CN112239782A

CN112239782A - Detection kit for clopidogrel metabolic marker

Info

Publication number: CN112239782A
Application number: CN201910644775.5A
Authority: CN
Inventors: 郑超; 吴祚达; 陈素华
Original assignee: Nky Bochang Wuhan Biotechnology Co ltd
Current assignee: Nky Bochang Wuhan Biotechnology Co ltd
Priority date: 2019-07-17
Filing date: 2019-07-17
Publication date: 2021-01-19

Abstract

The invention discloses a detection kit for a clopidogrel metabolic marker, wherein the clopidogrel metabolic marker is a CYP2C19 gene polymorphic site, the kit comprises two pairs of amplification primers and multiple PCR mixed reaction liquid, the amplification primers are used for amplifying the obesity gene polymorphic site, the amplification primers comprise an rs4244285 site and an rs4986893 site, and each pair of primers respectively corresponds to an upstream region and a downstream region of an SNP site. The detection kit for clopidogrel metabolic marker adopts a multiplex PCR amplification method, simultaneously amplifies two important SNP sites of CYP2C19, can flexibly select amplified site combinations according to actual requirements, can play a role of one kit for multiple purposes, and has the advantages of high primer specificity, low mismatch rate, primer length and T_mThe value design is reasonable, and the amplification product is convenient to detect and separate. Multiple target products are amplified at one time, so that the amplification efficiency is greatly improved, and the amplification cost is reduced. The detection kit can be expanded to other SNP locus detection, and has good popularization value.

Description

Detection kit for clopidogrel metabolic marker

Technical Field

The invention relates to a detection kit for a clopidogrel metabolic marker, and belongs to the field of detection kits.

Background

The incidence of Atherosclerosis (ASO) is a focus of medical attention with aging population, and the incidence of hypertension, hyperlipidemia, and diabetes increasing year by year. In the early disease stage of ASO, when the artery lumen is not obviously narrowed, a patient generally has no obvious symptoms and is easy to ignore by the patient and a clinician, the condition development is realized by the combined action of various risk factors, the fragility of blood vessels caused by the damage of artery tunica media tissues is increased, the ischemic symptoms caused by the blockage of atheromatous plaques on the lumen are gradually shown, the whole body artery is involved in the pathological process, the chronic ischemic symptoms of coronary artery and intracranial artery are easy to find, the arteriosclerosis patients of lower limbs mostly enter ASO late stage when in diagnosis, the stability of plaques of the LEAOD patients is poor generally, the lumen occlusion is serious, the atheroma is easy to break to cause acute arterial thromboembolism, at the moment, the patient needs to receive surgical treatment to rescue the affected limb, or the ischemic necrosis of the limb is difficult to.

Platelet aggregation inhibition is the basis for treatment of LEAOD, and reduction of platelet activity can effectively reduce the incidence of thrombotic events, whether or not surgery is performed. The traditional antiplatelet drug aspirin can not completely meet the treatment requirements of patients after lower limb arterial bypass operation, clopidogrel serving as a strong ADP receptor antagonist becomes another main role of antiplatelet treatment, and after verification, clopidogrel can greatly reduce the occurrence probability of re-embolism after bypass operation and stent implantation, and becomes a clinical common drug for cardiovascular department, vascular surgery, neurology and the like. However, the effect of clopidogrel is affected by many factors, the body's response to standard doses of clopidogrel drugs is low, and thrombotic events still occur after treatment, called clopidogrel hypo-response, i.e. clopidogrel resistance.

At present, related researches on the generation mechanism, influencing factors, examination means and alleviation modes of clopidogrel resistance are more and more, and objective research results are obtained, wherein the influence approved by broad scholars is caused by genetic factors, obesity, diabetes, poor compliance of taking medicines, antagonism of other medicines and the like. In terms of genetic factors, a plurality of drug metabolizing enzyme genes have been found to be associated with the action intensity of clopidogrel, such as cytochrome enzyme P450-2C19 genotype, cytochrome enzyme P450-3A4 and P450-3A5(CYP3A4/5), drug-resistant protein (MDR1), paraoxonase I (PON1, paraoxonase I). Among them, cytochrome enzyme CYP2C19 is the most well studied. The CYP2C19 genotypes reported at present have more than 30 mutant types, most of the mutant types code inactive proteins, only CYP2C19 x 1 type is a wild-type allele, and the mutant types code enzyme proteins with normal activity, which are called loss-of-function alleles (LOF), wherein CYP2C19 x 2(c.681G & gt A; rs4244285) and CYP2C19 x 3(c.636G & gt A; rs4986893) are the most common LOF in Asians. The response of different patients to clopidogrel is obviously different, and the appearance of Clopidogrel Resistance (CR) is closely related to the treatment effect of the patients. A large number of researches prove that the P450 function-loss allele loci CYP2C19 x 2 and CYP2C19 x 3 can weaken the metabolism of the liver on clopidogrel, which explains the phenomenon that some patients still have adverse clinical events after taking the clopidogrel according to the requirements of guidelines. At present, CYP2C19 (./3) allele codes P450-2C19 enzyme with low function, or codes tissues do not have enzyme activity, and the coding tissues carry P450-2C19 enzyme in endoplasmic reticulum of liver cells to have function loss, so that clopidogrel precursor entering liver is less than one of extremely important oxidase, although other few kinds of chromozymes can assist metabolism, the comprehensive transport metabolic capability is obviously reduced, and the conversion efficiency of all substances needing to be metabolized by the P450-2C19 chromozymes is reduced.

