High-throughput sequencing method for related genes of hypertension treatment medicine and application of high-throughput sequencing method

Abstract

The invention discloses a high-throughput sequencing method for related genes of hypertension treatment drugs and application of the high-throughput sequencing method, which comprises the following steps: 1, sampling: collecting 2-3ml of venous blood through a vacuum blood collection tube, and adding EDTA-K2 (Ethylene Diamine Tetraacetic Acid) for anticoagulation; 2, sample treatment: extracting DNA by using a genome DNA extraction kit, and detecting the DNA under a dark condition; 3, designing primers and probes, namely selecting nucleotide types of human gene loci needing to be detected to design sequencing primers, fluorescent PCR (Polymerase Chain Reaction) primers and probes; and 4, detecting, namely amplifying the extracted DNA by using the sequencing primer through a fluorescent quantitative PCR reaction, and marking the DNA by using a fluorescent substance. According to the method, Primer Express software is applied to design high-specificity primers and probes for human gene loci needing to be detected, a detection system is optimized and established, and the method is suitable for a two-channel fluorescent quantitative PCR instrument, has the advantages of simplicity, convenience, accuracy and rapidness, and can meet clinical conventional detection requirements.

Description

Technical Field

[0001] The present invention relates to the technical field of hypertension therapeutic drug-related gene detection, and in particular to a high-throughput sequencing method for hypertension therapeutic drug-related genes and applications thereof. Background Art

[0002] Blood pressure is one of the most common chronic diseases in my country. According to the 2019 "China Hypertension Prevention and Treatment Guidelines 2018 Revised Edition", the latest survey data on hypertension in my country shows that the crude prevalence of hypertension among residents aged 18 and above in my country was 27.9% from 2012 to 2015, and the prevalence of hypertension in the Chinese population is still on the rise. As one of the most important risk factors for cardiovascular and cerebrovascular diseases, hypertension has a high disability and mortality rate, a serious epidemic situation, and a heavy economic and mental burden on families and society. It has become an important public health issue in my country.

[0003] The five major categories of antihypertensive drugs commonly used in clinical practice are diuretics, beta-blockers, calcium channel blockers, angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers. There are many types of drugs available, but the genetic polymorphism of various antihypertensive drugs is an important influencing factor leading to individual differences in drug efficacy. Existing clinical detection methods include restriction fragment length polymorphism analysis, allele-specific PCR, DNA sequencing and gene chip technology, but these methods are cumbersome to operate and can hardly meet the needs of routine clinical testing. Summary of the invention

[0004] The present invention provides a high-throughput sequencing method for hypertension therapeutic drug-related genes and its application to solve the problems raised in the above background technology.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A high-throughput sequencing method for hypertension therapeutic drug-related genes and its application, comprising the following steps:

[0007] Step 1: Sampling: collect 2-3 ml of venous blood through a vacuum blood collection tube and add EDTA-K2 for anticoagulation;

[0008] The second step is sample processing, using a genomic DNA extraction kit to extract DNA and test the DNA under light-proof conditions;

[0009] The third step is to design primers and probes, select the nucleotide type of the human gene locus to be detected, and design sequencing primers and fluorescent PCR primers and probes;

[0010] The fourth step is detection. The sequencing primers amplify the extracted DNA through fluorescent quantitative PCR reaction, and then use fluorescent substances to label the DNA. At the same time, the PCR amplification product is detected using a PCR instrument to record the Ct value of each detection FAM channel and the Ct value of the VIC channel.

[0011] As a further improvement of the technical solution: in the first step, each sample is stored at 20-30 degrees Celsius, and the sample is repeatedly frozen and thawed no more than 3 times.

[0012] As a further improvement of the present technical solution: In the second step, the process of detecting DNA is to take 5uL of DNA and use a P-330-31 micro-ultra-micro spectrophotometer to determine the concentration and purity. When the zero degree of DNA is 5ng / L, it is necessary to use the genomic DNA extraction kit again to extract DNA and purify the DNA of the sample.

