CN106367479A - Detection composition for guiding hypertension medication, applications of detection composition, kit and detection method - Google Patents

Detection composition for guiding hypertension medication, applications of detection composition, kit and detection method Download PDF

Info

Publication number
CN106367479A
CN106367479A CN201610732578.5A CN201610732578A CN106367479A CN 106367479 A CN106367479 A CN 106367479A CN 201610732578 A CN201610732578 A CN 201610732578A CN 106367479 A CN106367479 A CN 106367479A
Authority
CN
China
Prior art keywords
seq
probe
gene
primer
polymorphism
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201610732578.5A
Other languages
Chinese (zh)
Other versions
CN106367479B (en
Inventor
林源吉
丁佳女
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hangzhou Hundred Biological Ltd By Share Ltd
Original Assignee
Hangzhou Hundred Biological Ltd By Share Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hangzhou Hundred Biological Ltd By Share Ltd filed Critical Hangzhou Hundred Biological Ltd By Share Ltd
Priority to CN201811169467.3A priority Critical patent/CN109706240A/en
Priority to CN201610732578.5A priority patent/CN106367479B/en
Priority to CN201810301466.3A priority patent/CN108690876A/en
Publication of CN106367479A publication Critical patent/CN106367479A/en
Application granted granted Critical
Publication of CN106367479B publication Critical patent/CN106367479B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to a detection composition, a kit and a detection method for guiding hypertension medication. According to the detection composition, the kit and the detection method, the genotypes of CYP2C9*3 (rs1057910), CYP2D6 (rs1065852), CYP3A5*3 (rs776746), ADRB1 (rs1801253), AGTR1 (rs5186), ACE (rs4646994) and NPPA (rs5065) are detected. The genotypes of the abovementioned seven sites are detected by utilizing the detection composition, the kit and the detection method, the method is simple and feasible, as well as rapid and efficient, the cost is low, and a novel path realizing simple and direct judgment is provided for the clinical medication of hypertension.

Description

Detection composition for guiding hypertension medication, application, kit and detection method
Technical Field
The invention relates to the fields of molecular biology and medicine, in particular to a detection composition, a kit and a detection method for guiding hypertension medication related SNP sites.
Background
With the development of social economy and the change of life style of residents, chronic non-infectious diseases (chronic diseases for short) become a great public health problem affecting the health of China and even residents all over the world, and hypertension is one of the chronic diseases with high morbidity and is also the most important risk factor of cardiovascular and cerebrovascular diseases. According to the statistics of the World Health Organization (WHO), 1700 thousands of cardiovascular deaths are found in 2012 in the world, accounting for 46% of chronic deaths, and 940 thousands of hypertension complications are the first risk factors affecting the global disease burden. 2011 world bank report of "create health harmony life and restrain Chinese epidemic of chronic diseases" indicates that: chronic diseases have become the first health threat in china. In about 1030 million dead patients caused by different reasons each year, the proportion of chronic diseases is over 80 percent, the death of cardiovascular and cerebrovascular diseases is the first cause of the chronic diseases, 50 to 75 percent of strokes and 40 to 50 percent of myocardial infarction are related to the rise of blood pressure. In 2010-2040, if the cardiovascular disease death rate can be reduced by 1% every year, the economic benefit is equivalent to 15% of the total domestic production value in 2010 ($ 2.34 trillion) every year, and if the cardiovascular disease death rate is reduced by 3%, the economic benefit reaches 34% of the total domestic production value in 2010 ($ 5.4 trillion) every year. Conversely, if not effective against chronic diseases, these diseases tend to exacerbate the economic and social impact of predictable aging of the population and a reduction in the population of the workforce.
