Primer group, kit and method for detecting hypertension individualized medication gene locus polymorphism

Abstract

The invention discloses a primer group and a kit for detecting genetic locus polymorphism of individualized medication of hypertension as well as a method for detecting the genetic locus polymorphism of the individualized medication of hypertension and application of the method. The primer group comprises a multiple PCR amplification primer and a UEP primer used for a single base extension reaction, multiple PCR amplification is firstly carried out, SAP purification is then carried out, the extension reaction is then carried out, nucleic acid mass spectrometry detection is carried out on an extension product, and related genotype information is obtained. The method has the advantages of high accuracy, high flux, low cost and short period, 96 samples or 384 samples can be detected in one batch, only 8-10 hours are needed, the time is short, and the price is low. The kit can detect 15 hypertension individualized medication gene loci, relates to 23 drugs, is high in sensitivity, can obtain a detection result only through a trace amount of genome DNA, and the detection result can guide clinical selection of proper drugs to achieve the purpose of precise medication.

Description

Technical Field

[0001] The present invention relates to a primer set, a kit and a method for detecting gene site polymorphism of hypertension individualized medication. Background Art

[0002] Hypertension is the most common chronic non-infectious disease. It can cause complications in the heart, brain, kidneys and other organs, and is the most important risk factor for cardiovascular disease. According to the 2018 China Cardiovascular Disease Report, the number of deaths from cardiovascular disease in my country accounts for more than 40% of the total deaths of residents, ranking first, higher than tumors. Studies have shown that hypertension is an important risk factor for many cardiovascular diseases. For example, 79.8% of strokes are related to hypertension. Controlling blood pressure at normal levels can reduce the occurrence of stroke by 40-50%. Therefore, controlling hypertension can effectively reduce the mortality rate of cardiovascular diseases.

[0003] The pathogenesis of hypertension is complex. In addition to family genetic factors, obesity, high-salt diet, lack of exercise, smoking, alcoholism, and mental stress are all related to it. In addition, there are many types of hypertension medications, and the drug metabolism genes of the population are also polymorphic. The sensitivity and metabolic rate of the same drug are different. Individual differences among patients will result in different drug metabolism capabilities, and the drug responses of different patients to the same drug will also vary greatly. Therefore, the current hypertension control rate is so low. In addition to reasons such as poor patient compliance with medication, the failure to personalize medication based on genetic polymorphism is also an important influencing factor.

[0004] There are many drugs available for hypertension, and the metabolism and transport processes of each drug are regulated by multiple different genes. Therefore, the detection of genes for personalized hypertension medication involves many sites, and common detection methods such as fluorescent PCR, high-throughput sequencing and other technologies all have their own shortcomings. Among them, fluorescent PCR has a small number of sites for a single detection, and although high-throughput sequencing technology has a high throughput, the cost of single sample detection is also high. With the continuous development of molecular detection technology, personalized medication testing projects that detect multiple sites / multiple drugs in one batch have become the mainstream detection trend. Therefore, it is necessary to develop a high-throughput, low-cost, and highly accurate detection solution for personalized medication gene site polymorphism. Summary of the invention

[0005] The purpose of the present invention is to provide a primer set and a kit for detecting polymorphisms of gene loci for personalized medication of hypertension. The gene detection kit for personalized medication of hypertension of the present invention uses nucleic acid mass spectrometry technology to detect multiple SNP sites related to drug sensitivity and metabolic capacity at one time, thereby guiding patients to use personalized medication and improving the control rate of hypertension.

[0006] The technical solution adopted by the present invention is:

[0007] A primer set for detecting polymorphism of gene loci for personalized hypertension medication, comprising multiple PCR amplification primers, the sequences of which are shown in SEQ NO. 1 to SEQ NO. 30, and used for amplifying 15 gene loci related to hypertension medication:

[0008]

[0009] The primer set also includes a UEP primer for single base extension reaction, the sequences of which are shown in SEQ NO31 to SEQ NO45:

[0010]

[0011] Furthermore, the present invention also provides a kit for detecting the polymorphism of gene loci for personalized medication of hypertension, and the kit comprises the above-mentioned multiple PCR amplification primers and UEP primers.

[0012] Furthermore, the kit includes a multiplex PCR amplification reaction system, a shrimp alkaline phosphatase (SAP) purification reaction system, and a single base extension reaction system.

[0013] The multiplex PCR amplification reaction solution comprises multiplex PCR amplification primers, PCR buffer, deoxynucleoside triphosphate mixture (dNTP), DNA polymerase, and MgCl2.

[0014] The SAP purification reaction system comprises SAP enzyme and SAP buffer.

