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CN110257506B - Polymorphism detection kit and method for hypertension accurate medication related gene - Google Patents

Polymorphism detection kit and method for hypertension accurate medication related gene Download PDF

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CN110257506B
CN110257506B CN201910614761.9A CN201910614761A CN110257506B CN 110257506 B CN110257506 B CN 110257506B CN 201910614761 A CN201910614761 A CN 201910614761A CN 110257506 B CN110257506 B CN 110257506B
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尹华立
裘惠良
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Hangzhou Meilian Medical Examination Institute Co ltd
Hangzhou Qianji Biotechnology Co ltd
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Abstract

The invention relates to the technical field of biological medicines, in particular to a kit and a method for detecting polymorphism of genes related to accurate administration of hypertension. The kit comprises a reaction solution I, a reaction solution II and a reaction solution III. The reaction solution I comprises a PCR buffer system and enzyme, the reaction solution II comprises ACE, CYP2D6 and ADRB1 gene detection primers and corresponding fluorescent probes, and the reaction solution III comprises NPPA, AGTR1, CYP3A5 and CYP2C9 gene detection primers and corresponding fluorescent probes. The kit and the method can detect the polymorphism of the gene related to clinical 5-class antihypertensive requirements quickly, sensitively and characteristically.

Description

Polymorphism detection kit and method for hypertension accurate medication related gene
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a kit and a method for detecting polymorphism of genes related to accurate administration of hypertension.
Background
Essential hypertension is one of the most common chronic diseases and is an important risk factor for cardiovascular diseases such as coronary heart disease, cerebral apoplexy and the like, and a large number of researches prove that essential hypertension is mainly influenced by both genetic factors and environmental factors. The blood pressure lowering drugs commonly used in clinic at present comprise diuretics, beta adrenoceptor blockers, calcium antagonists, ACE inhibitors, angiotensin II receptor antagonists and alpha adrenoceptor blockers, however, the drugs for treating hypertension in clinic have quite common individual response differences, mainly because of genetic variations of drug-related drug metabolic enzymes and receptors.
CYP2D6 only accounts for 1-2% of total CYP in liver, but the medicines catalyzed and metabolized by the CYP2D6 are more than 80, and the activity of CYP2D6 of different individuals can be different by 1000 times at most. The most common polymorphism in the chinese population is CYP2D6 x 10, and about 51.6% of the chinese population carry this genetic variation. This polymorphism results in a decrease in the enzymatic activity of CYP2D6 and is extremely unstable, thereby affecting drug metabolism.
The beta adrenergic receptor belongs to the superfamily of G protein-coupled receptors and is encoded by the ADRB1 gene, and the gene has polymorphism. Research shows that ADRB1 gene G1165C polymorphism is related to hypertension drug treatment, and after metoprolol, carvedilol and the like are used for treatment, C1165 homozygote individuals have higher effective rate.
Angiotensin converting enzyme is a key enzyme of renin-angiotensin system, and is coded by ACE gene, which is positioned in 3 zone of 2 region of long arm of 17 # chromosome and 21kb in sequence length. The ACE gene has a plurality of genetic polymorphisms, wherein polymorphism of an Alu insertion fragment (I) and a deletion fragment (D) of 287bp existing in a 16 th intron is related to hypertension drug treatment, research shows that the curative effect of a DD homozygote individual using benazepril and fosinopril is obvious, the curative effect of a II homozygote individual using enalapril and imidapril is obvious, and the curative effect of an ID heterozygote individual using angiotensin inhibitor drugs is effective.
