CN110863042A - Method for detecting hypertension related gene by using multiple PCR technology - Google Patents
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 39
- 206010020772 Hypertension Diseases 0.000 title claims abstract description 23
- 238000005516 engineering process Methods 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 title claims abstract description 11
- 108010001237 Cytochrome P-450 CYP2D6 Proteins 0.000 claims abstract description 23
- 102100021704 Cytochrome P450 2D6 Human genes 0.000 claims abstract description 23
- 102000017920 ADRB1 Human genes 0.000 claims abstract description 21
- 101000690425 Homo sapiens Type-1 angiotensin II receptor Proteins 0.000 claims abstract description 18
- 238000007403 mPCR Methods 0.000 claims abstract description 18
- 101000892264 Homo sapiens Beta-1 adrenergic receptor Proteins 0.000 claims abstract description 17
- 102000002269 Cytochrome P-450 CYP2C9 Human genes 0.000 claims abstract description 16
- 108010000543 Cytochrome P-450 CYP2C9 Proteins 0.000 claims abstract description 16
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- 102100026803 Type-1 angiotensin II receptor Human genes 0.000 claims abstract description 8
- 238000007480 sanger sequencing Methods 0.000 claims abstract description 5
- 101150072531 10 gene Proteins 0.000 claims abstract description 4
- 101150096290 ADRB1 gene Proteins 0.000 claims abstract description 4
- 101150059573 AGTR1 gene Proteins 0.000 claims abstract description 4
- 101150100998 Ace gene Proteins 0.000 claims abstract description 4
- 238000007400 DNA extraction Methods 0.000 claims abstract description 4
- 238000004458 analytical method Methods 0.000 claims description 5
- 239000003814 drug Substances 0.000 abstract description 18
- 229940079593 drug Drugs 0.000 abstract description 15
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- 239000002299 complementary DNA Substances 0.000 description 13
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- 102000004169 proteins and genes Human genes 0.000 description 5
- 208000024172 Cardiovascular disease Diseases 0.000 description 4
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- 230000000694 effects Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 101150053096 CYP2C9 gene Proteins 0.000 description 1
- 101150010738 CYP2D6 gene Proteins 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 208000031662 Noncommunicable disease Diseases 0.000 description 1
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- 230000036541 health Effects 0.000 description 1
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Abstract
The invention discloses a method for detecting hypertension related genes by using a multiple PCR technology, which comprises the following specific steps: step one, searching CYP2C9 × 3, CYP2D6 × 10, ADRB1, AGTR1 and ACE gene sequences, step two, respectively designing PCR primers of CYP2C9 × 3, CYP2D6 × 10, ADRB1 and AGTR1 genes by using Primer Premier 5 software according to a standard rule of multiplex PCR Primer design, step three, extracting whole blood genome DNA by using a blood/cell/tissue genome DNA extraction kit of TIANGEN, step four, respectively adding the components into a sterile PCR-EP tube in the following sequence in ice bath, carrying out single-tube amplification of CYP2D6 × 10, ADRB1 and AGTR1 genes, step five, carrying out necessary pairing on analytical results after Sanger sequencing, step six, carrying out multiplex PCR system search of CYP2C9 × 3, CYP2D6 × RB 10, ADTR 63 1 and AGTR1 genes. The invention has the beneficial effect of providing a safer, more effective and more economic reasonable medication way for clinic.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a method for detecting hypertension related genes by using a multiplex PCR technology.
Background
Hypertension is a chronic non-infectious disease, and is also a chronic disease with high morbidity, high disability rate and heavy disease burden in China. Data published by the national institutes of health in 2016 show: the prevalence rate of hypertension of adults 18 years old and older in China is 25.2%. Global disease burden studies show: the life-span of the Chinese population (DALY) for adjusting the disability caused by hypertension is up to 3794 ten thousand years, accounting for 12.0 percent of the total DALY and 63.5 percent of the total DALY for cardiovascular diseases; wherein the life-span loss (YLD) of the disabled is 3557 million people, the life-span loss (YLL) of the early time is 236.5 million people, which accounts for 50.1 percent and 64.5 percent of the weight of the cardiovascular diseases YLD and YLL and is the first risk factor of cardiovascular disease burden. The number of premature deaths caused by the increase of blood pressure in China is as high as more than 200 million each year, the direct medical cost reaches 366 hundred million each year, and the blood pressure standard reaching rate of the treated hypertension patients in China is 29.6 percent. Hypertension is taken as the most important risk factor of cardiovascular and cerebrovascular diseases, the prevalence situation is serious, the disability fatality rate of main complications such as stroke, myocardial infarction, heart failure, chronic kidney diseases and the like is high, medical and social resources are seriously consumed, heavy burden is caused to families and society, and the hypertension becomes an important public health problem in China. Despite the great progress made in the diagnosis and treatment of hypertension in recent years, and the number of hypertension treatment medicines is also infinite, there are many unreasonable treatments for hypertension, which also affects the treatment compliance, persistence and blood pressure control rate of patients.