[0015] SAP enzyme and SAP buffer can be purchased from Agena.

[0016] The single base extension reaction system includes UEP primer, iPLEX Buffer Plus, iPLEX Terminationmix and iPLEX Pro Enzyme.

[0017] The iPLEX Buffer Plus, iPLEX Termination mix and iPLEX Pro Enzyme were purchased from Agena.

[0018] The present invention also provides the use of the kit in detecting gene site polymorphism for individualized medication of hypertension.

[0019] The hypertension drugs include 23 kinds: atenolol, lisinopril, candesartan, furosemide, amiloride, enalapril, verapamil, benazepril, nitrendipine, spironolactone, telmisartan, losartan, bisoprolol, perindopril, amlodipine, bumetanide, metoprolol, folic acid, captopril, irbesartan, nifedipine, hydrochlorothiazide, and carvedilol.

[0020] Furthermore, the genes include 14 types: ADRB2, CACNA1C, ACY3, ACE, CYP2C9, AGTR1, CYP3A5, ADD1, NPPA-AS1, CYP4A11, GNB3, CYP2D6, ADRB1, and MTHFR.

[0021] The 15 gene loci include rs1042713, rs5063, rs1051375, rs1126742, rs2514036, rs5443, rs1799752, rs1065852, rs1057910, rs766507177, rs5186, rs1801253, rs776746, rs1801133, and rs4961.

[0022] The present invention also provides a method for detecting the polymorphism of the gene locus for individualized medication of hypertension using the kit, the method comprising the following steps:

[0023] (1) Multiplex PCR amplification reaction:

[0024] Extracting template DNA from samples, preparing a multiplex PCR amplification reaction system, performing multiplex PCR amplification reactions, and obtaining multiplex PCR amplification products;

[0025] The multiplex PCR amplification reaction system is a 5 μL reaction system, including the following components:

[0026] Ultrapure water 0.8μL, 10xPCR Buffer 0.5μL, 25mM MgCl2 0.4μL, dNTP 0.1μL, P Mix 1μL, PCR Enzyme 0.2μL, DNA template 2μL;

[0027] The P mix is ​​a primer sequence of SEQ NO1 to SEQ NO30, and the concentration of each primer is 0.5 μM;

[0028] The reaction conditions for the multiplex PCR amplification reaction were 95°C for 2 min; 95°C for 30 s, 56°C for 30 s, and 72°C for 60 s, for 45 cycles; and then 72°C for 5 min.

[0029] (2) Shrimp alkaline phosphatase (SAP) purification reaction:

[0030] The multiplex PCR amplification products were purified with SAP enzyme. The SAP purification reaction system included the following components: 1.53 μL ultrapure water, 0.17 μL SAP Buffer, 0.3 μL SAP Enzyme, 5 μL amplification product, a total of 7 μL reaction system; after the reaction, the purified product was obtained;

[0031] The reaction conditions for the purification reaction were 37 degrees for 40 minutes and 80 degrees for 5 minutes.

[0032] (3) Single base extension reaction

[0033] The purified product of step (2) is subjected to a single base extension reaction, wherein the single base extension reaction system comprises the following components: 0.62 μL of ultrapure water, 0.2 μL of iPLEX Buffer Plus, 0.2 μL of iPLEX Termination mix, 0.94 μL of UEP Mix, 0.04 μL of iPLEX Pro Enzyme, and 7 μL of purified product, for a total of 9 μL of reaction system; the reaction is performed to obtain an extension product;

[0034] UEP Mix contains primer sequences from SEQ NO31 to SEQ NO45, and the concentration of each primer is 0.7 μM;

[0035] The reaction procedure of the single base extension reaction is 94° C. for 30 s, {94° C. for 5 s, (41° C. for 5 s, 80° C. for 5 s) 5 internal cycles} 39 external cycles, 72° C. for 3 min.

[0036] (4) Nucleic acid mass spectrometry analysis

[0037] 35 μL of deionized water was added to the extension product, which was then detected using a nucleic acid mass spectrometer to obtain the gene site polymorphism for personalized hypertension medication.

[0038] The nucleic acid mass spectrometer is generally a MALDI-TOF MS matrix-assisted laser desorption ionization-time of flight mass spectrometer.

[0039] The nucleic acid mass spectrometer detector converts electrical signals into visual peak graphs. Each genotype has a unique molecular weight, and the software automatically analyzes the corresponding base type to obtain the final genotyping results.