The novel diuretic indapamide (Erbborn, indapamide) is marketed, so that the treatment status of the diuretic in hypertension is improved, and the novel diuretic indapamide is characterized in that the common dosage only shows slight diuretic effect and mainly shows vasodilatation effect, the effective rate for reducing blood pressure is about 80 percent, and the side effect of metabolic abnormality of the traditional diuretic is not existed, so that the novel indapamide is widely applied clinically. Atrial Natriuretic Peptide Precursor A (NPPA) is a "candidate gene" for studying genetic susceptibility to various diseases, is located on human chromosome 1p36, and encodes a precursor of ANP. ANP acts as a diuretic to control the balance of extracellular fluid volume and electrolytes, and increasing ANP levels can cause hypotension. Research shows that ANP (and NPPA gene) is related to various cardiovascular and cerebrovascular diseases, such as cerebral apoplexy, heart failure, left atrial hypertrophy, hypertension and the like. NPPA G664A is a single nucleotide mutation (G/A) located in exon 1 of the gene, resulting in a change of amino acid 7 of the ANP precursor protein to methionine, resulting in reduced ANP activity.
Cytochrome oxidase P450 (CYP 2C 9) is an important drug metabolizing enzyme in the human liver, accounting for about 20% of the total amount of CYP enzymes in the human liver. The distribution of CYP2C9 allele has obvious population, ethnicity and geographical difference, in Chinese population, the main mutant gene is CYP2C9 x 3, which is the mutation of A > C at 1075 th site of 7 th exon, resulting in the mutation of 359 nd isoleucine on CYP2C9 polypeptide chain into leucine (Ile 359> Leu 359), and the allele frequency is 1.0-3.5%. Losartan, irbesartan and other sartan are common blood pressure lowering drugs, are effective angiotensin receptor antagonists and are mainly metabolized by CYP2C9, and researches show that the sartan drugs are mainly metabolized by CYP2C9 in vivo, and the mutation of polymorphic CYP2C9 x 3 causes the remarkable reduction of enzyme activity, the increase of toxicity and the reduction of curative effect. Clinical studies show that CYP2C9 x 3 mutation can cause the reduction of the blood pressure reducing effect of losartan; the CYP2C9 x 3 mutation increases the blood concentration of valsartan and irbesartan, resulting in an enhanced hypotensive effect of valsartan and irbesartan. Therefore, when selecting a sartan drug for a hypertensive patient, CYP2C9 should be first subjected to genetic testing, and a sartan drug should be selected based on the genotype of CYP2C9 x 3.
Angiotensin II receptor antagonists (Angiotensin II receptor antagonists) are a class of drugs acting on the renin-Angiotensin system, and are mainly used for the treatment of hypertension, diabetic nephropathy and congestive heart failure. The type I Angiotensin II receptor (Angiotensin II receptor type 1, agtr1) is a receptor of Angiotensin II (Angiotensin, ang II), which is mainly distributed on vascular smooth muscle cells and mediates most of the biological effects of Ang II. It is a component of the renin-angiotensin system (RAS) and has important implications for the development of hypertension and for drug therapy. The human AGTR1 gene is located at 3q21-25, and contains only 1 exon, without intron region. The frequency of T573C, A1166C and A1062G substitution is the highest in AGTR1 gene polymorphism, and patients with the A1166C genotype show more significant blood pressure response when losartan and candesartan are used. The drug sensitivity of hypertensive patients to angiotensin II receptor blockers (such as losartan, irbesartan, telmisartan, etc.) is as follows: the drug sensitivity of the A1166A patient is normal, and the conventional dosage is recommended; A1166C patient has high drug sensitivity, and the dosage is recommended to be reduced; patients with C1166C have increased drug sensitivity and lower doses are recommended.
Calcium antagonists are chemical agents that lower blood pressure by blocking calcium channels. It can selectively inhibit Ca 2+ Enters into cells through calcium channels on cell membranes, relaxes vascular smooth muscle by expanding blood vessels and negative muscle strength, reduces peripheral vascular resistance, and thus has the function of reducing blood pressure. Cytochrome P450 (CYP) is a large class of drug metabolizing enzymes involved in the metabolism of many drugs, xenobiotics and endogenous substances. CYP3A5 is predominantly expressed in the liver and small intestine of adults with significant polymorphisms in its expression. Mutations in the CYP3A5 gene are the most important cause of differences in enzyme activity, with CYP3A5 x 3 being at intron3 (6959A)>G) The mutation(s) causes variable cleavage, resulting in an unstable protein, so that the mutagenized homozygous individual, i.e. the person carrying the gene CYP3A5 x 3/' 3, does not express CYP3A5. Research shows that calcium antagonist drugs are greatly influenced by CYP3A5 x 3 gene polymorphism, and patients with mutant genotypes have increased toxic and side effects of the drugs.
