Primer group and kit for detecting individualized medication gene locus polymorphism of statin lipid-lowering drugs

Abstract

The invention discloses a primer group and a kit for detecting individualized medication gene locus polymorphism of statin lipid-lowering drugs, a method for detecting the individualized medication gene locus polymorphism of the statin lipid-lowering drugs 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 six related medication gene loci of the statin lipid-lowering drugs, relates to seven 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 appropriate drugs to achieve the purpose of accurate medication.

Description

Technical Field

[0001] The present invention relates to a primer set and a kit for detecting the polymorphism of gene loci for individualized medication of statin lipid-lowering drugs. Background Art

[0002] As people's living standards continue to improve, their eating habits have also changed. Long-term and excessive fat intake leads to abnormally high blood lipid levels in the body. Abnormal lipid metabolism refers to abnormal levels of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), and total cholesterol (TC) in the blood. It is closely related to a variety of serious diseases such as coronary heart disease, atherosclerosis, myocardial infarction, and diabetic nephropathy, and is one of the important factors causing the occurrence and development of such diseases. The "Report on the Nutrition and Chronic Disease Status of Chinese Residents (2015)" shows that the overall prevalence of dyslipidemia in Chinese adults is as high as 40.40%, a significant increase from 2002. The increase in serum cholesterol levels in the population will lead to an increase of approximately 9.2 million cardiovascular disease events in my country between 2010 and 2030. Dyslipidemia plays an extremely important role in the occurrence and development of cardiovascular and cerebrovascular diseases and has become an important factor threatening human health.

[0003] Statins: Also known as 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, they can inhibit the rate-limiting enzyme HMG-CoA reductase in cholesterol synthesis, reduce cholesterol synthesis, and then upregulate cell surface LDL receptors to accelerate serum LDL catabolism. In addition, VLDL synthesis can also be inhibited. Therefore, statins can significantly reduce serum TC, LDL-C and ApoB levels, and can also reduce serum TG levels and slightly increase HDL-C levels. Therefore, statins can significantly reduce the risk of cardiovascular events in both primary and secondary prevention of atherosclerotic cardiovascular diseases. However, many research results also show that there are individual differences in the efficacy and side effects of statins. In addition to physiological and pathological factors, the influencing factors also include genetic factors.

[0004] With the rapid development of pharmacogenomics (PGx) in recent years, researchers have discovered some gene loci related to the efficacy and side effects of statins: such as SLCO1B1 rs4149056, SLCO1B1 rs2306283, APOErs7412, KIF6 rs20455, etc. These gene loci involve a series of functions such as encoding drug metabolizing enzymes, transporters, and targets. Therefore, changes in genetic polymorphisms will affect different individuals' sensitivity to drug efficacy, concentration, and target tissues. Therefore, detection and analysis of these gene loci will be able to help clinical selection of appropriate statin types and doses, which is very important for patients with dyslipidemia.

[0005] In addition, 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. For traditional gene mutation detection methods, such as Sanger sequencing, pyrophosphate sequencing, and real-time fluorescence PCR, due to their limited detection throughput, one batch can only detect mutations in a single gene or part of the exon of a single gene, and therefore are no longer applicable. The second-generation sequencing (NGS) can obtain hundreds of pharmacogenomics-related gene information in one test, but the disadvantage of the second-generation sequencing is that it is expensive, and its testing fee is difficult to be accepted by the public. Therefore, it is necessary to develop a high-throughput, low-cost, and highly accurate detection scheme for the polymorphism of gene sites for personalized medication of statins. Summary of the invention

[0006] The purpose of the present invention is to provide a primer set and a kit for detecting the polymorphism of gene loci for individualized medication of statins and lipid-lowering drugs.

[0007] The technical solution adopted by the present invention is:

[0008] A primer set for detecting polymorphism of gene loci for personalized medication of statins, comprising multiple PCR amplification primers, the sequences of which are shown in SEQ NO. 1 to SEQ NO. 12, and used for amplifying 6 gene loci related to statins:

[0009]

[0010] The primer set also includes a UEP primer for single base extension reaction, the sequences of which are shown in SEQ NO13 to SEQ NO18:

[0011]

[0012] Furthermore, the present invention also provides a kit for detecting the polymorphism of gene loci for personalized medication of statins, and the kit comprises the above-mentioned multiple PCR amplification primers and UEP primers.

