CN113604556A - Gene SNP detection kit for determining therapeutic effect correlation of GLP-1 receptor agonist - Google Patents
Gene SNP detection kit for determining therapeutic effect correlation of GLP-1 receptor agonist Download PDFInfo
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Abstract
The invention provides a gene SNP detection kit for determining the curative effect correlation of GLP-1 receptor agonist, which belongs to the technical field of genes and is used for determining the therapeutic effect correlation of GLP-1 receptor agonistGLP1RSequencing Single Nucleotide Polymorphisms (SNPs) of multiple sites of the gene to judge the expected curative effect on GLP-1 receptor agonist (GLP 1 RAs) treatment, thereby guiding the adjustment of the treatment scheme of the type 2 diabetic. Specifically, the SNP loci are rs3765467, rs10305420, rs6923761 and rs761386, and the forward primer and the reverse primer of each locus are designed to carry out PCR amplification, purification, re-amplification and sequencing on the collected sample. The kit provides a theoretical basis for clinical individualized medication and also provides a guide basis for research and development of new drugs based on pharmacogenomics concepts.
Description
Technical Field
The invention belongs to the technical field of genes, and relates to a detection kit for determining Single Nucleotide Polymorphism (SNP) related to curative effect of GLP-1 receptor agonist.
Background
Type 2 diabetes (T2 DM) is an endocrine and metabolic disease characterized by hyperglycemia, and controlling T2DM requires not only lowering blood sugar, but also formulating reasonable comprehensive management objectives of lowering blood fat, lowering blood pressure, losing weight, and the like. Glucagon-like peptide-1 (GLP-1) is an endogenous incretin, and can promote the release of insulin in a glucose concentration-dependent manner to achieve the purpose of reducing blood glucose. In addition, GLP-1 can slow down gastric emptying, control appetite, reduce weight, improve insulin resistance, and indirectly or directly play a role in protecting cardiovascular system, such as reducing blood pressure and improving blood lipid spectrum. However, the natural GLP-1 has short half-life of only 1-2 min, and is easily degraded by dipeptidyl peptidase IV (DPP-IV) in vivo and loses activity. GLP-1 receptor agonists (GLP 1 RAs), also known as GLP-1 analogues, are structurally modified on the basis of natural GLP-1, can exert the effect of GLP-1 and prolong the action time, and are novel therapeutic drugs for treating T2DM, particularly diabetes patients with atherosclerotic cardiovascular diseases, chronic kidney diseases or heart failure. In recent years, intensive research and clinical observation show that the clinical response of T2DM patients to GLP1RAs has obvious individualized difference, most patients show good curative effect and tolerance after taking the medicine, but part of patients have poor blood sugar control and no obvious weight reduction after the treatment of GLP1RAs, and part of patients have intolerable gastrointestinal tract response after taking the medicine. The molecular mechanism generated by the individual difference is searched for, and the accurate prediction is carried out on the molecular mechanism, so that the method has great clinical guiding significance.
GLP1RAs and endogenous GLP-1 have the same receptor (GLP 1R), and it is presumed that the variation of GLP 1R-encoding gene may be one of the important factors affecting the therapeutic effect of the drug. GLP1R is composed of a short arm 6p21.2 located on chromosome 6GLP1RGene coding has been demonstratedGLP1RMononucleotide of multiple sites of genePolymorphisms (SNPs) can affect the affinity and signal transduction processes of GLP1R with GLP1 RAs.GLP1RThe number of mutation sites is more, and the SNP site with the highest mutation frequency in the east Asian population is rs3765467 (G)>A) And rs10305420 (C)>T), minimum gene frequency was 22.7% and 13.7%, respectively. Researches show that rs3765467 wild type (GG type) can obtain better blood sugar-reducing and weight-reducing effects than mutant type (GA/AA type) patients after receiving GLP1RAs treatment. Another study showed that rs10305420 CT and TT genotypes were associated with reduced response to liraglutide in European obese and polycystic ovary syndrome women compared to CC types[1]. And the rs10305420T allele and the rs3765467A allele were confirmed to be associated with a decreased response to exenatide in Chinese T2DM patients[2]. Research has proved that the 2 SNP loci GLP1R rs6923761 and rs761386 also have influence on the curative effect of GLP1 RAs. rs6923761 is shown to be associated with basal GLP-1 levels of greater magnitude of weight and fat mass reduction and higher following liraglutide treatment[3-5]. Yet another study showed that the rs6923761 variation is associated with a decreased response to treatment with a DPP-IV inhibitor[6]. It has also been found that the rs6923761 GA/AA genotype can prolong the half-life of gastric emptying under the treatment with linagliptin[7]. Furthermore, rs761386 was shown to be significantly correlated with changes in blood glucose standard deviation[8]。
According to individual genetic characteristics (genotypes), the individual administration is carried out on the T2DM patients, so that an optimal administration scheme with both curative effect and safety can be conveniently formulated in clinical practice, and meanwhile, the medical cost is saved. In view of this, the invention is particularly proposed.