The detection methods of gene polymorphism are many, and the most common methods comprise a sequencing method, DHPLC, PCR-SSCP/RFLP, ARMS, taqman PCR, ME-PCR, gene chip and the like. The methods are complex to operate, long in detection period, and difficult to control, have a plurality of factors influencing detection results, and are difficult to meet the requirements of clinical reagent detection, and are only carried out in the field of scientific research at present.
Disclosure of Invention
The invention aims to provide a kit and a method for rapidly, accurately and systematically detecting hypertension medication related gene polymorphism aiming at the defects of the prior art.
In order to solve the problems, the invention adopts the following technical scheme:
a kit for detecting polymorphism of genes related to accurate medication of hypertension comprises a reaction liquid I, a reaction liquid II, a reaction liquid III, a positive control substance and a negative control substance. The reaction solution I comprises a PCR buffer system and enzyme, the reaction solution II comprises ACE, CYP2D6 and ADRB1 gene detection primers and corresponding fluorescent probes, the reaction solution III comprises NPPA, AGTR1, CYP3A5 and CYP2C9 gene detection primers and corresponding fluorescent probes, the positive control is a heterozygous plasmid of seven genes of ACE, CYP2D6, ADRB1, NPPA, AGTR1, CYP3A5 and CYP2C9, and the negative control is Tris salt without target genes.
The reaction solution I comprises 10 × Taq buffer I,5 × Taq buffer II, hotstart Taq enzyme, UDG enzyme, dNTP, dUTP and an enhancer; the 10 × taq buffer I comprises 200mM Tris-HCl (pH8.4), 200mM KCl,100mM (NH) 4 ) 2 SO 4 ，15mM MgCl 2 (ii) a The 5 × taq buffer II comprises 20mM Mg SO 4 The enhancer is a mixture comprising 0.5M betaine, 1% (V/V) formamide, 3% (V/V) glycerol and 0.2mg/ml BSA.
The reaction solution II contains ACE, CYP2D6 and ADRB1 detection primers and corresponding probes as follows:
ACE F1：TTTCTCTAGACCTGCTGCCTATAC
ACE R1：AGCTCAGAGAATTTCAGAGCTG
ACE P: CTGCTGCCTATACAGTCACTTTTATGTGG, end 5 labeled ROX and end 3 labeled BHQ2.
D6 F1：CCTCCCTCACCTGGTCGAAGC
D6 R1：TTCCTGCTCCTGGTGGACCTG
D6 P: CTGCACGCTACTCACCAGGCCC, end 5 labeled FAM, end 3 labeled BHQ1.
B1 F1：GCCTTCAACCCCATCATCT
B1 R1：GTCGTCGTCGTCGTCCGA
B1 P: TTCCAGCGACTGCTCTGCT, 5-end labeled HEX, 3-end labeled BHQ1.
The reaction solution II comprises the following detection primers in proportion: ACE F1: ACE R1=2pmol:20pmol; d6 F1: d6 R1=30pmol:3pmol; b1 F1: b1 R1=2pmol:20pmol; the amounts of the three detection probes added were all 5pmol.
The reaction solution III contains NPPA, AGTR1, CYP3A5, CYP2C9 detection primers and corresponding probes as follows:
R1 F1：TGAGTGACATGTTCGAAACC
R1 R1：CAGCCGTCATCTGTCTAATGC
R1P: CAAATGAGCATTAGCTACTTTTCAGA, end 5 labeled ROX and end 3 labeled BHQ2.
PA F1：GACACAAATGCAGCAGAGAC
PA R1：AGGATGGGCACACTCATAC
PA P: CTGTTATCTTCAGTACTGCAAAGAGAAC, 5-end labeled FAM, 3-end labeled BHQ1.
A5 F1：GAGAGTGGCATAGGAGATAC
A5 R1：CACAGCAAGAGTCTCACACAG
A5 P: TGTCTTTCAATATCTCTTCCCTGTTTG, end 5 is labeled CY5 and end 3 is labeled BHQ3.
