CN111961718B - Clopidogrel medication gene detection kit and use method thereof - Google Patents
Clopidogrel medication gene detection kit and use method thereof Download PDFInfo
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Abstract
The invention provides a clopidogrel drug gene detection kit, which comprises a primer pair, a probe and an inhibitor for detecting a CYP2C19 gene, PCR reaction liquid, a positive quality control product and a negative quality control product, wherein the primer pair is used for specifically detecting the polymorphism of the rs4244285 site and the rs4986893 site of the CYP2C19 gene. The invention also provides a use method of the clopidogrel medication gene detection kit. Solves the problems of long detection period, complex operation, high cost, weak specificity and the like of the gene detection kit.
Description
Technical Field
The invention is applied to the technical field of gene detection, and particularly relates to a clopidogrel medication gene detection kit and a using method thereof.
Background
Clopidogrel is a thienopyridine derivative, can be effectively used for antiplatelet therapy, and plays an important role in the secondary prevention of patients at risk of arterial thrombosis. The clopidogrel prodrug is a prodrug, has no antiplatelet activity in vitro, and can play a role only by being metabolized into active metabolites through cytochrome P450 enzymes (CYP450 enzymes) in the liver, wherein CYP2C19 is the main factor, is considered to be an independent factor influencing the clopidogrel reaction difference, and is the current research hotspot. The clopidogrel with the conventional dose can generate less active metabolite, reduce the platelet aggregation resistance and increase the thrombus risk in a slow metabolic patient, namely a patient with poor metabolism; in patients with ultrafast metabolism, the production of active metabolites is increased, the platelet aggregation inhibiting effect is enhanced, and the bleeding risk is increased.
The CYP2C19 has many alleles, but the proportion of the alleles is stable and the expression is higher in 4 in different populations, 1, 2, 3 and 17, and researches show that the incidence rate of the main adverse cardiovascular events of patients carrying CYP2C19 mutant genes (CYP2C19 x 2, CYP2C19 x 3) is obviously increased compared with that of non-carriers, and any mutation of CYP2C19 x 2 and CYP2C19 x 3 can lead the synthesis of coded protein to be terminated early so as to reduce the enzymatic activity, so that the enzymatic activity is lost, and the risk of the adverse cardiovascular events such as thrombus formation in a stent is higher.
At present, there are many technologies for detecting gene polymorphism, and the most commonly used technologies include polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP method), sequence specificity PCR, first-generation sequencing, second-generation sequencing, gene chip method and the like, but the technologies have respective application defects, some technologies are complex in operation, the detection period is long, and high-throughput multi-site simultaneous detection cannot be performed, so that the application of the polymorphism detection technology in clinic at present is still not ideal, and the clinical detection requirements cannot be met all the time.
Disclosure of Invention
The invention provides a clopidogrel drug gene detection kit and a using method thereof, aiming at the problems of high requirement on sample treatment, long detection period, complex operation, high cost, weak specificity and the like in the prior art when CYP2C19 gene polymorphism is detected. The kit has the characteristics of high sensitivity, low cost, high specificity, simple and convenient operation, short detection period and the like, and can be used for rapidly and accurately detecting the gene polymorphism of the clopidogrel personalized medicine.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
a clopidogrel drug gene detection kit comprises a primer pair, a probe and an inhibitor for detecting a CYP2C19 gene, PCR reaction liquid, a positive quality control product and a negative quality control product, wherein the primer pair specifically detects the polymorphism of the rs4244285 site and the rs4986893 site of the CYP2C19 gene.