[0040] The present invention firstly performs PCR amplification with self-designed primers, then SAP purification, and finally uses self-designed probes to perform extension reaction of target bases. The obtained extension products are subjected to nucleic acid mass spectrometry detection, and finally relevant genotype information is obtained.

[0041] The hypertension personalized medication gene site polymorphism detection kit of the present invention has the advantages of high accuracy, high throughput, low cost and short cycle. Compared with conventional Sanger sequencing, pyrophosphate sequencing and real-time fluorescence PCR technology, it has significant high throughput advantages, and can detect 96 samples (96-well plate) or 384 samples (384-well plate) in one batch, and each sample can measure 14 genes and 15 target sites; compared with the second-generation sequencing NGS, the present invention only needs 8-10 hours to complete a batch of samples, which is short in time, improves the detection efficiency, and also has a more affordable price. This kit can detect 15 hypertension personalized medication-related gene sites, involving 23 drugs, with high sensitivity, and only a trace amount of genomic DNA is needed to obtain the test results. The test results can guide the clinic to select appropriate drugs and use appropriate dosages, so that patients can obtain the best therapeutic effect and reduce adverse drug reactions, truly achieving the purpose of precision medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the mass spectrometry result of the rs5063 site of sample 257, and the result has been boxed.

[0043] Figure 2 This is the Sanger sequencing result of the rs5063 site of sample 257, and the result is boxed.

[0044] Figure 3 This is the mass spectrometry result of the rs1042713 site of sample 257, and the result has been boxed.

[0045] Figure 4 This is the Sanger sequencing result of the rs1042713 site of sample 257, and the result is boxed.

[0046] Figure 5 This is the mass spectrometry result of the rs1051375 site of sample 257, and the result has been boxed.

[0047] Figure 6 This is the Sanger sequencing result of the rs1051375 site of sample 257, and the result is boxed.

[0048] Figure 7 This is the mass spectrometry result of the rs5186 site of sample 257, and the result has been framed.

[0049] Figure 8 This is the Sanger sequencing result of the rs5186 site of sample 257, and the result is boxed.

[0050] Fig. 9 This is the mass spectrometry result of the rs1801253 site of sample 257, and the result has been boxed.

[0051] Fig.10 This is the Sanger sequencing result of the rs1801253 site of sample 257, and the result is boxed. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0053] In the present invention, SEQ NO1 to SEQ NO45 were all commissioned to be synthesized by Shanghai Bio-Tech Co., Ltd.

[0054] Example 1

[0055] Take one whole blood sample from each of the three volunteers and detect the 15 SNP sites of these three samples. First, extract the genomic DNA of the whole blood of the three volunteers, then perform multiple PCR reaction, SAP purification and single base extension reaction, and then place the product in the nucleic acid mass spectrometer for detection. Finally, after analyzing the offline data through relevant software, the genotype of the 15 SNP sites of each sample can be obtained. At the same time, the mass spectrometry results are verified by the first-generation sequencing method. The verification results show that the results of the method of the present invention and the first-generation sequencing method have a 100% consistency rate.

[0056] Detection method of the kit of the present invention: 1. Multiplex PCR amplification reaction

[0057] Single sample PCR amplification system: ultrapure water 0.8μL, 10xPCR Buffer 0.5μL (PCR Reagent And Spectro CHIP Kit, purchased from Agena Bioscience, catalog number: 21327M), 25mM MgCl2 0.4μL, dNTP0.1μL, P Mix 1μL, PCR Enzyme 0.2μL (PCR Reagent And Spectro CHIP Kit, purchased from Agena Bioscience, catalog number: 21327M), DNA template 2μL, a total of 5μL reaction system. The reaction conditions are 95 degrees for 2 minutes; 95 degrees for 30 seconds, 56 degrees for 30 seconds, 72 degrees for 60 seconds, 45 cycles; 72 degrees for 5 minutes. The primers of P Mix are SEQ NO1 to SEQ NO30 in Table 1, and the concentration of each primer is 0.5μM.

[0058] Each sample reaction yields an amplification product.

[0059] The DNA template was extracted using the CWE960 Blood DNA Kit from Kangwei Century. The multiplex PCR primer sequences are shown in Table 1:

[0060] Table 1

[0061]

[0062] In the present invention, the correspondence between individualized medication for hypertension and gene loci is shown in Table 2 below:

[0063] Table 2

[0064]

[0065]

[0066] The test only reports the variation results of 15 target sites in 14 genes. The specific sites are shown in Table 3. The reference genome is the human reference genome GRCh38 / hg38.