[0013] As a further improvement of the technical solution: in the third step, the human gene loci to be detected are selected by downloading the human gene loci to be detected from the NCBI gene bank in the United States.

[0014] As a further improvement of the present technical solution: in the third step, sequencing primers, fluorescent PCR primers and probes are designed for human gene loci using Primer Premier 5.0 and Primer Express 3.0 software.

[0015] As a further improvement of the present technical solution: in the fourth step, the PCR reaction amplification is carried out in a light-proof environment.

[0016] As a further improvement of the technical solution: in the fourth step, the PCR instrument is an ABI PRISM7500 fluorescence quantitative PCR instrument.

[0017] As a further improvement of the technical solution: in the fourth step, the total PCR system is 40uL, including MgCl24uL, 2U HS-Taq 0.4uL, UNG enzyme 0.2uL, 25mmol / L dNTP 0.32uL, 100umol / L upstream and downstream primers 0.08uL each, genomic DNA 20ng, and ddH2O is added to 40uL.

[0018] As a further improvement of the technical solution: in the fourth step, the cycle parameters of the PCR instrument detection are set to 95°C for 5 min, 95°C for 15 s, and 60°C for 40 s, for a total of 40 cycles, and the fluorescence signal is collected at 60°C using 7500Software Version 2.3 software.

[0019] As a further improvement of this technical solution: it is applied to the high-throughput sequencing of genes related to the preparation of hypertension treatment drugs.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] Finally, the Ct value of each detected FAM channel can be recorded as CtF, and the Ct value of the VIC channel can be recorded as CtV. Calculate the absolute value of the difference in Ct values between the FAM detection channel and the VIC detection channel, Ct = |CtF - CtV|; if △Ct < 3.0, this site is judged as a heterozygous mutant type; if △Ct > 3.0, it is judged in two cases: when CtF < CtV, this site is judged as a homozygous wild type; when CtV < CtF, this site is judged as a homozygous mutant type. Generally, the phenotypes of the effects of general antihypertensive drugs on the human body are three types, namely slow metabolism type, fast metabolism type, and intermediate metabolism type. Fast metabolism is mainly caused by defective alleles and is manifested as a homozygous type. People carrying this genotype metabolize drugs slowly and are prone to drug adverse reactions. Therefore, during clinical treatment, for patients with fast metabolism type, the drug dosage should be appropriately increased to reach the effective blood drug concentration, while patients with slow metabolism type are manifested as heterozygous type and the drug dosage should be reduced to prevent drug adverse reactions. This method uses Primer Express software to design highly specific primers and probes for the human gene loci to be detected, optimizes and establishes a detection system. Compared with the fluorescence PCR detection method, this method is applicable to a two-channel fluorescence quantitative PCR instrument and has the advantages of simplicity, accuracy, and rapidity, and can meet the needs of clinical routine detection.

[0022] The above description is only an overview of the technical solution of the present invention. In order to be able to more clearly understand the technical means of the present invention and implement it in accordance with the content of the specification, the following takes the preferred embodiments of the present invention and combines the accompanying drawings to elaborate in detail as follows. The specific implementation manners of the present invention are given in detail by the following embodiments and their accompanying drawings. Description of the Drawings

[0023] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0024] Figure 1 It is a schematic flow chart of a high-throughput sequencing method and its application for genes related to hypertension treatment drugs proposed by the present invention. Detailed Description of the Invention

[0025] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention. The present invention is described in more detail by way of example with reference to the accompanying drawings in the following paragraphs. The advantages and features of the present invention will become clearer according to the following description and claims. It should be noted that the drawings are all in a very simplified form and are not in precise proportions, and are only used to conveniently and clearly assist in explaining the purpose of the embodiments of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0027] See also Figure 1 In an embodiment of the present invention, a high-throughput sequencing method for hypertension therapeutic drug-related genes and its application include the following steps:

[0028] Step 1: Sampling: collect 2-3 ml of venous blood through a vacuum blood collection tube and add EDTA-K2 for anticoagulation;

[0029] The second step is sample processing, using a genomic DNA extraction kit to extract DNA and test the DNA under light-proof conditions;

[0030] The third step is to design primers and probes, select the nucleotide type of the human gene locus to be detected, and design sequencing primers and fluorescent PCR primers and probes;

[0031] The fourth step is detection. The sequencing primers amplify the extracted DNA through fluorescent quantitative PCR reaction, and then use fluorescent substances to label the DNA. At the same time, the PCR amplification product is detected using a PCR instrument to record the Ct value of each detection FAM channel and the Ct value of the VIC channel.