Further, the primer pair, the probe and the inhibitor for detecting the CYP2C19 gene comprise:
primer pairs, probes and inhibitors for detecting the site to be detected of CYP2C19 x 2(681G > A):
P227P-WF:CCACTATCATTGATTATTTCCCG SEQ IQ NO.1
P227P-MF:CCACTATCATTGATTATTTCCCA SEQ IQ NO.2
P227P-R:TTCTCCATTTTGATCAGGAAGCAAT SEQ IQ NO.3
P227P-P:FAM-CCATAACAAATTACTTAAAAACCTTGCTT-TRAMA SEQ IQ NO.4
P227P-WB:CCACTATCATTGATTATTTCCCAGGAACC-ddC SEQ IQ NO.5
P227P-MB:CCACTATCATTGATTATTTCCCGGGAACC-ddC SEQ IQ NO.6；
and a primer pair, a probe and an inhibitor for detecting the site to be detected of CYP2C19 x 3(636G > A):
W212X-WF:ACATCAGGATTGTAAGCACCCCCTGG SEQ IQ NO.7
W212X-MF:ACATCAGGATTGTAAGCACCCCCTGA SEQ IQ NO.8
W212X-R:TTTCCAGATATTCACCCCATGGCTGTCT SEQ IQ NO.9
W212X-P:VIC-TCTGGGAAATCCAAAATTCTATATTGACC-TRAMA SEQ IQ NO.10
W212X-WB:AACATCAGGATTGTAAGCACCCCCTGAATC-ddC SEQ IQ NO.11
W212X-MB:AACATCAGGATTGTAAGCACCCCCTGGATC-ddC SEQ IQ NO.12。
further, the fluorescent group at the 5 'end of the probe is a FAM, VIC, HEX, cy5 or ROX fluorescent reporter group suitable for fluorescent quantitative PCR analysis, and the quencher group at the 3' end is a TAMRA, BHQ1, BHQ2, MGB or Dabcy1 fluorescent quencher group suitable for fluorescent quantitative PCR analysis.
Further, the fluorescent group at the 5 'end of the probe is FAM or VIC, and the quenching group at the 3' end of the probe is TAMRA.
Further, the 3' end of the inhibitor is modified by C3spacer, phosphorylation, thio, MGB or dideoxycytidine (ddC).
Further, the 3' end of the inhibitor is modified by ddC.
Further, the PCR reaction solution contains a hot-start taq enzyme, a buffer solution, magnesium ions and dNTP substances required for PCR reaction.
Further, the method for obtaining the positive quality control product comprises the following steps: and (3) carrying out plasmid construction according to CYP2C19 x 2 and CYP2C19 x 3 gene sequences published by NCBI database to synthesize a sequence gene fragment, then inserting the fragment into a T vector, transforming by using an Escherichia coli DH5 alpha strain and extracting plasmids, and mixing all quality control product plasmids in equal proportion to obtain the positive quality control product.
Further, the negative quality control material is DEPC treated deionized water.
A use method of a clopidogrel medication gene detection kit comprises the following steps:
1) taking a vacuum blood sample containing an EDTA anticoagulant to perform nucleic acid extraction;
2) premixing and subpackaging hot start taq enzyme, buffer solution, magnesium ions and dNTP substance gene detection reagents to respectively obtain PCR reaction solution 1 and PCR reaction solution 2, and jointly forming a PCR reaction system;
3) adding the DNA obtained in the step 1) and each primer into the PCR reaction system in the step 2), and performing PCR amplification after uniformly mixing;
4) and (4) after the reaction is finished, performing genotyping and interpretation.
Furthermore, the site to be detected is rs4244285 site of CYP2C 19X 2(681G > A) gene and rs4986893 site of CYP2C 19X 3(636G > A) gene.
Further, the concentration of each component in the PCR reaction system in the step 3) is as follows: DNA, 1-200 ng; 2-3 mu M of each primer; hot start taq enzyme, 0.5U; magnesium ion, 5 Xbuffer; dNTP species, 20 mM; the total volume of the reaction was 20. mu.l.
Further, the conditions of the PCR amplification in the step 3) are as follows:
the conditions for pre-denaturation were: at 95 ℃ for 2 minutes;
the first phase consists of 5 amplification cycles with the conditions: denaturation: at 95 ℃ for 15 seconds; annealing: 30 seconds at 60 ℃; extension: 72 ℃ for 20 seconds;
the second phase consists of 40 amplification cycles with the conditions: denaturation: at 95 ℃ for 15 seconds; annealing: setting fluorescence signal collection at 60 ℃ for 30 seconds; extension: 72 ℃ for 30 seconds.
The mode of genotyping interpretation: under the conditions of the PCR reaction system and the circulation program, FAM and VIC fluorescent detection signals of positive quality control products No. 1-2 should form a logarithmic amplification S-shaped curve; no. 1-2 of the negative quality control product has no amplification curve or Ct value of 0; whether the FAM and VIC fluorescence detection signals of No. 1-2 form a logarithmic amplification "S" type curve or not is observed.