C9 F1：ATCAGCTAAAGTCCAGGAA
C9 R1：GAAACAAACTTACCTTGGGAAT
C9 P: CGAGGTCCAGAGATACATTGACC, end 5 labeled HEX, and end 3 labeled BHQ1.
The reaction solution III contains the following detection primers in proportion: R1F 1: r1=4pmol:40pmol; PA F1: PA R1=3pmol:30pmol; a5 F1: a5 R1=2pmol:20pmol; c9 F1: c9 R1=40pmol:4pmol, and the amounts of the four detection probes were 5pmol each.
A method for detecting polymorphism of related genes of accurate medication of hypertension comprises the following detection steps:
1) The reaction solution I was mixed in an amount of 19 ul: 2ul of reaction liquid II is prepared into an amplification detection reagent I; the reaction solution I was measured in 19 ul: 2ul of reaction solution III is prepared into an amplification detection reagent II.
2) Absorbing 4ul of each DNA of a sample to be detected, respectively adding the DNA into the amplification detection reagent I and the amplification detection reagent II, and respectively mixing uniformly.
3) And placing the reaction tube with the DNA-added amplification detection reagent I and the amplification detection reagent II in a real-time fluorescent PCR instrument for PCR amplification detection.
4) The real-time fluorescent PCR amplification program was set up as follows:
Figure BDA0002123565650000051
a method for detecting polymorphism of related genes of accurate medication of hypertension is disclosed, wherein the interpretation of results of 7 genotypes is as follows:
Figure BDA0002123565650000052
Figure BDA0002123565650000061
compared with the prior art, the invention has the following characteristics:
1) The invention provides a kit and a method for detecting polymorphism of related genes of five major antihypertensive drugs, which can effectively guide clinical application of mainstream antihypertensive drugs. Compared with the existing products on the market, the coverage is more complete.
2) The invention adopts a single probe combined with a melting curve method to detect gene polymorphism, and simultaneously adopts two-tube multiplex PCR reaction to detect 7 related gene polymorphisms of the hypertension drug, thereby having the advantages of rapidness, high sensitivity, strong specificity, simple method, accurate detection result and the like.
3) The kit can detect polymorphism of 10pg genome DNA.
Drawings
FIG. 1 shows the result of detecting polymorphism at CYP2D6 x 10 locus.
FIG. 2 shows the result of detecting ADRB1 (1165G > C) site polymorphism.
FIG. 3 shows the detection results of ACE (I/D) site polymorphism.
FIG. 4 shows the result of detecting the polymorphism of NPPA (664G > A) site.
FIG. 5 shows the result of detecting polymorphism at CYP2C9 x 3 site.
FIG. 6 shows the result of detecting polymorphisms of AGTR1 (1166A > C) sites.
FIG. 7 shows the results of detecting polymorphisms at CYP3A5 x 3 locus.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
Example 1: design of primers and probes of hypertension individualized medication related gene polymorphism detection kit
Detection probes and primers are designed according to seven gene related detection sites of ACE (rs 4646994), CYP2D6 (rs 1065852), ADRB1 (rs 1801253), NPPA (rs 5065), AGTR1 (rs 5186), CYP3A5 (rs 776746) and CYP2C9 (rs 56165452), and related sequences are as follows:
ACE F1：TTTCTCTAGACCTGCTGCCTATAC
ACE R1：AGCTCAGAGAATTTCAGAGCTG
ACE P: CTGCTGCCTATACAGTCACTTTTATGTGG, end 5 marked ROX and end 3 marked BHQ2.
D6 F1：CCTCCCTCACCTGGTCGAAGC
D6 R1：TTCCTGCTCCTGGTGGACCTG
D6 P: CTGCACGCTACTCACCAGGCCC, end 5 labeled FAM, end 3 labeled BHQ1.
B1 F1：GCCTTCAACCCCATCATCT
B1 R1：GTCGTCGTCGTCGTCCGA
B1 P: TTCCAGCGACTGCTCTGCT, end 5 labeled HEX, and end 3 labeled BHQ1.