The individual difference of drug response is the main factor of low blood pressure control rate in clinic, and the reasons for the difference are many, for example, various factors such as gene, height, sex, weight and disease condition can cause the difference of the response of different individuals to the drug, however, the genetic factor is the most fundamental factor, and the gene polymorphism of drug metabolism, transport and receptor directly determines the function difference of the encoded protein, so that the difference of drug metabolism and absorption is caused, and the significant difference of human blood drug concentration is caused. The 5 major classes of drugs commonly used in clinical practice at present and the corresponding major gene polymorphisms and functional influences are as follows:
the gene mutations are more likely to be related to the response of the hypertension drug in different individuals, the drug with the same dosage generates obvious difference in different individuals, some individuals are effective, some individuals are ineffective, and even some people generate drug response. Therefore, the effective gene polymorphism detection technology is used for carrying out gene detection on the hypertension patient, the medicine type or the dosage of the patient can be adjusted according to the detection result, the medicine can play the best effect, and meanwhile, the toxic and side effect of the medicine can be prevented. The method provides the best scientific practical argument for individual medical treatment in precise medical treatment and is an ongoing continuous effort for meeting wider individual medical age.
At present, there are many technologies for detecting gene polymorphism, and the most commonly used technologies include polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP method), sequence specificity PCR, first-generation sequencing, second-generation sequencing and the like, but the technologies have respective application defects, some of which have complicated operation and long detection period, and cannot perform high-throughput multi-site simultaneous detection, so that the application of the polymorphism detection technology in clinic is still not ideal at present, and the clinical detection requirements cannot be met all the time.
Generally, PCR only uses one pair of primers, and a nucleic acid fragment is generated through PCR amplification, and is mainly used for identification of a single pathogenic factor and the like. Multiplex PCR (multiplex PCR) is an improved PCR technique based on general PCR, in which multiple pairs of specific primers are added to a PCR reaction system to amplify multiple target fragments for multiple DNA templates or different regions of the same template. The multiplex PCR can amplify a plurality of target genes simultaneously, has the advantages of saving time, reducing cost, improving efficiency and saving precious experimental samples, and is rapidly developed in various fields of life science.
At present, the single-gene amplification of the project is relatively perfect, and the multiplex PCR technology is relatively mature, but the project is not applied to the aspect of gene detection related to hypertension at present. The invention uses multiplex PCR technology to detect hypertension related gene, is used for individual medication, and provides safer, effective and more economic reasonable medication approach for clinic.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for detecting hypertension-related genes by using a multiple PCR technology, which is safer, more effective and more economical in clinic and reasonable in medication path.
The technical scheme adopted by the invention for realizing the purpose is as follows: a method for detecting hypertension related genes by using a multiplex PCR technology is characterized by comprising the following specific steps:
searching CYP2C9 x 3, CYP2D6 x 10, ADRB1, AGTR1 and ACE gene sequences;
secondly, PCR primers of CYP2C9 x 3, CYP2D6 x 10, ADRB1 and AGTR1 genes are respectively designed by using Primer Premier 5 software according to the standard rule of multiplex PCR Primer design;
step three, extracting whole blood genome DNA by using a TIANGEN blood/cell/tissue genome DNA extraction kit;
step four, in ice bath, adding the components into a sterile PCR-EP tube according to the following sequence respectively, and carrying out single-tube amplification on CYP2D6 x 10, ADRB1 and AGTR1 genes;
fifthly, carrying out essential pairing on analysis results after Sanger sequencing;
step six, searching the multiplex PCR system and amplification conditions of CYP2C9 × 3, CYP2D6 × 10, ADRB1, and AGTR1 genes.