Observing an amplification curve of the reaction tube of the sample to be detected under the condition that the conditions are met, and if a logarithmic amplification S-shaped curve is formed, the sample to be detected contains corresponding base mutation; if the S-shaped curve of the logarithmic amplification is absent or the Ct value is 0, the corresponding base mutation is absent.
Compared with the prior art, the invention has the beneficial effects that:
(1) according to the invention, 2 related genes are respectively arranged in 8-linked PCR reaction strips, each reaction strip comprises 4 copies of detection reagent, and the detection is convenient due to the premixed packaging, so that the multi-gene variation analysis of a reaction system is realized, the cost is reduced, and the detection efficiency is improved;
(2) the method has the characteristics of high sensitivity, low cost, high specificity, simple and convenient operation, short detection period and the like, and can be used for quickly and accurately detecting related genes of clopidogrel medication.
Drawings
FIG. 1 is a positive quality control product amplification curve of the clopidogrel drug gene detection kit and the use method thereof.
FIG. 2 is a negative quality control product amplification curve of the clopidogrel drug gene detection kit and the use method thereof.
Detailed Description
The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
In addition, unless otherwise specifically indicated, various starting materials, reagents, instruments and equipment used in the present invention may be commercially available or prepared by existing methods.
The inventor screens 2 polymorphic sites of genes closely related to the metabolism and the drug effect of clopidogrel by analyzing the individual difference of the use, the metabolism and the drug effect of clopidogrel clinically, prepares a composition for detecting the 2 polymorphic sites and a detection kit using the composition, and establishes an accurate detection method for realizing effective medication guidance for clopidogrel.
Example 1: primer and probe combination design and use
1. Primer and probe combinations for identifying CYP2C19 x 2(681> G):
P227P-WF:CCACTATCATTGATTATTTCCCG SEQ IQ NO.1
P227P-R:TTCTCCATTTTGATCAGGAAGCAAT SEQ IQ NO.3
P227P-P:FAM-CCATAACAAATTACTTAAAAACCTTGCTT-TRAMA SEQ IQ NO.4
P227P-WB:CCACTATCATTGATTATTTCCCAGGAACC-ddC SEQ IQ NO.5
primer and probe combinations for identifying CYP2C19 x 2(681> a):
P227P-MF:CCACTATCATTGATTATTTCCCA SEQ IQ NO.2
P227P-R:TTCTCCATTTTGATCAGGAAGCAAT SEQ IQ NO.3
P227P-P:FAM-CCATAACAAATTACTTAAAAACCTTGCTT-TRAMA SEQ IQ NO.4
P227P-MB:CCACTATCATTGATTATTTCCCGGGAACC-ddC SEQ IQ NO.6
the 5 'end fluorescent group of the probe is a conventionally used fluorescent reporter group suitable for fluorescent quantitative PCR analysis, preferably FAM, VIC, HEX, cy5 or ROX, the 3' end quenching group is a conventionally used fluorescent quenching group suitable for fluorescent quantitative PCR analysis, preferably TAMRA, BHQ1, BHQ2, MGB or Dabcy1, and more preferably the 5 'end fluorescent group is FAM and the 3' end quenching group is TAMRA. The 3 'end of the inhibitor is specially modified, preferably C3spacer, phosphorylation, thio, MGB, dideoxycytidine (ddC) and the like, and more preferably the 3' end of the inhibitor for specific template amplification is modified by ddC.