Reference to the literature
[1] Jensterle, M., et al., Genetic variability in GLP-1 receptor is associated with inter-individual differences in weight lowering potential of liraglutide in obese women with PCOS: a pilot study. European Journal of Clinical Pharmacology, 2015. 71(7): p. 817-824.
[2]Yu, M.J., et al., GLP1R variant is associated with response to exenatide in overweight Chinese Type 2 diabetes patients. Pharmacogenomics, 2019. 20(4):p. 273-278.
[3]de Luis, D.A., et al., Relation of the rs6923761 gene variant in glucagon-like peptide 1 receptor with weight, cardiovascular risk factor, and serum adipokine levels in obese female subjects. JOURNAL OF CLINICAL LABORATORYANALYSIS, 2015. 29(2): p. 100-105.
[4]de Luis, D.A., et al., Role of rs6923761 gene variant in glucagon-like peptide 1 receptor in basal GLP-1 levels, cardiovascular risk factor and serum adipokine levels in naive type 2 diabetic patients. Journal of Endocrinological Investigation, 2015. 38(2): p. 143-147.
[5]de Luis, D.A., et al., Evaluation of weight loss and metabolic changes in diabetic patients treated with liraglutide, effect of RS 6923761 gene variant of glucagon-like peptide 1 receptor. JOURNAL OF DIABETES AND ITS COMPLICATIONS, 2015. 29(4): p. 595-598.
[6]Javorsky, M., et al., A missense variant in GLP1R gene is associated with the glycaemic response to treatment with gliptins. DIABETES OBESITY & METABOLISM, 2016. 18(9): p. 941-944.
[7]Chedid, V., et al., Allelic variant in the glucagon-like peptide 1 receptor gene associated with greater effect of liraglutide and exenatide on gastric emptying: A pilot pharmacogenetics study. NEUROGASTROENTEROLOGY AND MOTILITY, 2018. 30(7): p. e13313.
[8]Lin CH, Lee YS, Huang YY, Hsieh SH, Chen ZS, Tsai CN. Polymorphisms of GLP-1 receptor gene and response to GLP-1 analogue in patients with poorly controlled type 2 diabetes. J Diabetes Res. 2015;2015:176949。
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a gene SNP detection kit for determining the drug curative effect correlation of GLP1 RAs; in particular to the method for correlating the curative effect of GLP1RAs by utilizing the Polymerase Chain Reaction (PCR) technologyGLP1RA method for detecting gene polymorphism sites and a corresponding kit.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, the invention relates to a primer group for detecting gene polymorphism sites related to GLP1RAs drug treatment effect, which comprises a forward primer SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7, and a reverse primer SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6 and SEQ ID No. 8.
The primer is designed and synthesized on an automatic DNA synthesizer, and is more efficient, accurate and high-flux compared with the prior art.
In a second aspect, the invention relates to a kit for SNP detection of GLP1RAs personalized medicine related genes, which comprises the primer set.
As one embodiment of the kit, the kit further comprises a detection reagent, a detection chip and a detection carrier.
The detection reagent comprises Taq DNA polymerase, dNTP mixture, Buffer DE-A, Buffer DE-B, Buffer W1, Buffer W2, Eluent, BigDye (2.5X) and BigDye Seq Buffer (5X).
As an embodiment of the kit, the kit also comprises a resin for purification, a target sheet for spotting and mass spectrometry and a human genome DNA extraction reagent.
In a third aspect, the present invention further relates to a method for the non-diagnostic purpose use of a kit for SNP detection of GLP1RAs personalized medicine related genes, said method comprising the steps of:
(1) carrying out PCR amplification on a sample by using the amplification primers in the primer group to obtain a first amplification product;
(2) purifying the first amplification product;
(3) re-amplifying the purified first amplification product to obtain a second amplification product;
(4) sequencing the second amplification product.
In the step (1) of the present invention, the PCR amplification is carried out in the following system:
in the step (1) of the present invention, the procedure for performing PCR amplification is as follows:
in the step (3) of the present invention, the system for carrying out the re-amplification reaction is as follows:
in the step (3) of the present invention, the procedure for carrying out the re-amplification reaction is as follows:
compared with the prior art, the invention has the following beneficial effects:
(1) to pairGLP1RThe rs3765467, rs10305420, rs6923761 and rs761386 polymorphism detection has the characteristics of low cost, simple result interpretation, simple operation and the like.