[0067] Table 3

[0068] Gene rs number Gene rs number ADRB2 rs1042713 NPPA-AS1 rs5063 ACY3 rs2514036 GNB3 rs5443 ACE rs1799752 CYP2D6 rs1065852 CYP2C9 rs1057910 CYP2D6 rs766507177 AGTR1 rs5186 ADRB1 rs1801253 CYP3A5 rs776746 MTHFR rs1801133 ADD1 rs4961

[0069] 2. Shrimp alkaline phosphatase (SAP) purification reaction

[0070] 2 μL of SAP mixed solution was added to the above amplification reaction wells, including: 1.53 μL ultrapure water, 0.17 μL SAP Buffer, 0.3 μL SAP Enzyme (iPLEX Pro kit, purchased from Agena Bioscience, catalog number: 10212), to obtain a SAP purification system: 1.53 μL ultrapure water, 0.17 μL SAP Buffer, 0.3 μL SAP Enzyme, 5 μL first step PCR amplification product, a total of 7 μL reaction system. The reaction conditions for the purification reaction were 37 degrees for 40 minutes and 80 degrees for 5 minutes.

[0071] 3 Single base extension reaction

[0072] After the purification reaction, 2 μL of iPLEX reaction system was added to the reaction well, and the extension reaction system for each well was: 0.62 μL of ultrapure water, 0.2 μL of iPLEX Buffer Plus (iPLEX Pro kit, purchased from Agena Bioscience Catalog No.: 10212), 0.2 μL of iPLEX Termination mix (iPLEX Pro kit, purchased from Agena Bioscience Catalog No.: 10212), 0.94 μL of UEP Mix, 0.04 μL of iPLEX Pro Enzyme (iPLEX Pro kit, purchased from Agena Bioscience Catalog No.: 10212), 7 μL of the second step purified product, a total of 9 μL of reaction system. The extended UEP Mix sequence is SEQ NO31 to SEQ NO45 in Table 4, and the concentration of each primer is 0.7 μM. The reaction conditions are shown in Table 5:

[0073] Table 4

[0074]

[0075]

[0076] Table 5

[0077]

[0078] 4. Nucleic acid mass spectrometry analysis

[0079] Add 35 μL of deionized water to the extension product. Place the sample in the MALDI-TOF MS matrix-assisted laser desorption ionization-time of flight mass spectrometer, and set the detection parameters: Wells to Process is set to Automatic, Start Disoense Condition is set to 1000, Resin Volume is set to 13, Shot is set to 30, Maximum Acquisitions is set to 9, Minimum Good Spectra and Maximum Good Spectra are both set to 5, and 5 items need to be checked, including Tum OffHy After Analysis, Transfer Resin to MTP, Dispense Samples onto Chips, Transferchips to Anayzer, and AnalyzeChips. Then the mass spectrometer spotter spots the extension product onto the SpectroCHIP (chip) for detection. After the detection, the detector converts the electrical signal into a visible peak graph. Each genotype has a unique molecular weight. The software automatically analyzes the corresponding base type to obtain the final genotyping result. The results of the three test samples are shown in Tables 6 to 8.

[0080] Tables 6 to 8 are the test results of 3 samples:

[0081] Table 6

[0082]

[0083]

[0084] Table 7

[0085]

[0086] Table 8

[0087]

[0088]

[0089] It can be seen from the test results that the kit of the present invention can accurately detect the 15 SNP genotypes.

[0090] The same sample was subjected to Sanger sequencing, and the sequencing results and mass spectrometry results were exactly the same. The detection spectrum of some gene loci of the representative sample (No. 257) was compared as follows: Figures 1 to 10 shown.

[0091] Figure 1This is the mass spectrometry result of the rs5063 site of sample 57, and the result is boxed. Figure 2 This is the Sanger sequencing result of the rs5063 site of sample 257, and the result is boxed. Figure 3 This is the mass spectrometry result of the rs1042713 site of sample 257, and the result is boxed. Figure 4 This is the Sanger sequencing result of the rs1042713 site of sample 257, and the result is boxed. Figure 5 This is the mass spectrometry result of the rs1051375 site of sample 257, and the result is boxed. Figure 6 This is the Sanger sequencing result of the rs1051375 site of sample 257, and the result is boxed. Figure 7 This is the mass spectrometry result of the rs5186 site of sample 257, and the result is boxed. Figure 8 This is the Sanger sequencing result of the rs5186 site of sample 257, and the result is boxed. Fig. 9 This is the mass spectrometry result of the rs1801253 site of sample 257, and the result is boxed. Fig.10 This is the Sanger sequencing result of the rs1801253 site of sample 257, and the result is boxed.

[0092] Figures 1 to 10 The results showed that the sequencing method of the present invention was completely consistent with the results of Sanger sequencing.

