CN112391457A

CN112391457A - Reagent kit

Info

Publication number: CN112391457A
Application number: CN201910737761.8A
Authority: CN
Inventors: 张辉
Original assignee: Xinkaiyuan Huicheng Wuhan Medical Technology Co ltd
Current assignee: Xinkaiyuan Huicheng Wuhan Medical Technology Co ltd
Priority date: 2019-08-12
Filing date: 2019-08-12
Publication date: 2021-02-23

Abstract

The invention discloses a kit, which comprises two pairs of amplification primers and multiple PCR mixed reaction liquid, wherein the amplification primers are used for amplifying clopidogrel pharmacodynamic gene polymorphic sites, including an rs4244285 site and an rs1045642 site, and each pair of primers respectively corresponds to an upstream region and a downstream region of one SNP site. The invention adopts a multiplex PCR amplification method to simultaneously amplify two important SNP sites of clopidogrel pharmacodynamic genes, can flexibly select amplified site combinations according to actual requirements, can play a role of one box 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 extended to other SNP locus detection and hasGood popularization value.

Description

Reagent kit

Technical Field

The invention relates to a kit, and belongs to the field of clinical detection kits.

Background

Clopidogrel is a novel antiplatelet drug, is an ADP P2Y12 receptor antagonist, and plays an important role in preventing and treating thrombus in patients with coronary heart disease. However, it has been clinically found that some patients cannot effectively prevent the occurrence of arterial thrombotic events even taking conventional doses of clopidogrel for a long time, which is called Clopidogrel Resistance (CR). Although the mechanism of clopidogrel resistance cannot be completely elucidated at present, current researches have found that the occurrence of clopidogrel resistance is a result of the common occurrence of various factors, and genetic factors may play a very important role in the occurrence of clopidogrel resistance.

The ABCB1(ATP-binding cassette subset B member 1) gene plays an important role in the intestinal metabolism of clopidogrel. Clopidogrel is a prodrug, has no antiplatelet effect, and can realize the platelet inhibition effect only by converting the clopidogrel into an active metabolite through cytochrome P450(CYP 450). Studies have shown that CYP2C19 x2 is associated with clopidogrel hyporeactivity. And (4) fitting. The maintenance dose group and the loading dose group were set, blood was taken 7 days after continuous administration, and plasma concentrations of clopidogrel and clopidogrel active metabolite (clopi-H4) and inactive metabolite (CLPM) in plasma were measured using HPLC-MS/MS method. Research results show that CYP2C19 gene status is a determinant factor for clopidogrel antiplatelet effect, the genetic variant of ABCB 1C 3435T plays an important role in clopidogrel absorption, gene polymorphism reduces clopidogrel antiplatelet aggregation curative effect, and gene polymorphism and clopidogrel pharmacodynamic relationship are confirmed, so that gene level guidance can be provided for clinical medication. However, no rapid detection kit for the two related gene polymorphisms and clopidogrel exists in the market.

Disclosure of Invention

In view of the above problems in the prior art, the present invention aims to obtain a kit for rapidly detecting the pharmacodynamic relationship between two related gene polymorphisms and clopidogrel.

In order to realize one of the above purposes, the technical scheme of the kit and the application thereof adopted by the invention is as follows:

the kit comprises two pairs of amplification primers and multiple PCR mixed reaction liquid, wherein the amplification primers are used for amplifying clopidogrel pharmacodynamic gene polymorphic sites, including an rs4244285 site and an rs1045642 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 rs1045642 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 amplification kit of the invention generally adopts a treated DNA sample as an amplification template, the sample is generally derived from human whole blood or tissues, and the treatment of the sample can reduce the interference of immunoglobulin G, hemoglobin and lactoferrin in the blood on a PCR reaction system.

Preferably, the amplification 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.

The invention also provides a clopidogrel pharmacodynamic gene polymorphism detection method, which sequentially comprises the following steps: multiplex PCR, amplification product verification and amplification product detection.

Preferably, the amplification system at least comprises a combination of rs4244285 and rs1045642, wherein the addition amount of the amplification primer and the template of each SNP site is the same.

Preferably, an amplification system for amplifying clopidogrel pharmacodynamic gene rs1045642 and rs4244285 is a 50 mu L PCR system, and the system simultaneously contains amplification primers of rs1045642 and rs 4244285.

The amplification system is specifically as follows:

2×Platinum Multiplex PCR Master Mix 25μL；

2 mu L of template;

1 mu L of each of two pairs of upstream and downstream primers (10 mu mol/L) of rs1045642 and rs 4244285; sterile double distilled water is filled to 50 mu L.

The multiple PCR reaction conditions recommended by the kit are as follows: pre-denaturation at 95 ℃ for 10 min; then the mixture is denatured at 96 ℃ for 30s, annealed at 55 ℃ for 90s, and extended at 72 ℃ for 60s, after 35 cycles, the mixture is extended again at 72 ℃ for 10min and then 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 application of the kit, and the kit is applied to detection of clopidogrel metabolic capacity.

The invention also provides application of another kit, and the kit is applied to detection of clopidogrel resistance risk caused by clopidogrel pharmacodynamics.