(2) The invention aims to research the group of type 2 diabetes in ChinaGLP1RThe relation between the gene polymorphism and the GLP1RAs clinical medication effectiveness lays a foundation, provides a theoretical basis for clinical individualized medication, and provides a guidance basis for research and development of new drugs based on the pharmacogenomics concept.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1: primer design and Synthesis
Gene polymorphism site related to GLP1RAs medicationGLP1Rrs3765467, rs10305420, rs6923761 and rs761386, corresponding specific PCR primer sequences (SEQ ID Nos: 1-8) are designed, specifically as follows:
example 2: sample DNA extraction
The method comprises the steps of collecting peripheral whole blood by using an EDTA (ethylene diamine tetraacetic acid) anticoagulation blood collection tube, and immediately reversing and uniformly mixing the blood for 5-8 times after blood collection so as to ensure that the blood is fully mixed with anticoagulant. Extracting DNA by using a whole blood DNA extraction kit (Beijing Tiangen company); DNA concentration was determined using a semer femorodrop 4000 spectrophotometer. When the sample is measured, three values of concentration, 260/280 and 260/230 need to be recorded, so that the concentration does not meet the requirement of searching possible pollution reasons, and simultaneously, laboratory staff is required to split the DNA sample, so that the times of sample freeze thawing are reduced in the experimental process, and the high quality of the DNA is ensured. And then diluting the extracted sample to 10-20 ng/mu L by using deionized water so as to meet the basic requirement of quality control of a subsequent genotyping sample.
Example 3: gene detection
Using ABI3730XL PCR sequencer, as described in the Specification pairsGLP1RAnd (3) detecting the genetic polymorphisms of rs3765467, rs10305420, rs6923761 and rs 761386.
3.1 PCR product amplification
(1) Amplification system
(2) Conditions for amplification reaction
3.2 PCR product purification
(1) The agarose containing the DNA of interest was cut in an ultra-clean bench under irradiation of an ultraviolet lamp, and then the liquid on the surface of the agarose gel was blotted with a clean paper towel and cut up. The weight of the agarose gel was calculated (1.5 mL EP tube weight was recorded in advance) and the weight of the gel was taken as one gel volume (e.g. 100 mg = 100 μ L);
(2) adding 3 volumes of Buffer DE-A, uniformly mixing the Buffer DE-A and the gel, heating at 75 ℃ (heating low-melting point agarose gel at 40 ℃), and discontinuously mixing uniformly (every 2-3 min); until the gel block is completely melted (about 6-8 min);
(3) adding 0.5 Buffer DE-A volume of Buffer DE-B after the gel block is completely melted, and fully and uniformly mixing;
(4) transferring the mixed solution prepared in the step 3 into a DNA preparation tube, placing the DNA preparation tube into a 2 mL EP tube, placing the tube into a centrifuge for 1 min at 12000r/min, and discarding the filtrate;
(5) placing the preparation tube back into a 2 mL EP tube, adding 500 μ L Buffer W1 into the preparation tube, placing the preparation tube into a centrifuge for centrifugation at 12000r/min for 30 s, and discarding the filtrate;
(6) placing the preparation tube back into a 2 mL EP tube, adding 700 mu L of Buffer W2, centrifuging at 12000r/min for 30 s, and pouring off the filtrate; adding 700 μ L Buffer W2, placing in a centrifuge at 12000r/min, centrifuging for 1 min, and discarding the filtrate;
(7) placing the preparation tube back into the 2 mL EP tube, then placing the preparation tube into a centrifuge for 1 min at 12000r/min, taking out the preparation tube, and discarding the 2 mL EP tube and filtrate;
(8) placing the preparation tube in a clean EP tube with the volume of 1.5 mL, dropwise adding 25-30 mu L of Eluent or deionized water in the center of the prepared membrane, standing for 1 min at room temperature, and then placing the membrane in a centrifuge for 1 min at 12000 r/min;
(9) the preparation tube was discarded to obtain purified DNA.