[0013] 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.

[0014] The multiplex PCR amplification reaction solution comprises multiplex PCR amplification primers, PCR buffer, deoxynucleoside triphosphate mixture (dNTP), DNA polymerase, and MgCl2.

[0015] The SAP purification reaction system comprises SAP enzyme and SAP buffer.

[0016] SAP enzyme and SAP buffer can be purchased from Agena.

[0017] The single base extension reaction system includes UEP primer, iPLEX Buffer Plus, iPLEX Terminationmix and iPLEX Pro Enzyme.

[0018] The iPLEX Buffer Plus, iPLEX Termination mix and iPLEX Pro Enzyme were purchased from Agena.

[0019] The present invention also provides the use of the kit in detecting the polymorphism of gene sites for individualized medication of statins and lipid-lowering drugs.

[0020] The statins include seven types: atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.

[0021] Furthermore, the genes include 4 types: SLCO1B1, ABCG2, APOE, and KIF6.

[0022] The gene loci include rs2231142, r7412, rs4149056, rs20455, rs429358, and rs2306283.

[0023] The present invention also provides a method for detecting the polymorphism of the gene loci for individualized medication of statins lipid-lowering drugs using the kit, the method comprising the following steps:

[0024] (1) Multiplex PCR amplification reaction:

[0025] Extracting template DNA from samples, preparing a multiplex PCR amplification reaction system, performing multiplex PCR amplification reactions, and obtaining multiplex PCR amplification products;

[0026] The multiplex PCR amplification reaction system is a 5 μL reaction system, including the following components:

[0027] 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;

[0028] The P mix is ​​the primer sequence of SEQ NO1 to SEQ NO12, and the concentration of each primer is 0.5 μM;

[0029] 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.

[0030] (2) Shrimp alkaline phosphatase (SAP) purification reaction:

[0031] 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;

[0032] The reaction conditions for the purification reaction were 37 degrees for 40 minutes and 80 degrees for 5 minutes.

[0033] (3) Single base extension reaction

[0034] 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;

[0035] UEP Mix contains primer sequences from SEQ NO13 to SEQ NO18, and the concentration of each primer is 0.7 μM;

[0036] 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.

[0037] (4) Nucleic acid mass spectrometry analysis

[0038] 35 μL of deionized water was added to the extension product, which was then detected using a nucleic acid mass spectrometer to obtain the polymorphism of the gene loci for personalized medication of statins.

[0039] The nucleic acid mass spectrometer is generally a MALDI-TOF MS matrix-assisted laser desorption ionization-time of flight mass spectrometer.

[0040] 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.

[0041] 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.

[0042] The statin lipid-lowering drug personalized medication gene locus 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 6 gene loci; compared with the second-generation sequencing NGS, a batch of samples of the present invention only needs 8-10 hours to complete, which is short in time and improves the detection efficiency, while also having a more affordable price. This kit can detect 6 statin lipid-lowering drug-related gene loci, involving 7 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. Description of the drawings:

[0043] Figure 1 This is the mass spectrometry result of the rs2231142 site of sample 41, and the result has been boxed.

[0044] Figure 2 This is the Sanger sequencing result of the rs2231142 site of sample 41, and the result is boxed.

[0045] Figure 3 This is the mass spectrometry result of the rs4149056 site of sample 41, and the result has been boxed.

[0046] Figure 4 This is the Sanger sequencing result of the rs4149056 site of sample 41, and the result is boxed.

[0047] Figure 5 This is the mass spectrometry result of the rs20455 site of sample 41, and the result has been framed.

[0048] Figure 6 This is the Sanger sequencing result of the rs20455 site of sample 41, and the result is boxed.

[0049] Figure 7 This is the mass spectrometry result of the rs7412 locus of sample 41, and the result has been boxed.

[0050] Figure 8 This is the Sanger sequencing result of the rs7412 site of sample 41, and the result is boxed.