Since the establishment of new China, 4 surveys of the prevalence of hypertension of residents of 15 years old and older, the nutrient and health condition of the residents of China in 2002, 4 field surveys of Chinese chronic diseases and risk factor monitoring in 2004-2013, the nutrient surveys of the residents of China in 2010-2012 and the like, which are respectively developed in 1959, 1979, 1991 and 2002, obtain a large amount of hypertension disease and control data. These data show that the prevalence of hypertension in adults in China is increasing from 5.11% in 1959 to 17.65% in 2002, the newly published reports on nutrition and chronic disease states of residents in China (2015) show that the prevalence of hypertension in residents 18 years old and older in 2012 is 25.2%, males are higher than females, cities are higher than rural areas, and the number of adult hypertension patients in China is estimated to be about 2.6 hundred million; compared with 2002, the prevalence rate of hypertension is obviously increased, and the increase in rural areas is more rapid. But the prevalence rate of adult hypertension in China is only 46.5%, the treatment rate is 41.1%, and the control rate is 13.8%. Meanwhile, hypertension risk factors (such as smoking, excessive drinking, high salt and high fat food intake, lack of activity, overweight, obesity, and high total cholesterol) are ubiquitous in the population and are increasing or persistent, and become potential threats to cardiovascular and cerebrovascular diseases such as hypertension, myocardial infarction, and stroke. The awareness rate, the treatment rate and the control rate of the hypertension in 2011-2012 in the United states reach 82.7%, 75.6% and 51.8% respectively. Compared with developed countries, the number of people suffering from hypertension is large, the awareness rate, treatment rate and control rate of hypertension are improved in recent years, but the awareness rate, treatment rate and control rate of hypertension are still at a low level, regional differences of hypertension control rate are large, and great challenges are brought to the situation of chronic disease prevention and control in China.
In order to effectively control chronic diseases, in 2013, the WHO issued 'global non-infectious disease prevention and control action plans (2013-2020)', and in conjunction with fifteen committees in China, 'Chinese chronic disease prevention and control work plans (20152012-2015)', aiming at controlling the growth of risk factors of chronic diseases, inhibiting or reducing the incidence and death rate of the chronic diseases and reducing the disease burden caused by the chronic diseases through cooperation of multiple fields and multiple departments. Therefore, in order to strengthen the prevention and treatment work of hypertension of residents in China, multiple departments should participate in making relevant policies, such as making price reduction of low sodium salt, food salt addition amount limitation, increase of physical exercise facilities, environment improvement and the like, advocating a healthy life style of the whole population and reducing the prevalence level of hypertension risk factors; the method has the advantages that the screening of the hypertension is strengthened through the first diagnosis of blood pressure and the health examination of residents by medical institutions, and the awareness rate of the hypertension of the residents is improved, so that early discovery, early diagnosis and early treatment are facilitated; the guiding function of a large hospital on a basic medical institution is fully exerted in the aspect of drug treatment, the treatment process of the hypertension drug is standardized, the number and the technical level of professional technicians of the basic health service institution are continuously improved, meanwhile, the health management and the standardized treatment of hypertension patients in basic public health service are further enhanced, and the reasonable medication level of the hypertension and the control rate of the hypertension are improved.
The antihypertensive drugs commonly used in clinical practice at present are of 5 types: diuretics, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, beta-blockers, calcium antagonists. The drug is related to a plurality of drug metabolizing enzymes, transporters and receptors in the in vivo metabolic process, and the existence of gene polymorphism of the drugs finally causes obvious individual difference in the treatment effect, the generated poor development, the drug tolerance and the like of patients taking the same drug in the same way.
At present, hypertension research based on pharmacogenomics mainly focuses on the relationship between gene diversity and drug antihypertensive effect, the influence of gene difference caused by different race, age, sex, diet and living environment on drug antihypertensive effect, and the like, and less focuses on the long-term influence of gene mutation on drug antihypertensive effect. The future research of the hypertension pharmacogenomics group is a multidisciplinary, including the system research engineering of the joint collaboration of molecular biology, clinical medicine, genetics, mathematics, sociology and the like, not only pays attention to the relationship between genes and physiology and pathology, but also pays attention to the relationship between genes and environment, humanity and society, and provides basis for the final prevention and treatment of diseases such as hypertension and the like.
Due to the interaction of various factors such as genetic difference of different individuals, race, age, sex, diet and living environment, the susceptibility of different individuals and groups to hypertension and the reaction of antihypertensive drugs show different effects in terms of sex, race and age. The treatment for hypertension should be prevention and cure. With the coming of the age of gene control, the gene test is carried out to carry out the early prevention of different individuals for different susceptible people, and the gene test and the evaluation of comprehensive factors are carried out to provide suitable individualized treatment for the susceptible people, so that the administration of the susceptible people is safe, effective and economical.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a detection composition for guiding hypertension medication, which comprises reagents for respectively detecting polymorphism of CYP2C9 x 3 gene, CYP2D6 gene, CYP3A5 x 3 gene, ADRB1 gene, AGTR1 gene, ACE gene and NPPA gene.