[0032] Preferably, in the first step, each sample is stored at 20-30 degrees Celsius, and the sample is repeatedly frozen and thawed no more than three times, thereby further ensuring the quality of the sample.

[0033] Preferably, in the second step, the process of detecting DNA is to take 5uL of DNA and use a P-330-31 micro-ultra-micro spectrophotometer to determine the concentration and purity. When the zero degree of DNA is 5ng / L, it is necessary to re-use the genomic DNA extraction kit to extract DNA and purify the DNA of the sample. The process of detecting DNA ensures the purity of the DNA of the sample, thereby greatly improving the accuracy of the sample.

[0034] Preferably, in the third step, the human gene loci to be detected can be selected by downloading the human gene loci to be detected from the NCBI gene library in the United States, and the gene loci that undergo metabolic reactions with antihypertensive drugs can be selected.

[0035] Preferably, in the third step, sequencing primers and fluorescent PCR primers and probes are designed for human gene loci using Primer Premier 5.0 and Primer Express 3.0 software.

[0036] Preferably, in the fourth step, the PCR amplification is carried out in a light-proof environment, preferably in a dark room with red light, as strong light will affect the PCR amplification reaction.

[0037] Preferably, in the fourth step, the PCR instrument is an ABI PRISM 7500 fluorescence quantitative PCR instrument.

[0038] Preferably, in the fourth step, the total PCR system is 40uL, including MgCl24uL, 2U HS-Taq 0.4uL, UNG enzyme 0.2uL, 25mmol / L dNTP 0.32uL, 100umol / L upstream and downstream primers 0.08uL each, genomic DNA 20ng, and ddH2O is added to 40uL.

[0039] Preferably, in the fourth step, the cycle parameters of the PCR instrument detection are set to 95°C for 5 min, 95°C for 15 s, and 60°C for 40 s, for a total of 40 cycles, and the fluorescence signal is collected at 60°C using 7500Software Version 2.3 software.

[0040] The method is applied to prepare high-throughput sequencing of genes related to hypertension therapeutic drugs.

[0041] The working principle of the present invention is:

[0042] In the above-mentioned fifth step, the Ct value of each detected FAM channel can be denoted as CtF, and the Ct value of the VIC channel can be denoted as CtV. Calculate the absolute value of the difference in Ct values between the FAM detection channel and the VIC detection channel, Ct = |CtF - CtV|. If △Ct < 3.0, this locus is judged as a heterozygous mutant type; if △Ct > 3.0, it is judged in two cases: when CtF < CtV, this locus is judged as a homozygous wild type; when CtV < CtF, this locus is judged as a homozygous mutant type. Generally, the phenotypes of the effects of antihypertensive drugs on the human body are of three types, namely slow metabolism type, fast metabolism type, and intermediate metabolism type. Fast metabolism is mainly caused by defective alleles and is manifested as the homozygous type. The population carrying this genotype metabolizes drugs slowly and is prone to drug adverse reactions. Therefore, during clinical treatment, the drug dosage should be appropriately increased for fast metabolism type patients to reach the effective blood drug concentration, while slow metabolism type patients are manifested as the heterozygous type and the drug dosage should be reduced to prevent drug adverse reactions. Compared with the method of PCR combined with restriction fragment length polymorphism, after the PCR reaction, a restriction endonuclease is added, and the corresponding genotype is determined according to different fragments after agarose gel electrophoresis. Compared with this method, the risk of aerosol contamination in electrophoresis after PCR can be avoided; compared with the allele-specific amplification method, this method does not require setting a PCR reaction for each genotype, and the operation is simple. This method uses Primer Express software to design highly specific primers and probes for the human gene loci to be detected, optimizes and establishes a detection system. Compared with the fluorescence PCR detection method, this method is applicable to two-channel fluorescence quantitative PCR instruments and has the advantages of simplicity, accuracy, and rapidity.