2. Primer and probe combinations for identifying CYP2C19 x 3(636> G):
W212X-WF:ACATCAGGATTGTAAGCACCCCCTGG SEQ IQ NO.7
W212X-R:TTTCCAGATATTCACCCCATGGCTGTCT SEQ IQ NO.9
W212X-P:VIC-TCTGGGAAATCCAAAATTCTATATTGACC-TRAMA SEQ IQ NO.10
W212X-WB:AACATCAGGATTGTAAGCACCCCCTGAATC-ddC SEQ IQ NO.11
primer and probe combinations for identifying CYP2C19 x 3(636> a):
W212X-MF:ACATCAGGATTGTAAGCACCCCCTGA SEQ IQ NO.8
W212X-R:TTTCCAGATATTCACCCCATGGCTGTCT SEQ IQ NO.9
W212X-P:VIC-TCTGGGAAATCCAAAATTCTATATTGACC-TRAMA SEQ IQ NO.10
W212X-MB:AACATCAGGATTGTAAGCACCCCCTGGATC-ddC SEQ IQ NO.12
the fluorescent group at the 5 'end of the probe is a conventionally used fluorescent reporter group suitable for fluorescent quantitative PCR analysis, preferably FAM, VIC, HEX, cy5 or ROX, the quenching group at the 3' end is a conventionally used fluorescent quenching group suitable for fluorescent quantitative PCR, preferably TAMRA, BHQ1, BHQ2, MGB or Dabcy1, and more preferably the fluorescent group at the 5 'end is VIC and the quenching group at the 3' end is TAMRA. The 3 'end of the inhibitor is specially modified, preferably C3spacer, phosphorylation, thio, MGB, dideoxycytidine (ddC) and the like, and more preferably the 3' end of the inhibitor for specific template amplification is modified by ddC.
The primers and the probes can be used for specifically detecting the mutation type of the related gene of clopidogrel medication in a sample, as shown in table 1.
TABLE 1
| Numbering | Gene | RS number | Variation of nucleic acid |
| 1 | CYP2C19*2 | rs4244285 | c.681G>A |
| 2 | CYP2C19*3 | rs4986893 | c.636G>A |
Example 2: acquisition of Positive quality control
The method for obtaining the positive quality control product comprises the following steps: and (3) carrying out plasmid construction according to CYP2C19 x 2 and CYP2C19 x 3 gene sequences published by NCBI database to synthesize a sequence gene fragment, then inserting the fragment into a T vector, transforming by using an Escherichia coli DH5 alpha strain and extracting plasmids, and mixing all quality control product plasmids in equal proportion to obtain the positive quality control product.
Example 3: configuration of PCR reaction system
The kit adopts 8-linked PCR reaction strip design, each reaction strip comprises 4 human detection reagents, and the tubes 1-2 consist of gene detection reagents and respectively indicate different genotypes of CYP2C19 x 2 and CYP2C19 x 3. The reaction solutions of tubes 1-2 correspond to PCR reaction solutions 1-2, respectively, and the compositions of PCR reaction solutions 1-2 are shown in tables 2-3.
TABLE 2 PCR reaction System 1
TABLE 3 PCR reaction System 2
Instrument channel and reaction volume selection:
firstly, FAM (report: FAM, Quencher: TAMRA) and VIC (report: VIC, Quencher: TAMRA) channels are selected to detect amplification conditions;
② the reaction Volume (Sample Volume) is 20. mu.L.
Reference fluorescence (Reference Dye): if ABI series PCR instrument is used, please choose "none" from passive reference; the specific detection channel arrangement can refer to the use instructions of each instrument.
The detection sites in the PCR reaction system are shown in the following table.
Example 4: use of Gene detection kit
1. Sample collection
The embodiment collects peripheral blood of a patient, uses a vacuum blood sampling vessel containing EDTA anticoagulant to collect 5ml of blood sample, stands at room temperature for 30 minutes, centrifuges at 1500-2000 rpm for 10 minutes, and respectively collects cells in blood and plasma in a sterile screw plastic tube.
2. Sample processing
The samples were subjected to nucleic acid extraction using a commercial Kit, and the present invention was carried out using HiPure Blood DNA Mini Kit (cat # D3111) produced by Meiji organisms, and the experimental procedures were carried out with reference to the instructions.