R1 F1：TGAGTGACATGTTCGAAACC
R1 R1：CAGCCGTCATCTGTCTAATGC
R1P: CAAATGAGCATTAGCTACTTTTCAGA, end 5 marked ROX and end 3 marked BHQ2.
PA F1：GACACAAATGCAGCAGAGAC
PA R1：AGGATGGGCACACTCATAC
PA P: CTGTTATCTTCAGTACTGCAAAGAGAAC, 5-end labeled FAM, 3-end labeled BHQ1.
A5 F1：GAGAGTGGCATAGGAGATAC
A5 R1：CACAGCAAGAGTCTCACACAG
A5 P: TGTCTTTCAATATCTCTTCCCTGTTTG, end 5 is labeled CY5 and end 3 is labeled BHQ3.
C9 F1：ATCAGCTAAAGTCCAGGAA
C9 R1：GAAACAAACTTACCTTGGGAAT
C9 P: CGAGGTCCAGAGATACATTGACC, end 5 labeled HEX, and end 3 labeled BHQ1. Example 2: preparation of hypertension individualized medication related gene polymorphism detection kit
The reaction solution I is a mixture containing 10 × Taq buffer I,5 × Taq buffer II, hotstart Taq enzyme, UDG enzyme, dNTP, dUTP and an enhancer, and provides a buffer system for efficient amplification for real-time fluorescent PCR. Wherein the 10 × taq buffer I comprises 200mM Tris-HCl(pH8.4)，200mM KCl，100mM(NH 4 ) 2 SO 4 ，15mM MgCl 2 (ii) a The 5 × taq buffer II comprises 20mM Mg SO 4 The enhancer is a mixture comprising 0.5M betaine, 1% (V/V) formamide, 3% (V/V) glycerol and 0.2mg/ml BSA.
Reaction solution II contains ACE, CYP2D6 and ADRB1 gene detection primers and corresponding fluorescent probes, wherein CE F1: ACE R1=2pmol:20pmol; d6 F1: d6 R1=30pmol:3pmol; b1 F1: b1 R1=2pmol:20pmol; the amount of the three detection probes added was 5pmol each.
The reaction solution III contains NPPA, AGTR1, CYP3A5, CYP2C9 gene detection primers and related fluorescent probes, wherein R1F 1: r1=4pmol:40pmol; PA F1: PA R1=3pmol:30pmol; a5 F1: a5 R1=2pmol:20pmol; c9 F1: c9 R1=40pmol:4pmol, and 5pmol for each of the four detection probes.
The positive control is a hybrid plasmid of seven genes of ACE, CYP2D6, ADRB1, NPPA, AGTR1, CYP3A5 and CYP2C 9.
The negative control is Tris salt without target gene.
Example 3: method for using hypertension individualized medication related gene polymorphism detection kit
1) And extracting the genome DNA in the sample to be detected.
2) The reaction solution I was mixed in an amount of 19 ul: 2ul of reaction liquid II is prepared into an amplification detection reagent I; the reaction solution I was mixed in an amount of 19 ul: 2ul of reaction solution III is prepared into an amplification detection reagent II.
3) And (3) sucking the DNA of the sample to be detected, the positive reference substance and the negative reference substance, respectively adding the DNA, the positive reference substance and the negative reference substance into the amplification detection reagent I and the amplification detection reagent II respectively, and uniformly mixing.
4) And placing the reaction tube of the amplification detection reagent I and the amplification detection reagent II which are added with the DNA into a real-time fluorescent PCR instrument for PCR amplification detection.
Wherein the amplification procedure is as follows:
Figure BDA0002123565650000081
Figure BDA0002123565650000091
interpretation criteria of results:
Figure BDA0002123565650000092
100 clinical blood samples were collected, genomic DNA was extracted using a commercial blood extraction kit, and then the kit of the present invention was used for detecting the relevant gene polymorphisms, and the sanger sequencing method was used for comparison, all of which were consistent.
Sequence listing
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HANGZHOU BOXIN BIOTECHNOLOGY Co.,Ltd.