Compared with the prior art, the invention adopts a multiplex PCR amplification method to simultaneously amplify two important SNP sites of the clopidogrel pharmacodynamic gene, can flexibly select the amplified site combination according to the actual requirement, can play a role of one box 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 kit provided by the present invention and the use thereof will be described in further detail and in full with reference to the following examples. 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

the rs1045642 site recorded in NCBI is an A/C/T base mutation and is represented by an H, and the specific sequence is as follows:

ATTAGCAACC TTACATCTAC TACTTTAGTT TCTTTTTGCC ATGTAACATA ACACATTCAC AGGATCCAGG GATTAGGACA CAGATGTCTT GTGGGAGAGG GAACATTATT CTGCCTACCA CATGCATACA TCAGAAACCA TGGTTGAAAC ACAGGAAACA TGACAGTTCC TCAAGGCATA CAATTATGAC CTTGTTGGGT TAACCTTCAC TATCCAAATT TTAATCACAC AAACTTTTCC TTAATCTCAC AGTAACTTGG CAGTTTCAGT GTAAGAAATA ATGATGTTAA TTGTGCTACA TTCAAAGTGT GCTGGTCCTG AAGTTGATCT GTGAACTCTT GTTTTCAGCT GCTTGATGGC AAAGAAATAA AGCGACTGAA TGTTCAGTGG CTCCGAGCAC ACCTGGGCAT CGTGTCCCAG GAGCCCATCC TGTTTGACTG CAGCATTGCT GAGAACATTG CCTATGGAGA CAACAGCCGG GTGGTGTCAC AGGAAGAGAT

H

GTGAGGGCAG CAAAGGAGGC CAACATACAT GCCTTCATCG AGTCACTGCC TAATGTAAGT CTCTCTTCAA ATAAACAGCC TGGGAGCATG TGGCAGCCTC TCTGGCCTAT AGTTTGATTT ATAAGGGGCT GGTCTCCCAG AAGTGAAGAG AAATTAGCAA CCAAATCACA CCCTTACCTG TATACAAGCA TCTGGCCACA CTTCCTGTTT GGGTTAGTTG TTACCTTTAC CTGATCACCT GACCCTCCTT GTGAGGAAGG GATGAAAGTG TTCGACCACT TCAGGTTTAG GAGAGAGGAA CATTTCTGGG ATAGGAGAAC TGGAACAATT GTCTTGATCC AAAGCTATAG GCTTGAGGCT CCACCTTTGT CAGCCTTAGG GGTAAGTACA ATATCTGGAA AGCCTTTCAC TTTAAGTCCA AGTACAGAGT CTGGGTCCCC ACCTGCACAT GCTGCTTCTG GCCTGCTGAG GAAGTAGGCA TGACTGTCTC TCCCCATGTC

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, multiplex PCR was used to amplify two SNP sites simultaneously or in any combination for detection.

II, kit composition

The rapid amplification kit in the embodiment is a multiplex PCR kit for simultaneously amplifying two higher SNP sites of a clopidogrel pharmacodynamic gene, and comprises upstream and downstream amplification primers of two pairs of SNP sites, an amplification template extraction reagent and a multiplex PCR mixed reaction solution, wherein the reaction solution comprises 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;

two pairs of upstream and downstream primers (10. mu. mol/L) of rs1045642 and rs4244285 respectively account for 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 the mixture is denatured at 96 ℃ for 30s, annealed at 55 ℃ for 90s, and extended at 72 ℃ for 60s, after 35 cycles, the mixture is extended again at 72 ℃ for 10min and then restored to 4 ℃.

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 combined and amplified, the amplification product is verified, 2% agarose gel electrophoresis is selected for a PCR product, and the position of an 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 clopidogrel pharmacodynamic gene

The two SNP sites are detected, and the statistics of detection data of each genotype are shown in the following table 1:

TABLE 1

By the clinical randomized detection and, Hardy-Weinberg test, the observed values of the individual mutation sites correspond to the theoretical values, are in accordance with the genetic equilibrium and are therefore population-representative. The correlation between clopidogrel and the two gene sites is verified by detecting the drug metabolism of clopidogrel, i.e. inactive carboxylic acid metabolites. However, the conclusion is only limited to the sampling detection, and whether the conclusion is accurate or not needs to be further researched.

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 kit, characterized in that: the kit comprises two pairs of amplification primers and multiple PCR mixed reaction liquid, wherein the amplification primers are used for amplifying clopidogrel pharmacodynamic gene polymorphic sites, including an rs4244285 site and an rs1045642 site, and each pair of primers respectively corresponds to an upstream region and a downstream region of one SNP site.

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

3. The kit of claim 1, wherein: the amplification primer sequence of the rs1045642 site is shown in a sequence table SEQ ID NO: 3 to 4.

4. The kit of claim 1, wherein: the multiple PCR mixed reaction solution comprises DNA polymerase and Mg²⁺And dNTPs.

5. The kit of claim 1, wherein: the amplification kit also comprises a whole blood sample PCR buffer solution.

6. The kit of claim 5, wherein: the buffer solution contains 100mmol/L Tris-HCl, 50mmol/L KCl and has the pH value of 9.3-9.5.

7. A clopidogrel pharmacodynamic gene polymorphism detection method is characterized by sequentially comprising the following steps: multiplex PCR, amplification product verification and amplification product detection.

8. The method for detecting clopidogrel pharmacodynamic gene polymorphism according to claim 7, wherein the amplification system is: 2 XPlatin Multiplex PCR Master Mix25 μ L;

2 mu L of template;

9. The method for detecting clopidogrel pharmacodynamic gene polymorphism of claim 7, wherein the amplification system comprises a combination of rs4244285 and rs1045642, wherein the addition amount of the amplification primer and the template of each SNP site is the same.

10. Use of a kit according to any one of claims 1 to 8, wherein: the kit is applied to detection of clopidogrel resistance risk caused by clopidogrel pharmacodynamics.