[0043] As described above, it is only a preferred embodiment of the present invention and does not impose any form of limitation on the present invention; any ordinary technician in the industry can smoothly implement the present invention as shown in the accompanying drawings of the specification and described above; however, any minor changes, modifications, and equivalent variations made by those skilled in the art within the scope of the technical solution of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications, and variations made to the above embodiments based on the essential technology of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A high-throughput sequencing method for genes related to hypertension therapeutic drugs and its application, characterized in that: The following steps are involved: Step 1: Sampling: collect 2-3 ml of venous blood through a vacuum blood collection tube and add EDTA-K2 for anticoagulation; The second step is sample processing, using a genomic DNA extraction kit to extract DNA and test the DNA under light-proof conditions; The third step is to design primers and probes, select the nucleotide type of the human gene locus to be detected, and design sequencing primers and fluorescent PCR primers and probes; The fourth step is detection. The sequencing primers amplify the extracted DNA through fluorescent quantitative PCR reaction, and then use fluorescent substances to label the DNA. At the same time, the PCR amplification product is detected using a PCR instrument to record the Ct value of each detection FAM channel and the Ct value of the VIC channel.

2. The high-throughput sequencing method for hypertension therapeutic drug-related genes and its application according to claim 1, characterized in that: In the first step, each sample is stored at 20-30 degrees Celsius, and the sample is repeatedly frozen and thawed no more than 3 times.

3. The high-throughput sequencing method for hypertension therapeutic drug-related genes and its application according to claim 1, characterized in that: In the second step, the process of DNA detection is to take 5uL of DNA and use a P-330-31 micro-ultra-micro spectrophotometer to measure the concentration and purity. When the zero degree of DNA is 5ng / L, it is necessary to use the genomic DNA extraction kit again to extract DNA and purify the sample DNA.

4. The high-throughput sequencing method for hypertension therapeutic drug-related genes and its application according to claim 1, characterized in that: In the third step, the human gene loci to be detected are selected and the human gene loci to be detected can be downloaded from the NCBI gene bank in the United States.

5. The high-throughput sequencing method for hypertension therapeutic drug-related genes and its application according to claim 1, characterized in that: In the third step, sequencing primers, fluorescent PCR primers and probes are designed for human gene loci using Primer Premier 5.0 and Primer Express 3.0 software.

6. The high-throughput sequencing method for hypertension therapeutic drug-related genes and its application according to claim 1, characterized in that: In the fourth step, the PCR amplification is carried out in a light-proof environment.

7. The high-throughput sequencing method for hypertension therapeutic drug-related genes and its application according to claim 1, characterized in that: In the fourth step, the PCR instrument is an ABI PRISM 7500 fluorescence quantitative PCR instrument.

8. The high-throughput sequencing method for hypertension therapeutic drug-related genes and its application according to claim 1, characterized in that: In the fourth step, the total PCR system is 40uL, including MgCl24uL, 2U HS-Taq 0.4uL, UNG enzyme 0.2uL, 25mmol / L dNTP 0.32uL, 100umol / L upstream and downstream primers 0.08uL each, genomic DNA 20ng, and ddH2O is added to 40uL.

9. The high-throughput sequencing method for hypertension therapeutic drug-related genes and its application according to claim 1, characterized in that: In the fourth step, the cycle parameters of the PCR instrument detection were set to 95°C for 5 min, 95°C for 15 s, and 60°C for 40 s, for a total of 40 cycles, and the fluorescence signal was collected at 60°C using 7500 Software Version 2.3 software.

10. A high-throughput sequencing method for hypertension therapeutic drug-related genes and its application according to any one of claims 1 to 9, characterized in that: Applied to high-throughput sequencing of genes related to the preparation of hypertension therapeutic drugs.

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