Furthermore, the polymorphisms of the rs1057910 site of the CYP2C9 x 3 gene, the rs1065852 site of the CYP2D6 gene, the rs776746 site of the CYP3a5 x 3 gene, the rs1801253 site of the ADRB1 gene, the rs5186 site of the AGTR1 gene, the rs 464646994 site of the ACE gene, and the rs5065 site of the NPPA gene were detected.
The reagents for detecting the above gene polymorphisms can be selected from different reagents as detection compositions according to different detection principles and methods. One type of the reagent of the detection composition comprises an amplification primer and a probe designed based on the gene polymorphic site, wherein the probe is two probes of a wild type and a mutant type designed respectively for the gene polymorphic site.
The detection composition is applied to a detection kit for guiding hypertension medication, wherein the composition comprises reagents for detecting polymorphism of CYP2C9 x 3 gene, CYP2D6 gene, CYP3A5 x 3 gene, ADRB1 gene, AGTR1 gene, ACE gene and NPPA gene.
The invention also provides a detection kit for guiding hypertension medication, which is convenient and fast to operate and comprises reagents for respectively detecting the polymorphism of the CYP2C 9X 3 gene, the CYP2D6 gene, the CYP3A 5X 3 gene, the ADRB1 gene, the AGTR1 gene, the ACE gene and the NPPA gene.
The above-mentioned amplification primers and probes are respectively selected from:
primer and probe sequences for amplifying CYP2C9 x 3(rs 1057910):
CYP2C9 x 3(rs1057910) upstream primer: ATTTAATGTCACAGGTCACTGC (SEQ NO1)
CYP2C9 x 3(rs1057910) downstream primer: CACATGCCCTACACAGAT (SEQ NO2)
1, probe 1: AGATACATTGACCTT (SEQ NO16)
And (3) probe 2: AGATACCTTGACCTT (SEQ NO17)
Primer and probe sequences for amplifying CYP2D6(rs 1065852):
CYP2D6(rs1065852) upstream primer: TGCTCCTGGTGGACCTGA (SEQ NO3)
CYP2D6(rs1065852) downstream primer: AGTCCACATGCAGCAGGT (SEQ NO4)
And 3, probe 3: CGCTACTCACCAG (SEQ NO18)
And 4, probe 4: CGCTACCCACCAG (SEQ NO19)
Primer and probe sequences for amplifying CYP3a5 x 3(rs 776746):
CYP3a5 x 3(rs776746) upstream primer: AGCTTAACGAATGCTCTAC (SEQ NO5)
CYP3a5 × 3(rs776746) downstream primer: AGCAAGAGTCTCACACAGG (SEQ NO6)
And 5, probe: CTTTCAATATCTCTT (SEQ NO20)
And 6, probe 6: CTTTCAGTATCTCTT (SEQ NO21)
Primer and probe sequences for amplification of ADRB1(rs 1801253):
ADRB1(rs1801253) upstream primer: CCTTCAACCCCATCATCT (SEQ NO7)
ADRB1(rs1801253) downstream primer: GGTCTCCGTGGGTCGCG (SEQ NO8)
And (7) probe: TTCCAGGGACTGC (SEQ NO22)
And (3) probe 8: TTCCAGCGACTGC (SEQ NO23)
Primer and probe sequences for amplification of AGTR1(rs 5186):
AGTR1(rs5186) upstream primer: CATTCCTCTGCAGCACTTCACT (SEQ NO9)
AGTR1(rs5186) downstream primer: CGGTTCAGTCCACATAATGCAT (SEQ NO10)
And (3) probe 9: AATGAGCATTAGCTAC (SEQ NO24)
A probe 10: AATGAGCCTTAGCTAC (SEQ NO25)
Primer and probe sequences for amplifying ACE (rs 4646994):
ACE (rs4646994) upstream primer: TTTCTCCCATTTCTCTAGACCT (SEQ NO11)
ACE (rs4646994) downstream primer 1: ATCCCGCCACTGCACT (SEQ NO12)
ACE (rs4646994) downstream primer 2: TCAGAGAATTTCAGAGCTG (SEQ NO13)
A probe 11: CGCTCTGTCGCCCAGGC (SEQ NO26)
The probe 12: CTATACAGTCACTTTTATGTGGTTT (SEQ NO27)
Primer and probe sequences for amplification of NPPA (rs 5065):
NPPA (rs5065) upstream primer: CCAGCCCTGCTTGT (SEQ NO14)
NPPA (rs5065) downstream primer: AGGATGGGCACACTCAT (SEQ NO 15).