[0051] Fig. 9 This is the mass spectrometry result of the rs429358 site of sample 41, and the result has been boxed.

[0052] Fig.10 This is the Sanger sequencing result of the rs429358 site of sample 41, and the result is boxed.

[0053] Fig.11 This is the mass spectrometry result of the rs2306283 site of sample 41, and the result has been boxed. Fig.12 This is the Sanger sequencing result of the rs2306283 site of sample 41, and the result is boxed. DETAILED DESCRIPTION

[0054] 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.

[0055] In the present invention, SEQ NO. 1 to SEQ NO. 18 were all commissioned to be synthesized by Shanghai Bio-Tech Co., Ltd.

[0056] Example 1

[0057] Take one whole blood sample from each of the three volunteers and detect the six 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 six 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.

[0058] Detection method of the kit of the present invention: 1. Multiplex PCR amplification reaction

[0059] 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 NO12 in Table 1, and the concentration of each primer is 0.5μM.

[0060] The DNA template was extracted using the CWE960 Blood DNA Kit from Kangwei Century.

[0061] Each sample reaction yields an amplification product.

[0062] The multiplex PCR primer sequences are shown in Table 1:

[0063] Table 1

[0064]

[0065] In the present invention, the correspondence between individualized medication of statins and gene loci is shown in Table 2 below:

[0066] Table 2

[0067]

[0068]

[0069]

[0070] 2. Shrimp alkaline phosphatase (SAP) purification reaction

[0071] 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.

[0072] 3 Single base extension reaction

[0073] 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 NO13 to SEQ NO18 in Table 3, and the concentration of each primer is 0.7 μM. The reaction conditions are shown in Table 4:

[0074] Table 3

[0075]

[0076]

[0077] Table 4

[0078]

[0079] 4. Nucleic acid mass spectrometry analysis

[0080] 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 5 to 7.

[0081] Tables 5 to 7 are the test results of 3 samples respectively:

[0082] Table 5

[0083]

[0084]

[0085] Table 6

[0086]

[0087] Table 7

[0088]

[0089] It can be seen from the test results that the kit of the present invention can accurately detect the 6 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 the representative sample (No. 41) is compared as follows Figures 1 to 12 shown.

[0091] Figure 1 This is the mass spectrometry result of the rs2231142 site of sample 41, and the result is boxed; Figure 2This is the Sanger sequencing result of the rs2231142 locus of sample 41, and the result is boxed; Figure 3 This is the mass spectrometry result of the rs4149056 site of sample 41, and the result is boxed; Figure 4 This is the Sanger sequencing result of the rs4149056 site of sample 41, and the result is boxed; Figure 5 This is the mass spectrometry result of the rs20455 site of sample 41, and the result is boxed; Figure 6 This is the Sanger sequencing result of the rs20455 site of sample 41, and the result is boxed; Figure 7 This is the mass spectrometry result of the rs7412 locus of sample 41, and the result is boxed; Figure 8 This is the Sanger sequencing result of the rs7412 locus of sample 41, and the result is boxed; Fig. 9 This is the mass spectrometry result of the rs429358 locus of sample 41, and the result is boxed; Fig.10 This is the Sanger sequencing result of the rs429358 site of sample 41, and the result is boxed; Fig.11 This is the mass spectrometry result of the rs2306283 site of sample 41, and the result is boxed; Fig.12 This is the Sanger sequencing result of the rs2306283 site of sample 41, and the result is boxed.

[0092] Figures 1 to 12 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 statins, characterized in that: The primer set includes multiple PCR amplification primers, the sequences of which are shown in SEQ NO. 1 to SEQ NO. 12, and are used to amplify 6 statin lipid-lowering medication-related gene loci:

2. The primer set for detecting the polymorphism of the gene locus for personalized medication of statins 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 NO13 to SEQ NO18:

3. A kit for detecting polymorphism of gene loci for personalized medication of statins, characterized in that: The kit comprises 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 statins; the statins include seven types of lipid-lowering drugs: atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.

7. A method for detecting the polymorphism of the gene loci for personalized medication of statins 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 ​​the primer sequence of SEQ NO1 to SEQ NO12, 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 NO13 to SEQ NO18, 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 statins.

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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