Therefore, the important role of CYP2C19 in clopidogrel and the detection requirement of clopidogrel in clinic are aimed at. It is very important to design a detection kit for detecting CYP2C19 to indicate clopidogrel.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to obtain a detection kit for a clopidogrel metabolic marker.

In order to realize the purpose, the technical scheme of the detection kit for the clopidogrel metabolic marker adopted by the invention is as follows:

the kit comprises two pairs of amplification primers and a multiplex PCR mixed reaction solution, wherein the amplification primers are used for amplifying obesity gene polymorphic sites including an rs4244285 site and an rs4986893 site, and each pair of primers respectively corresponds to an upstream region and a downstream region of one SNP site.

Preferably, the sequence of the amplification primer at the rs4244285 locus is shown as a sequence table SEQ ID NO: 1 to 2.

Preferably, the amplification primer sequence of the rs4986893 site is shown in a sequence table SEQ ID NO: 3 to 4.

Preferably, the mixed reaction solution for multiplex PCR comprises DNA polymerase and Mg²⁺And dNTPs. More preferably, the multiplex PCR mixture further comprises a PCR stabilizer and an enhancer.

Preferably, in the amplification system, the addition amount of the amplification primer and the template at each SNP site is the same.

The detection kit generally adopts a treated DNA sample as an amplification template, the sample is generally derived from human whole blood or tissues, and the interference of immunoglobulin G, hemoglobin and lactoferrin in the blood on a PCR reaction system can be reduced by treating the sample.

Preferably, the detection kit further comprises a whole blood sample PCR buffer solution, wherein the buffer solution contains 100mmol/L Tris-HCl, 50mmol/L KCl and has pH of 9.3-9.5. Whole blood sample PCR buffer is a preferred embodiment of the present invention, and other types of whole blood PCR reagents can be used without limitation to the buffer and buffer formulations of the present invention.

Preferably, the kit amplification system is a 50-microliter PCR system, and the system simultaneously contains amplification primers of rs4244285 site and rs4986893 site.

The amplification system is specifically as follows:

2×Platinum Multiplex PCR Master Mix 25μL；

2 mu L of template;

rs4244285, rs4986893 and upstream and downstream primers (10. mu. mol/L) respectively in an amount of 1. mu.L; sterile double distilled water is filled to 50 mu L.

And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 10 min; then denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 90s, and extension at 72 ℃ for 60s, after 40 cycles, extension at 72 ℃ is carried out again for 10min, and the temperature is restored to 4 ℃.

The kit generally adopts a purified DNA sample for amplification detection. However, the kit also comprises a whole blood amplification buffer solution, so that the collected human whole blood sample can be directly amplified.

The invention also aims to provide a detection method of the clopidogrel metabolic marker detection reagent, wherein the detection kit adopts a multiple PCR detection method to simultaneously amplify the rs4244285 site and the rs4986893 site and detect an amplified product after amplification.

The third purpose of the invention is to provide the application of the detection kit for the clopidogrel metabolic marker, and the application of the detection kit in the clinical clopidogrel medication detection.

Compared with the prior art, the detection kit adopting the clopidogrel metabolic marker adopts a multiplex PCR amplification method, simultaneously amplifies two important SNP sites of CYP2C19, can flexibly select amplified site combinations according to actual requirements, can play a role of one kit for multiple purposes, and has the advantages of high primer specificity, low mismatch rate, primer length and T_mThe value design is reasonable, and the amplification product is convenient to detect and separate. Multiple target products are amplified at one time, so that the amplification efficiency is greatly improved, and the amplification cost is reduced. The detection kit can be expanded to other SNP locus detection, and has good popularization value.

Detailed Description

The detection kit for clopidogrel metabolic marker provided by the invention is further detailed and completely described in the following by combining the embodiment. The following examples are illustrative only and are not to be construed as limiting the invention.

The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all commercially available unless otherwise specified.