3.3 post-purification PCR product Re-amplification
(1) Standard reaction System
(2) Sequencing reaction PCR amplification conditions
3.4 sequencing of PCR products after purification
(1) Adding 30 mu L of purified PCR amplification product into a 96-well plate, sequentially adding 2 mu L of EDTA (ethylene diamine tetraacetic acid) and 3mol/L of NaAc and 50 mu L of 100% alcohol into each well of the 96-well plate, covering the 96-well plate, shaking for 4 times, and standing at room temperature for 15 min;
(2) putting the 96-hole plate into a plate centrifuge, centrifuging for 30 min at 4 ℃ at 3000 r/min, immediately inverting the 96-hole plate, centrifuging to 185 r/min, turning off a power supply of the centrifuge, and stopping centrifuging;
(3) adding 70 μ L70% ethanol into 96-well plate, centrifuging at 4 deg.C at 3000 r/min for 15 min, immediately inverting 96-well plate, centrifuging to 185 r/min, turning off power supply of centrifuge, and stopping centrifuging; this step was repeated 1 time;
(4) placing the 96-well plate at room temperature for a period of time, adding 10 mu L of Hi-Di Formamide to dissolve DNA after the alcohol naturally volatilizes completely;
(5) the pretreated sample was placed on a PCR instrument for denaturation: 4 min at 95 ℃ and 4 min at 4 ℃;
(6) placing the processed sample plate in an ABI3730XL sequencer;
(7) opening software '3730 Data Collection v 3.0' in an ABI3730XL sequencer, and selecting a 'Sequencing Analysis' type starting program;
(8) obtaining a sequencing result file after electrophoresis is finished;
(9) the sequencing result file is analyzed and calibrated.
The genotyping results using this kit are given in the following table:
through the analysis, the results show that,GLP1Rthe rs3765467, rs10305420, rs6923761 and rs761386 gene polymorphism has significant difference on the effect of GLP1RAs treatment effect:
in conclusion, the invention is suitable for the type 2 diabetes mellitus people in ChinaGLP1RThe gene polymorphism and clinical guidance of GLP1RAs administration provide theoretical and prognostic foundation, and establish genetic foundation for individualized pharmacogenomics.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by those skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Sequence listing
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<160> 8
<170> SIPOSequenceListing 1.0
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<213> Artificial Sequence (Artificial Sequence)
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gcagggatag ccctcagaat 20
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<213> Artificial Sequence (Artificial Sequence)
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tgttccaagg ccagagaggt 20
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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tgcgcttcct gattccca 18
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<213> Artificial Sequence (Artificial Sequence)
<400> 4
agcggtctcg gagtttcg 18
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gggaaggaag attgtgggaa gagg 24
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ggttggtggt gcttgcgtgg tatt 24
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<213> Artificial Sequence (Artificial Sequence)
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ctgtgggtcc atgggactgg tttt 24
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<213> Artificial Sequence (Artificial Sequence)
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tagaaggaaa ggggcacttg ctga 24
Claims (10)
1. A gene SNP detection kit for determining the therapeutic effect correlation of GLP-1 receptor agonist is characterized by comprising a forward primer and a reverse primer for determining polymorphic site rs3765467, a forward primer and a reverse primer of rs10305420, a forward primer and a reverse primer of rs6923761 and a forward primer and a reverse primer of rs 761386;
wherein the content of the first and second substances,
2. the kit of claim 1, wherein the kit further comprises a detection reagent, a detection chip and a detection carrier.
3. The kit of claim 2, wherein the detection reagents comprise Taq DNA polymerase, dNTP mix, Buffer DE-A, Buffer DE-B, Buffer W1, Buffer W2, Eluent, BigDye (2.5X) and BigDye Seq Buffer (5X).
4. The kit of claim 1 or 2, wherein the kit further comprises a resin for purification, a target for spotting and mass spectrometric detection, and a human genomic DNA extraction reagent.
5. The kit according to claim 1, wherein the method of using the kit specifically comprises the steps of:
(1) carrying out PCR amplification on a sample by using the amplification primers in the primer group to obtain a first amplification product;
(2) purifying the first amplification product;
(3) re-amplifying the purified first amplification product to obtain a second amplification product;
(4) sequencing the second amplification product.
6. The kit according to claim 5, wherein in the step (1), the PCR amplification system comprises:
7. the kit according to claim 5, wherein in the step (1), the PCR amplification procedure is as follows:
8. the kit according to claim 5, wherein in the step (3), the system for performing the re-amplification reaction is as follows:
9. the kit according to claim 5, wherein in step (3), the procedure for performing the re-amplification reaction is as follows:
10. use of a kit according to claim 1, for sequencing a gene encoding GLP1R for use in a non-disease diagnostic method.
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| CN114317710A (en) * | 2021-12-24 | 2022-04-12 | 廊坊诺道中科医学检验实验室有限公司 | Primer probe combination for detecting SNP typing of polymorphic site of GLP1R gene, kit and application thereof |
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