the annealing extension temperature of two sites of rs662 and rs1045642 is detected to be 62 ℃.
Preferably, the reaction system is slowly programmed to heat up in a fluorescent quantitative PCR analyzer, and the fluorescent signal is recorded every time the temperature rises by 0.2 ℃.
In order to achieve the third object, the invention adopts the technical scheme that: the primer combination is applied to preparation of a reagent or a kit for multiple HRM analysis.
The reagent or the kit is used for detecting the clopidogrel drug resistance gene.
A reagent kit for detecting clopidogrel drug resistance genes based on multiple HRM analysis comprises magnesium ions, dNTP, rTaq enzyme, Syto9 dye, primers with base sequences shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, wherein the first six primers and the last four primers are separately arranged.
The invention has the advantages that:
1. the detection method is an HRM typing method aiming at a plurality of SNP sites and is used for analyzing 5 SNP sites rs4244285, rs4986893, rs12248560, rs662 and rs1045642 related to accurate medication of clopidogrel.
2. The detection method can detect three sites of rs4244285, rs4986893 and rs12248560 and/or two sites of rs1045642 and rs662 in the same reaction system, so that the dosage of reagents is reduced, and the detection time is shortened.
3. The detection method provided by the invention is used for detecting samples of 316 patients subjected to percutaneous coronary intervention radiography operation and verifying the samples by a Sanger sequencing method, and the detection accuracy rates of five sites of rs4244285, rs4986893, rs12248560, rs1045642 and rs662 are respectively 100%, 99.37%, 100% and 100%, so that the accuracy rate is high.
Drawings
FIG. 1: high-resolution melting curve negative derivative graph of rs4986893 and rs12248560 fragment multiplex PCR reaction products. The rs4986893 primer in curve 1 does not contain an additional GC sequence, and it can be seen that the melting peak of rs4986893 coincides with the melting peak of rs12248560 and interferes with each other. The rs4986893 primer in curve 2 is added with a GC sequence, and the melting peak (right) and the melting peak (left) of rs12248560 can be well separated.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
Example 1 clopidogrel drug resistance gene detection method of the present invention
Material
Rotor-Gene Q real-time fluorescent quantitative PCR analyzer (qiagen), rTaq enzyme (Takara), Syto9 fluorescent dye (Invitrogen), primers (Shanghai Biopsis), human genomic DNA samples (extracted from patients undergoing percutaneous coronary intervention).
Method of producing a composite material
1. Designing a primer: aiming at five SNP loci related to clopidogrel drug resistance: rs4244285, rs4986893, rs12248560, rs1045642 and rs662, and five pairs of PCR amplification primers are designed, so that each pair of primers generates an amplicon containing a corresponding SNP site. Wherein, in order to increase the Tm value of the rs4986893 amplicon, the melting peak of the rs12248560 amplicon is separated on the HRM curve, and a GC sequence (underlined) is added to the 5' end of the rs4986893 primer. The primer sequences are shown in Table 1.
2. PCR amplification System: the amplification reaction volume was 20. mu.L, including 1.5mM magnesium ion, 200. mu.M dNTP, 0.5U rTaq enzyme, 50pmol of Syto9 dye, and 1. mu.L of genomic DNA sample. The reaction system for detecting three sites of rs4244285, rs4986893 and rs12248560 further comprises: the F, R primer of rs4244285 is 0.35. mu.M each, the F, R primer of rs4986893 is 0.1. mu.M each, and the F, R primer of rs12248560 is 0.35. mu.M each. The reaction system for detecting rs662 and rs1045642 also comprises F, R primers of rs662 of 0.15. mu.M each, and F, R primers of rs1045642 of 0.35. mu.M each.
3. PCR amplification conditions: pre-denaturation at 95 ℃ for 3min, and then 35 reaction cycles, wherein each cycle comprises denaturation at 95 ℃ for 15s and annealing extension at 30s (the temperature of three sites of rs4244285, rs4986893 and rs12248560 is detected as 60 ℃, and the temperature of two sites of rs662 and rs1045642 is detected as 62 ℃), and the amplification reaction is carried out in a fluorescent quantitative PCR analyzer.
4. HRM analysis: after the PCR reaction is finished, slowly raising the temperature of the reaction system in a fluorescent quantitative PCR analyzer in a program way, recording a fluorescent signal once when the temperature rises by 0.2 ℃, and forming a high-resolution melting curve by the change of the fluorescent signal along with the temperature. Melting peaks of different amplicons in the same system are distributed in different temperature intervals due to different Tm values. And analyzing different temperature intervals by using HRM analysis software to obtain the genotype of the corresponding SNP locus. The amplicon melting peak distribution intervals for the different SNPs are shown in table 2.