First, design primer

Primer design is carried out according to polymorphic sites published in NCBI, and rs4244285 site is 'A/C/G' base mutation and is represented by 'V'. The specific sequence is as follows:

CTCCCACTTC TAAAATACTA ATTCAATTTC AGAGGCTGCT TGATAGAAAT CAATATAGCA

GGGACTATCT TTGTAGTATC AATCAGGTTG TGCAAACTCT TTTAACCTAT GCTATCATCT

CCAAAATGTT AATGTAGTAA TTCATACCAT CTTATATTTC AAGATTGTAG AGAAGAATTG

TTGTAAAAAG TAAGAGAATT AATATAAAGA TGCTTTTATA CTATCAAAAG CAGGTATAAG

TCTAGGAAAT GATTATCATC TTTGATTCTC TTGTCAGAAT TTTCTTTCTC AAATCTTGTA

TAATCAGAGA ATTACTACAC ATGTACAATA AAAATTTCCC CATCAAGATA TACAATATAT

TTTATTTATA TTTATAGTTT TAAATTACAA CCAGAGCTTG GCATATTGTA TCTATACCTT

TATTAAATGC TTTTAATTTA ATAAATTATT GTTTTCTCTT AGATATGCAA TAATTTTCCC

ACTATCATTG ATTATTTCCC

V

GGAACCCATA ACAAATTACT TAAAAACCTT GCTTTTATGG AAAGTGATAT TTTGGAGAAA

GTAAAAGAAC ACCAAGAATC GATGGACATC AACAACCCTC GGGACTTTAT TGATTGCTTC

CTGATCAAAA TGGAGAAGGT AAAATGTTAA CAAAAGCTTA GTTATGTGAC TGCTTGCGTA

TTTGTGATTC ATTGACTAGT TTTGTGTTTA CTACGGATGT TTAACAGGTC AAGGAGTAAT

GCTTGAGAAG CATATTTAAG TTTTTATTGT ATGCATGAAT ATCCAGTAAG CATCATAGAA

AATGTAAAAT TAAATTGTTA AATAATTAGA ATACATAGAA GAAATTGTTT AGATAAATAT

AATCTATCTG AACAATAAGG ATGTCAGGAT AGGAAAAGCT CTGTTCTGCA GCTTCCAGTG

AGATCAGCAC AGGAGGAACT TAAATTTAAA AGAAAATAAA AAACATCTCC ATCAAAAAGT

GAGTGAAGGA TATGAACAGA

primer design is carried out according to polymorphic sites published in NCBI, and rs4986893 site is 'A/G' base mutation and is represented by 'R'. The specific sequence is as follows:

TTGTCTACAG ACTTTGCAGA CTGATGTGAT TCCCTCTGAA ACTTGAATTA TTTGGTTTCT

AAAAAAGTCT CTTTTTTTCT TTCCAAAGTA AAAGACAAAT AGGCCGGGAA TGTAAATTTA

GCATTTGAGC AACCATTATT TAACCAGCTA GGCTGTAATT GTTAATTCGA GATTAATGTA

AAAGTGATGT GTTGATTTTA TGCATGCCAA ACTCTTTTTT GCTTTTAAGG GAATTCATAG

GTAAGATATT ACTTAAAATT TCTAAACTAT TATTATCTGT TAACAAATAT GAAGTGTTTT

ATATCTAATG TTTACTCATA TTTTAAAATT GTTTCCAATC ATTTAGCTTC ACCCTGTGAT

CCCACTTTCA TCCTGGGCTG TGCTCCCTGC AATGTGATCT GCTCCATTAT TTTCCAGAAA

CGTTTCGATT ATAAAGATCA GCAATTTCTT AACTTGATGG AAAAATTGAA TGAAAACATC

AGGATTGTAAGCACCCCCTG

R

ATCCAGGTAA GGCCAAGTTT TTTGCTTCCT GAGAAACCAC TTACAGTCTT TTTTTCTGGG

AAATCCAAAA TTCTATATTG ACCAAGCCCT GAAGTACATT TTTGAATACT ACAGTCTTGC

CTAGACAGCC ATGGGGTGAA TATCTGGAAA AGATGGCAAA GTTCTTTATT TTATGCACAG

GAAATGAATA TCCCAATATA GATCAGGCTT CTAAGCCCAT TAGCTCCCTG ATCAGTGTTT

TTTCCACTAA ACTCCAAAGC CCTGTTTCTA TAAAGTACTT TGGTGACAGC CCCAAAGCGT

GCTTATATCA CTCCATGGAC ATCCAGGCAC TTTGGAGTCT TCCATTACTC ACAAGGCTTG

TCCTTCAATT CACACTTTGT CATATTGTGT GACAGAAATA TCCTAATCTA AAAGACATTA

TCTCCTTCAA GGACAGAGAA TATTTGGAAC CACAGAAGCT GCCAAGAAAC ACTGAATAGG

GCAGAGGTGT TTGATGTCTC

specific amplification primers are designed according to the sequences, so that rapid and efficient amplification is facilitated. The primer sequences are as follows:

in this example, a multiplex PCR method was used to simultaneously amplify two SNP sites and then detect them.