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<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
cgaggtccag agatacattg acc 23

Claims (2)

1. A polymorphism detection kit for a gene related to accurate administration of hypertension is characterized by comprising a reaction liquid I, a reaction liquid II, a reaction liquid III, a positive control substance and a negative control substance; the reaction solution I comprises a PCR buffer system, a reinforcing agent and an enzyme, the reaction solution II comprises ACE, CYP2D6 and ADRB1 gene detection primers and corresponding fluorescent probes, the reaction solution III comprises NPPA, AGTR1, CYP3A5 and CYP2C9 gene detection primers and corresponding fluorescent probes, the positive control is a heterozygous plasmid of seven genes of ACE, CYP2D6, ADRB1, NPPA, AGTR1, CYP3A5 and CYP2C9, and the negative control is Tris salt without a target gene;
the reaction solution I comprises 10 Xtaq buffer I,5 Xtaq buffer II, hotspot Taq enzyme, UDG enzyme, dNTP, dUTP and an enhancer; the 10 × taq buffer I contains 200mM Tris-HCl,200mM KCl,100mM (NH) with pH8.4 4 ） 2 SO 4 ，15mM MgCl 2 (ii) a The 5 Xtaq buffer II contains 20mM Mg SO 4 The enhancer is a mixture containing 0.5M betaine, 1% (V/V) formamide, 3% (V/V) glycerol and 0.2mg/ml BSA;
the reaction solution II comprises the following detection primers and fluorescent probes:
ACE F1：TTTCTCTAGACCTGCTGCCTATAC
ACE R1：AGCTCAGAGAATTTCAGAGCTG
ACE P: CTGCTGCCTATACAGTCACTTTTATGTGG, end 5 labeled ROX, end 3 labeled BHQ2;
D6 F1：CCTCCCTCACCTGGTCGAAGC
D6 R1：TTCCTGCTCCTGGTGGACCTG
d6 P: CTGCACGCTACTCACCAGGCCC, end 5 labeled FAM, end 3 labeled BHQ1;
B1 F1：GCCTTCAACCCCATCATCT
B1 R1：GTCGTCGTCGTCGTCCGA
b1 P: TTCCAGCGACTGCTCTGCT,5 end labeled HEX,3 end labeled BHQ1;
the reaction solution III comprises the following detection primers and fluorescent probes:
R1 F1：TGAGTGACATGTTCGAAACC
R1 R1：CAGCCGTCATCTGTCTAATGC
R1P: CAAATGAGCATTAGCTACTTTTCAGA, end 5 labeled ROX, end 3 labeled BHQ2;
PA F1：GACACAAATGCAGCAGAGAC
PA R1：AGGATGGGCACACTCATAC
PA P: CTGTTATCTTCAGTACTGCAAAGAGAAC, end 5 labeled FAM, end 3 labeled BHQ1;
A5 F1：GAGAGTGGCATAGGAGATAC
A5 R1：CACAGCAAGAGTCTCACACAG
a5 P: TGTCTTTCAATATCTCTTCCCTGTTTG, end 5 marker CY5, end 3 marker BHQ3;
C9 F1：ATCAGCTAAAGTCCAGGAA
C9 R1：GAAACAAACTTACCTTGGGAAT
c9 P: CGAGGTCCAGAGATACATTGACC, end 5 labeled HEX, and end 3 labeled BHQ1.
2. The kit for detecting the polymorphism of the gene related to the accurate administration of hypertension according to claim 1, wherein the ratio of the detection primers in the reaction solution II is as follows: ACE F1: ACE R1=2pmol:20pmol; d6 F1: d6 R1=30pmol:3pmol; b1 F1: b1 R1=2pmol:20pmol; the addition amount of the three detection probes is 5pmol;
the proportion of the detection primers in the reaction solution III is as follows: R1F 1: r1=4pmol:40pmol; PA F1: PA R1=3pmol:30pmol; a5 F1: a5 R1=2pmol:20pmol; c9 F1: c9 R1=40pmol:4pmol, and 5pmol for each of the four detection probes.
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