And (3) probe 13: TTATCTTCAGTACTG (SEQ NO28)
The probe 14: TTATCTTCGGTACTG (SEQ NO 29).
The invention also provides a detection method for guiding hypertension medication, which comprises the following steps:
(a) extracting genomic DNA in a sample;
(b) amplifying and detecting polymorphism of rs1057910 site of CYP2C9 x 3 gene, polymorphism of rs1065852 site of CYP2D6 gene, polymorphism of rs776746 site of CYP3A5 x 3 gene, polymorphism of rs1801253 site of ADRB1 gene, polymorphism of rs5186 site of AGTR1 gene, polymorphism of rs4646994 site of ACE gene and polymorphism of rs5065 site of NPPA gene.
The hypertension medicine of the present invention includes diuretic, angiotensin converting enzyme inhibitor, angiotensin receptor blocker, beta receptor blocker and calcium ion antagonist.
The technical scheme of the invention has the following advantages: the invention contains the analysis of gene polymorphism sites related to five major hypertension drugs, and can effectively guide the clinical use of the mainstream antihypertensive drugs (the five major drugs are respectively diuretic, angiotensin converting enzyme inhibitor, angiotensin receptor blocker, beta receptor blocker and calcium ion antagonist) through the detection and analysis of 7 site polymorphism, and the coverage type is wider than that of similar products on the market. And the double-probe design of a wild probe and a mutant probe with high specificity is adopted during detection, so that the use of an internal reference primer and a probe required by the current fluorescent quantitative PCR detection is avoided. This not only enhances the specificity of the assay, but also reduces the operational errors and reduces the cost of the assay. After the Taqman probe is adopted, the detection specificity is greatly improved, and the detection time is effectively shortened from the original 2.5-3 hours to about 1 hour, so that the accuracy of a detection result and the detection efficiency are greatly improved.
Drawings
FIG. 1 Primary sequencing results for ADRB1(rs1801253) wild type (GG).
FIG. 2 typing results of an embodiment of the present invention of ADRB1(rs1801253) wild type (GG).
FIG. 3 Primary sequencing results for ADRB1(rs1801253) mutant heterozygotes (GC).
FIG. 4 typing results of an embodiment of the present invention of ADRB1(rs1801253) mutant heterozygotes (GC).
FIG. 5 results of one-generation sequencing of ADRB1(rs1801253) mutant homozygotes (CCs).
FIG. 6 typing results of an embodiment of the present invention wherein ADRB1(rs1801253) mutations are homozygous (CC).
Detailed Description
The inventor screens 7 polymorphic sites of genes closely related to five major antihypertensive drugs by analyzing the use, metabolism and individual difference of drug effect of common drugs of patients with clinical hypertension, prepares a composition for detecting the 7 polymorphic sites and a detection kit using the composition, and establishes an accurate detection method for realizing effective medication guidance for the five major antihypertensive drugs. The five major antihypertensive drugs are diuretics, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, beta receptor blockers and calcium antagonists. The 7 polymorphic sites (specific RS numbers) aiming at the five major antihypertensive drugs are CYP2C9 x 3(RS1057910), CYP2D6(RS1065852), CYP3A5 x 3(RS776746), ADRB1(RS1801253), AGTR1(RS5186), ACE (RS 46994) and NPPA (RS 5065). The curative effect and the risk of hypertension medication of the 7-site genotype mutation individual are different from those of the common population.