To a 1.5ml centrifuge tube, 25. mu.l of protease K is added and 10-250. mu.l of anticoagulated blood, serum, plasma, milk, saliva, or other liquid sample is transferred to the centrifuge tube containing the protease. Mix by gentle shaking, add 250 μ l Buffer AL to the sample, reverse 3-5 times mix, vortex mix at maximum speed for 30 seconds, water bath at 70 ℃ for 10 minutes, vortex mix once in between. Add 250. mu.l of absolute ethanol to the sample, vortex for 30 seconds and mix, and centrifuge briefly to collect droplets on the tube wall. The DNA column was loaded into a fresh collection tube and the mixture was transferred to the column. Centrifugation at 10000 Xg for 1 min, discarding the collection tube and the effluent. The DNA column was loaded into a fresh collection tube, 500. mu.l Buffer DW1 was added to the column, the mixture was inverted and mixed several times, centrifuged at 10000 Xg for 30-60 seconds, the effluent was decanted, and the column was reloaded into the collection tube. 650. mu.l Buffer DW2 (diluted with ethanol) was added to the column, centrifuged at 10000 Xg for 30-60 seconds, the effluent was decanted, and the column was replaced in the collection tube. Centrifugation at 10000 Xg for 2 minutes allowed complete removal of residual ethanol from the column. The column was transferred to a new 1.5ml centrifuge tube. 30-100. mu.l of Elution Buffer or Buffer TE preheated to 70 ℃ was added to the center of the membrane of the column, left for 3 minutes, and then centrifuged at 10000 Xg for 1 minute. Adding 30-100 μ l of solution Buffer or Buffer TE preheated to 70 deg.C to the center of the membrane of the column, standing for 3 min, centrifuging at 10000 Xg for 1 min, discarding the DNA binding column, storing the DNA at 2-8 deg.C, and storing at-20 deg.C for long term.
3. Preparation of reaction System
Reaction System preparation A reaction system was prepared in accordance with the PCR reaction systems 1 to 2 in example 3.
4. PCR reaction
DNA extracted from No. 1-10 samples is added into a prepared reaction system, and the amount of the added template is 1-200 ng. When PCR reaction is carried out, a sample to be detected, a positive quality control product and a negative quality control product are required to be tested on a parallel machine, and each sample is required to be added with a No. 1-2 tube for reaction.
5. Instrument channel and reaction volume selection
Firstly, FAM (report: FAM, Quencher: TAMRA) and VIC (report: VIC, Quencher: TAMRA) channels are selected to detect amplification conditions;
② the reaction Volume (Sample Volume) is 20. mu.L.
Reference fluorescence (Reference Dye): if ABI series PCR instrument is used, please choose "none" from passive reference; the specific detection channel arrangement can refer to the use instructions of each instrument.
6. PCR reaction procedure
The conditions for pre-denaturation were: at 95 ℃ for 2 minutes;
the first phase consists of 5 amplification cycles with the conditions: denaturation: at 95 ℃ for 15 seconds; annealing: 30 seconds at 60 ℃; extension: 72 ℃ for 20 seconds;
the second phase consists of 40 amplification cycles with the conditions: denaturation: at 95 ℃ for 15 seconds; annealing: setting fluorescence signal collection at 60 ℃ for 30 seconds; extension: 72 ℃ for 30 seconds.
7. Results of the experiment
And (5) storing and interpreting the result after the reaction program is finished.
Referring to the attached figure 1, FAM and VIC fluorescence detection signals in a reaction tube of a positive quality control product No. 1-2 form a logarithmic amplification S-shaped curve;
see FIG. 2, tubes No. 1-2 of negative quality control, no amplification curve.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-described embodiments. It will be understood by those skilled in the art that various changes, substitutions of equivalents, and alterations can be made without departing from the spirit and scope of the invention.