The invention has the beneficial effects that: the invention uses multiplex PCR technology to detect the hypertension related gene, is used for individual medication, provides safer, effective and more economic reasonable medication approach for clinic, provides guidance for the medication of the hypertension patient for the clinician, provides a new sensitive, efficient, systematic, economic and simple detection method, and provides research application of the hypertension resistant related gene in the aspect of molecular biology.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The method comprises the following steps of carrying out sequence search on CYP2C9 x 3, CYP2D6 x 10, ADRB1, AGTR1 and ACE gene, wherein the full length of CYP2C9 gene is about 55kb, the gene containing 8 introns and 9 exons codes for a protein with 490 amino acid residues, the CYP2C9 x 3(1075A > C, I359L) site is on exon 7, the gene coding protein containing the mutation site is selected, the gene upstream and downstream sequences of the gene coding for the protein are designed as primer, CYP2D6 gene is located on chromosome 22 (22q13.1), the gene contains 9 exons and 8 introns, the gene is 7kb in total length, the cDNA thereof has 1 bases, the protein consists of 497 amino acids, CYP2D6 (100C > T, P34S) is the main gene mutation type in Chinese, the gene is located on the first exon, the gene sequence containing the mutated site of RB 15242T, the mutated gene containing the mutated gene sequence of RB 11626C 75, the cDNA of the mutated site is designed as a codon of a codon 11624 bp, the cDNA of the cDNA, the cDNA of the cDNA, the cDNA of the cDNA, the cDNA of the cDNA, the cDNA of the cDNA for the cDNA of the;
the gene sequence of CYP2C9 × 3 located on exon 7 is as follows:
the gene sequence of CYP2D6 × 10 located on exon 1 is as follows:
the gene sequence of ADRB1 is as follows:
the gene sequence of the exon where the AGTR1 mutation point is located is as follows:
example 2
PCR primers of CYP2C9 x 3, CYP2D6 x 10, ADRB1 and AGTR1 genes are respectively designed by using Primer Premier 5 software according to the standard rule of multiplex PCR Primer design, the melting temperatures of 4 pairs of primers corresponding to the genes are similar, complementary sequences are avoided, and more than 3 continuous G or C at the 3' end is avoided. Verifying each pair of primers, determining whether the primers can amplify a single primer and the temperature range in which all the primers can act, and redesigning and verifying the primers with larger differences;
CYP2C9*3：Sense:5’-ATGCAAGACAGGAGCCACATG-3’
Anti-Sense:5’-GGGGACTTCGAAAACATGGA-3’
CYP2D6*10：Sense:5’-CCATTTGGTAGTGAGGCAGGTAT-3’
Anti-Sense:5’-CACCATCCATGTTTGCTTCTGGT-3’
ADRB1：Sense:5’-GGCTGGGCTACGCCAACT-3’
Anti-Sense:5’-CGTCGTCGTCGTCCGAGG-3’
AGTR1：Sense:5’-CACCATGTTTTGAGGTTG-3’
Anti-Sense:5’-CGAGTTTCTGACATTGTT-3’
example 3
Extracting whole blood genome DNA by using a TIANGEN blood/cell/tissue genome DNA extraction kit;
example 4
In ice bath, adding the components into a sterile PCR-EP tube in the following order, and carrying out single-tube amplification on CYP2D6 x 10, ADRB1 and AGTR1 genes;
add ddH2O to 50. mu.l
Adjusting the reaction program, and placing the reaction program on a PCR instrument, wherein the PCR reaction parameters are as follows: circulating for 35 times at 94 ℃ for 45s, 52-67 ℃ for 45s and 72 ℃ for 30 s. Finally, the temperature is kept for 5min at 72 ℃.
Taking the PCR product for electrophoresis detection. The 4 pairs of primers corresponding to CYP2C9 x 3, CYP2D6 x 10, ADRB1 and AGTR1 all can obtain a single target band at 52-56 ℃.
Example 5
The analysis result after Sanger sequencing is compared with the result of Beijing and Heijiao medical diagnosis technology GmbH, certainly.
Example 6
And step six, searching a multiplex PCR system of CYP2C9 x 3, CYP2D6 x 10, ADRB1 and AGTR1 genes and amplification conditions, wherein primers of the CYP2D6 x 10, ADRB1 and AGTR1 genes can be placed in a PCR reaction system for multiplex PCR amplification.
The PCR system was as follows:
add ddH2O to 50. mu.l
The reaction program was adjusted and placed on a PCR instrument. The PCR reaction parameters are as follows: the cycle was 35 times at 94 ℃ for 45s, 54 ℃ for 45s and 72 ℃ for 30 s. Finally, the temperature is kept for 5min at 72 ℃. Three target bands, namely CYP2D6 x 10, ADRB1 and AGTR1, can be simultaneously amplified in a PCR system by agarose gel electrophoresis. The analysis result after Sanger sequencing is compared with the single-tube amplification sequencing result of CYP2D6 x 10, ADRB1 and AGTR1 genes.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (1)
1. A method for detecting hypertension related genes by using a multiplex PCR technology is characterized by comprising the following specific steps:
searching CYP2C9 x 3, CYP2D6 x 10, ADRB1, AGTR1 and ACE gene sequences;
secondly, PCR primers of CYP2C9 x 3, CYP2D6 x 10, ADRB1 and AGTR1 genes are respectively designed by using Primer Premier 5 software according to the standard rule of multiplex PCR Primer design;
step three, extracting whole blood genome DNA by using a TIANGEN blood/cell/tissue genome DNA extraction kit;
step four, in ice bath, adding the components into a sterile PCR-EP tube according to the following sequence respectively, and carrying out single-tube amplification on CYP2D6 x 10, ADRB1 and AGTR1 genes;
fifthly, carrying out essential pairing on analysis results after Sanger sequencing;
step six, searching the multiplex PCR system and amplification conditions of CYP2C9 × 3, CYP2D6 × 10, ADRB1, and AGTR1 genes.
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