The rs1057910 site of CYP2C9 x 3 gene, rs1065852 site of CYP2D6 gene, rs776746 site of CYP3a5 x 3 gene, rs1801253 site of ADRB1 gene, rs5186 site of AGTR1 gene, rs4646994 site of ACE gene, and rs5065 site of NPPA gene may be respectively expressed as CYP2C9 x 3(rs1057910), CYP2D6(rs1065852), CYP3a5 x 3(rs776746), ADRB1(rs1801253), AGTR1(rs5186), ACE (rs 46994), and NPPA (rs 5065).
Example 1 detection kit Using fluorescent quantitative PCR detection System
The detection kit comprises an amplification primer, a probe and other matched reagents for carrying out fluorescent quantitative PCR detection.
Amplification primers and probes were designed against 7 polymorphic sites CYP2C9 x 3(rs1057910), CYP2D6(rs1065852), CYP3a5 x 3(rs776746), ADRB1(rs1801253), AGTR1(rs5186), ACE (rs 46994) and NPPA (rs 5065).
First, design and synthesis of amplification primer
Based on the region of the rs1057910 site in CYP2C9 x 3 gene, the region of the rs1065852 site in CYP2D6 gene, the region of the rs776746 site in CYP3A5 x 3 gene, the region of the rs1801253 site in ADRB1 gene, the region of the rs5186 site in AGTR1 gene, the region of the rs4646994 site in ACE gene and the region of the rs5065 site in NPPA gene, an amplification primer is designed, wherein,
primer sequences for amplifying CYP2C9 x 3(rs 1057910):
CYP2C9 x 3(rs1057910) upstream primer: ATTTAATGTCACAGGTCACTGC (SEQ NO1)
CYP2C9 x 3(rs1057910) downstream primer: CACATGCCCTACACAGAT (SEQ NO2)
Primer sequences for amplifying CYP2D6(rs 1065852):
CYP2D6(rs1065852) upstream primer: TGCTCCTGGTGGACCTGA (SEQ NO3)
CYP2D6(rs1065852) downstream primer: AGTCCACATGCAGCAGGT (SEQ NO4)
Primer sequences for amplifying CYP3a5 x 3(rs 776746):
CYP3a5 x 3(rs776746) upstream primer: AGCTTAACGAATGCTCTAC (SEQ NO5)
CYP3a5 × 3(rs776746) downstream primer: AGCAAGAGTCTCACACAGG (SEQ NO6)
Primer sequence for amplification of ADRB1(rs 1801253):
ADRB1(rs1801253) upstream primer: CCTTCAACCCCATCATCT (SEQ NO7)
ADRB1(rs1801253) downstream primer: GGTCTCCGTGGGTCGCG (SEQ NO8)
Primer sequence for amplification of AGTR1(rs 5186):
AGTR1(rs5186) upstream primer: CATTCCTCTGCAGCACTTCACT (SEQ NO9)
AGTR1(rs5186) downstream primer: CGGTTCAGTCCACATAATGCAT (SEQ NO10)
Primer sequence for amplifying ACE (rs 4646994):
ACE (rs4646994) upstream primer: TTTCTCCCATTTCTCTAGACCT (SEQ NO11)
ACE (rs4646994) downstream primer 1: ATCCCGCCACTGCACT (SEQ NO12)
ACE (rs4646994) downstream primer 2: TCAGAGAATTTCAGAGCTG (SEQ NO13)
Primer sequence for amplification of NPPA (rs 5065):
NPPA (rs5065) upstream primer: CCAGCCCTGCTTGT (SEQ NO14)
NPPA (rs5065) downstream primer: AGGATGGGCACACTCAT (SEQ NO15)
Since the polymorphism of ACE (rs4646994) is not single point mutation but insertion/deletion mutation, in order to ensure the amplification effectiveness, the invention designs an upstream primer and two downstream primers. Experiments show that the primers of SEQ NO.11 to 13 and the probes of SEQ NO.26 to 27 can be used for detecting all ACE (rs4646994) polymorphism, the amplification efficiency is high, and the specificity and the accuracy are good.