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Claims (6)
1. A clopidogrel drug gene detection kit is characterized in that: the kit comprises a primer pair, a probe and an inhibitor for detecting the CYP2C19 gene, PCR reaction liquid, a positive quality control product and a negative quality control product, wherein the primer pair specifically detects the polymorphism of the rs4244285 site and the rs4986893 site of the CYP2C19 gene;
primer pairs, probes and inhibitors for detecting the CYP2C19 gene include:
primer pairs, probes and inhibitors for detecting the site to be detected of CYP2C19 x 2(681G > A):
P227P-WF:CCACTATCATTGATTATTTCCCG SEQ IQ NO.1
P227P-MF:CCACTATCATTGATTATTTCCCA SEQ IQ NO.2
P227P-R:TTCTCCATTTTGATCAGGAAGCAAT SEQ IQ NO.3
P227P-P:FAM-CCATAACAAATTACTTAAAAACCTTGCTT-TRAMA SEQ IQ NO.4
P227P-WB:CCACTATCATTGATTATTTCCCAGGAACC-ddC SEQ IQ NO.5
P227P-MB:CCACTATCATTGATTATTTCCCGGGAACC-ddC SEQ IQ NO.6；
and a primer pair, a probe and an inhibitor for detecting the site to be detected of CYP2C19 x 3(636G > A):
W212X-WF:ACATCAGGATTGTAAGCACCCCCTGG SEQ IQ NO.7
W212X-MF:ACATCAGGATTGTAAGCACCCCCTGA SEQ IQ NO.8
W212X-R:TTTCCAGATATTCACCCCATGGCTGTCT SEQ IQ NO.9
W212X-P:VIC-TCTGGGAAATCCAAAATTCTATATTGACC-TRAMA SEQ IQ NO.10
W212X-WB:AACATCAGGATTGTAAGCACCCCCTGAATC-ddC SEQ IQ NO.11
W212X-MB:AACATCAGGATTGTAAGCACCCCCTGGATC-ddC SEQ IQ NO.12。
2. the gene detection kit for clopidogrel administration according to claim 1, wherein: the fluorescent group at the 5 'end of the probe is FAM, VIC, HEX, cy5 or ROX fluorescent reporter group suitable for fluorescent quantitative PCR analysis, and the quenching group at the 3' end is TAMRA, BHQ1, BHQ2, MGB or Dabcy1 fluorescent quenching group suitable for fluorescent quantitative PCR analysis.
3. The gene detection kit for clopidogrel administration according to claim 1, wherein: the 3' end of the inhibitor is modified by C3spacer, phosphorylation, thio, MGB or dideoxycytidine (ddC).
4. The gene detection kit for clopidogrel administration according to claim 1, wherein: the PCR reaction solution contains a hot start taq enzyme, a buffer solution, magnesium ions and dNTP substances required by the PCR reaction.
5. The gene detection kit for clopidogrel administration according to claim 1, wherein: the method for obtaining the positive quality control product comprises the following steps: and (3) carrying out plasmid construction according to CYP2C19 x 2 and CYP2C19 x 3 gene sequences published by NCBI database to synthesize a sequence gene fragment, then inserting the fragment into a T vector, transforming by using an Escherichia coli DH5 alpha strain and extracting plasmids, and mixing all quality control product plasmids in equal proportion to obtain the positive quality control product.
6. The gene detection kit for clopidogrel administration according to claim 1, wherein: the negative quality control material is deionized water treated by DEPC.
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| CN202010886407.4A CN111961718B (en) | 2020-08-28 | 2020-08-28 | Clopidogrel medication gene detection kit and use method thereof |
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| WO2019164885A1 (en) * | 2018-02-20 | 2019-08-29 | William Marsh Rice University | Systems and methods for allele enrichment using multiplexed blocker displacement amplification |
| CN110241231A (en) * | 2019-06-26 | 2019-09-17 | 湖南健基生物技术有限公司 | Detect composition, kit, method and the application of CYP2C19 gene pleiomorphism |
| CN111334573A (en) * | 2020-04-28 | 2020-06-26 | 重庆浦洛通基因医学研究院有限公司 | Gene detection kit for hypertension medication and use method |
| CN111534574A (en) * | 2020-06-02 | 2020-08-14 | 北京鑫诺美迪基因检测技术有限公司 | Gene enrichment method for enhancing sequencing sensitivity |
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| WO2019164885A1 (en) * | 2018-02-20 | 2019-08-29 | William Marsh Rice University | Systems and methods for allele enrichment using multiplexed blocker displacement amplification |
| CN110241231A (en) * | 2019-06-26 | 2019-09-17 | 湖南健基生物技术有限公司 | Detect composition, kit, method and the application of CYP2C19 gene pleiomorphism |
| CN111334573A (en) * | 2020-04-28 | 2020-06-26 | 重庆浦洛通基因医学研究院有限公司 | Gene detection kit for hypertension medication and use method |
| CN111534574A (en) * | 2020-06-02 | 2020-08-14 | 北京鑫诺美迪基因检测技术有限公司 | Gene enrichment method for enhancing sequencing sensitivity |
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