Claims

1. A primer set for detecting polymorphism of gene loci for personalized medication of hypertension, characterized in that The primer set includes multiple PCR amplification primers, the sequences of which are shown in SEQ NO. 1 to SEQ NO. 30, and are used to amplify 15 gene loci related to hypertension medication:

2. The primer set for detecting the polymorphism of the gene locus for personalized medication of hypertension according to claim 1, characterized in that The primer set also includes a UEP primer for single base extension reaction, the sequences of which are shown in SEQ NO31 to SEQ NO45:

3. A kit for detecting polymorphism of gene loci for personalized medication of hypertension, the kit comprising the multiplex PCR amplification primers as claimed in claim 1 and the UEP primers as claimed in claim 2.

4. The kit according to claim 3, characterized in that The kit comprises a multiplex PCR amplification reaction system, a shrimp alkaline phosphatase purification reaction system and a single base extension reaction system.

5. The kit as claimed in claim 4, characterized in that The multiplex PCR amplification reaction solution comprises multiplex PCR amplification primers, PCR buffer, deoxynucleoside triphosphate mixture, DNA polymerase and MgCl2; The SAP purification reaction system comprises SAP enzyme and SAP buffer; The single base extension reaction system includes UEP primer, iPLEX Buffer Plus, iPLEX Termination mix and iPLEX Pro Enzyme.

6. Use of the kit as claimed in any one of claims 3 to 5 in detecting polymorphisms of gene loci for personalized medication of hypertension; the hypertension drugs include 23 kinds: atenolol, lisinopril, candesartan, furosemide, amiloride, enalapril, verapamil, benazepril, nitrendipine, spironolactone, telmisartan, losartan, bisoprolol, perindopril, amlodipine, bumetanide, metoprolol, folic acid, captopril, irbesartan, nifedipine, hydrochlorothiazide, and carvedilol.

7. A method for detecting a polymorphism of a gene locus for personalized medication for hypertension using the kit according to any one of claims 3 to 5, characterized in that: The method comprises the following steps: (1) Multiplex PCR amplification reaction: Extracting template DNA from samples, preparing a multiplex PCR amplification reaction system, performing multiplex PCR amplification reactions, and obtaining multiplex PCR amplification products; The multiplex PCR amplification reaction system is a 5 μL reaction system, including the following components: Ultrapure water 0.8μL, 10xPCR Buffer 0.5μL, 25mM MgCl2 0.4μL, dNTP 0.1μL, P Mix 1μL, PCREnzyme 0.2μL, DNA template 2μL; The P mix is ​​a primer sequence of SEQ NO1 to SEQ NO30, and the concentration of each primer is 0.5 μM; (2) Shrimp alkaline phosphatase purification reaction: The multiplex PCR amplification products were purified with SAP enzyme. The SAP purification reaction system included the following components: 1.53 μL ultrapure water, 0.17 μL SAP Buffer, 0.3 μL SAP Enzyme, 5 μL amplification product, a total of 7 μL reaction system; after the reaction, the purified product was obtained; (3) Single base extension reaction The purified product of step (2) is subjected to a single base extension reaction, wherein the single base extension reaction system comprises the following components: 0.62 μL of ultrapure water, 0.2 μL of iPLEX Buffer Plus, 0.2 μL of iPLEX Termination mix, 0.94 μL of UEP Mix, 0.04 μL of iPLEX Pro Enzyme, and 7 μL of purified product, a total of 9 μL of reaction system; after the reaction, an extension product is obtained; UEP Mix contains primer sequences from SEQ NO31 to SEQ NO45, and the concentration of each primer is 0.7 μM; (4) Nucleic acid mass spectrometry analysis 35 μL of deionized water was added to the extension product, and then the product was detected by a nucleic acid mass spectrometer to obtain the polymorphism of the gene site for personalized medication of hypertension.

8. The method according to claim 7, characterized in that The reaction conditions of the multiplex PCR amplification reaction were 95° C. for 2 minutes; 95° C. for 30 seconds, 56° C. for 30 seconds, 72° C. for 60 seconds, 45 cycles; and then 72° C. for 5 minutes.

9. The method according to claim 7, characterized in that In the step (2), the reaction conditions of the purification reaction are 37 degrees for 40 minutes and 80 degrees for 5 minutes.

10. The method according to claim 7, characterized in that In the step (3), the reaction procedure of the single base extension reaction is 94°C for 30s, {94°C for 5s, (41°C for 5s, 80°C for 5s) for 5 internal cycles} 39 external cycles, and 72°C for 3min.

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