II, kit composition

The rapid detection kit in the embodiment aims at two higher SNP sites of CYP2C19The multiple PCR kit for simultaneous amplification includes upstream and downstream amplification primers for two SNP sites, amplification template extracting reagent, and multiple PCR mixed reaction liquid including DNA polymerase and Mg²⁺dNTPs, PCR stabilizers and enhancers.

The DNA template used in the kit of this embodiment is a human blood DNA template extracted after processing, and the collected human whole blood or tissue needs to be processed in advance. If the sample to be amplified is an unprocessed whole blood sample, a buffer solution for PCR of the whole blood sample is adopted, wherein the buffer solution contains 100mmol/L Tris-HCl, 50mmol/L KCl and has the pH value of 9.3-9.5.

Triple, multiplex PCR

The Multiplex PCR selects Platinum Multiplex PCR Master Mix as a reaction system, which comprises DNA polymerase and Mg²⁺dNTPs, PCR stabilizer and enhancer, wherein the Invitrogen Platinum Multiplex PCR Master Mix is selected in the experiment, and the PCR system is 50 mu L, wherein

2×Platinum Multiplex PCR Master Mix 25μL；

2 mu L of template;

Fourth, detection of amplification result

1. Amplification product detection

Because multiple PCR has more influence factors, reaction conditions, primer sites, amplification products and other information need to be comprehensively considered during amplification, and the amplification primer combination in the embodiment is repeatedly verified through multiple experiments, and the multiple PCR can be realized with the minimum mutual influence.

After the upstream and downstream primers in the embodiment are used for combined amplification, amplification products are verified, 2% agarose gel electrophoresis is selected for PCR products, bands 1 and 2 in the electrophoresis result are the amplification product sequence of rs4986893, bands 3 and 4 are the amplification product sequence of rs17817449, and the position of the electrophoresis band indicates that the amplification result is correct.

And recovering the electrophoresis result gel and then sequencing, wherein the sequencing result is consistent with the sequence of the target product.

2. SNP locus genotype frequency of CYP2C19 gene

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Claims

1. A detection kit for a clopidogrel metabolic marker is characterized in that the clopidogrel metabolic marker is a CYP2C19 gene polymorphic site, the kit comprises two pairs of amplification primers and multiple PCR mixed reaction liquid, wherein the amplification primers are used for amplifying the obesity gene polymorphic site, the amplification primers comprise an rs4244285 site and an rs4986893 site, and each pair of primers respectively corresponds to an upstream region and a downstream region of one SNP site.

2. The detection kit for the clopidogrel metabolic marker according to claim 1, wherein the amplification primer sequence of the rs4244285 site is shown as a sequence table SEQ ID NO: 1 to 2.

3. The detection kit for the clopidogrel metabolic marker according to claim 1, wherein the amplification primer sequence of the rs4986893 site is as shown in a sequence table SEQ ID NO: 3 to 4.

4. The detection kit for clopidogrel metabolic marker according to claim 1, wherein the multiple PCR mixed reaction solution comprises DNA polymerase and Mg²⁺And dNTPs.

5. The kit for detecting the clopidogrel metabolic marker according to claim 4, wherein the multiplex PCR mixed reaction solution further comprises a PCR stabilizer and an enhancer.

6. The detection kit for the clopidogrel metabolic marker according to claim 1, characterized in that the detection kit further comprises a whole blood sample PCR buffer solution, wherein the buffer solution contains 100mmol/L Tris-HCl, 50mmol/L KCl and has a pH value of 9.3-9.5.

7. The detection kit for the clopidogrel metabolic marker as claimed in claim 1, wherein the kit amplification system is a 50 μ L PCR system, and the system simultaneously contains amplification primers of rs4244285 site and rs4986893 site.

8. The detection kit for the clopidogrel metabolic marker according to claim 1, which is characterized in that an amplification system of the kit specifically comprises:

2×Platinum Multiplex PCR Master Mix25μL；

2 mu L of template;

9. The detection kit for the clopidogrel metabolic marker according to claim 1, wherein the reaction condition of the kit is pre-denaturation at 95 ℃ for 10 min; then denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 90s, and extension at 72 ℃ for 60s, after 40 cycles, extension at 72 ℃ is carried out again for 10min, and the temperature is restored to 4 ℃.

10. The detection kit for the clopidogrel metabolic marker according to claim 1, wherein the detection kit is applied to the monitoring of clinical clopidogrel medication.