Results
The research establishes an HRM typing method aiming at a plurality of SNP sites, and is used for analyzing 5 SNP sites rs4244285, rs4986893, rs12248560, rs662 and rs1045642 related to accurate medication of clopidogrel. The addition of GC sequences at the 5' end of the primers allows better distribution of the regions under investigation of different SNP sites over different temperature ranges (FIG. 1).
Table 1: primer sequences for different SNP sites
Table 2: amplicon melting peak distribution intervals of different SNPs
Example 2 accuracy verification of clopidogrel drug resistance gene detection method of the present invention
316 samples with unknown genotypes are detected by using the clopidogrel drug-resistant gene detection method. All samples are from continuous patients who need aspirin and clopidogrel double antiplatelet therapy and are subjected to percutaneous coronary intervention operation in chest distress and chest pain admission at 4-12 months of 2015 in kernel-based hospitals, 77 samples are female, 239 samples are male and the age range is 35-94 years. The detection result is compared with a Sanger sequencing method, the detection accuracy of the five loci rs4244285, rs4986893, rs12248560, rs1045642 and rs662 is respectively 100%, 99.37%, 100% and 100%, and the accuracy is obviously higher than the conventional level.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
Claims (5)
1. A primer combination for detecting clopidogrel drug resistance gene by HRM analysis is characterized in that the primer combination is used for detecting three sites of rs4244285, rs4986893 and rs12248560, the upstream primer sequence of the site of rs4244285 is shown as SEQ ID NO:1,
the sequence of the downstream primer of the rs4244285 locus is shown as SEQ ID NO. 2;
the sequence of the upstream primer of the rs4986893 site is shown as SEQ ID NO. 3,
the sequence of the downstream primer of the rs4986893 site is shown as SEQ ID NO. 4;
the sequence of the upstream primer at the rs12248560 site is shown as SEQ ID NO. 5,
the sequence of the downstream primer of the rs12248560 site is shown as SEQ ID NO. 6.
2. A primer combination for detecting clopidogrel drug resistance gene by HRM analysis is characterized in that the primer combination is used for detecting rs4244285, rs4986893, rs12248560, rs662 and rs1045642 loci,
the sequence of the upstream primer of the rs4244285 site is shown as SEQ ID NO:1,
the sequence of the downstream primer of the rs4244285 locus is shown as SEQ ID NO. 2;
the sequence of the upstream primer of the rs4986893 site is shown as SEQ ID NO. 3,
the sequence of the downstream primer of the rs4986893 site is shown as SEQ ID NO. 4;
the sequence of the upstream primer at the rs12248560 site is shown as SEQ ID NO. 5,
the sequence of the downstream primer of the rs12248560 site is shown as SEQ ID NO. 6;
the sequence of the upstream primer at the rs662 site is shown as SEQ ID NO. 7,
the sequence of the downstream primer of the rs662 site is shown as SEQ ID NO. 8;
the sequence of the upstream primer at site rs1045642 is shown as SEQ ID NO. 9,
the sequence of the downstream primer of rs1045642 site is shown in SEQ ID NO. 10.
3. Use of a primer combination according to claim 1 or 2 in the preparation of a reagent or kit for multiplex HRM analysis for the detection of clopidogrel drug resistance genes.
4. A clopidogrel drug-resistant gene detection kit based on multiple HRM analysis is characterized in that the kit comprises magnesium ions, dNTP, rTaq enzyme, Syto9 dye and primers with base sequences shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
5. A clopidogrel drug-resistant gene detection kit based on multiple HRM analysis is characterized in that the kit comprises magnesium ions, dNTP, rTaq enzyme, Syto9 dye, primers with base sequences shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, and the first six primers and the last four primers are arranged separately.
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| CN109055526A (en) * | 2018-08-17 | 2018-12-21 | 潍坊德诺泰克生物科技有限公司 | For detecting the primed probe group and its application of rs662 |
| CN108949995A (en) * | 2018-08-23 | 2018-12-07 | 山东德诺生物科技有限公司 | For detecting the primed probe group and its application of rs4986893 |
| CN109486933A (en) * | 2018-09-20 | 2019-03-19 | 中国科学院苏州生物医学工程技术研究所 | The detection kit and detection method of clopidogrel medication related gene parting |
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