II, designing and synthesizing a probe
Based on the characteristics of CYP2C9 x 3(rs1057910), CYP2D6(rs1065852), CYP3A5 x 3(rs776746), ADRB1(rs1801253), AGTR1(rs5186), ACE (rs 46994) and NPPA (rs5065) locus gene polymorphism, a high-specificity probe for fluorescent quantitative PCR analysis is designed, and the probe is a wild-type probe and a mutant-type probe which are respectively designed for gene polymorphism loci. Wherein,
the probe sequence for detecting CYP2C9 x 3(rs1057910) polymorphism comprises:
1, probe 1: AGATACATTGACCTT (SEQ NO16)
And (3) probe 2: AGATACCTTGACCTT (SEQ NO17)
The probe sequence for detecting CYP2D6(rs1065852) polymorphism comprises:
and 3, probe 3: CGCTACTCACCAG (SEQ NO18)
And 4, probe 4: CGCTACCCACCAG (SEQ NO19)
The probe sequence for detecting the CYP3A5 x 3(rs776746) polymorphism comprises:
and 5, probe: CTTTCAATATCTCTT (SEQ NO20)
And 6, probe 6: CTTTCAGTATCTCTT (SEQ NO21)
The probe sequence for detecting ADRB1(rs1801253) polymorphism comprises:
and (7) probe: TTCCAGGGACTGC (SEQ NO22)
And (3) probe 8: TTCCAGCGACTGC (SEQ NO23)
The probe sequence for detecting AGTR1(rs5186) polymorphism comprises:
and (3) probe 9: AATGAGCATTAGCTAC (SEQ NO24)
A probe 10: AATGAGCCTTAGCTAC (SEQ NO25)
The probe sequence for detecting ACE (rs4646994) polymorphism comprises:
a probe 11: CGCTCTGTCGCCCAGGC (SEQ NO26)
The probe 12: CTATACAGTCACTTTTATGTGGTTT (SEQ NO27)
The probe sequence for detecting NPPA (rs5065) polymorphism comprises:
and (3) probe 13: TTATCTTCAGTACTG (SEQ NO28)
The probe 14: TTATCTTCGGTACTG (SEQ NO 29).
In one embodiment, the probes are TaqMan probes, each probe having a fluorescent reporter group at the 5 'end and a quencher group at the 3' end. Wherein the fluorescent reporter group can be selected from FAM, TET, VIC, JOE, HEX, Cy3, Cy3.5, Cy5, Cy5.5, TAMRA, ROX, Texas Red, LC RED640, LC RED705 and the like; the quenching group can be selected from MGB, BHQ0, BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, NFQ and the like.
Third, PCR amplification reaction reagent
The PCR amplification reaction reagent comprises the amplification primer and the probe, and also comprises other matched reagents required by a PCR amplification reaction system.
The other matched reagents comprise PCR Buffer and Mg2+dNTP and Taq enzyme. In a more preferred embodiment, the other kit further comprises DMSO and ROX. With respect to the choice of ROX, not all instruments or experiments need to have ROX added, depending on the fluorescent quantitative PCR instrument used in the experiment and the needs of the experiment. ROX is chosen to correct for background signal, so that it is not necessary to correct for background signal, but other background signal correcting functions or reagents may be included, or the design of the experimentIn all cases, the background signal correction may be performed without using ROX.
In one specific example of a PCR amplification reaction, a quantitative fluorescent PCR assay was performed using the reagents and amounts described in Table 1:
table 1:
components Dosage of
10×buffer(Mg2+) 2μl
MgCl2(25mM) 0-5μl
dNTP(10mM) 0.2-1μl
rTaq enzyme (5U/ul) 0.1-0.5μl
Upstream primer (10uM) 0.2-1μl
Downstream primer (10uM) 0.2-1μl
Probe 1 or 3 or 5 or 7 or 9 or 11 or 13(10uM) 0.2-1μl
Probe 2 or 4 or 6 or 8 or 10 or 12 or 14(10uM) 0.2-1μl
DMSO 0-2μl
ROX 0.05-1μl
Template 1 μ l of genomic DNA
ddH2O Sample addition to 20ul
In a preferred embodiment, the MgCl is2The amount of (25mM) used is 3 μ l, the amount of dNTP (10mM) used is 0.5 μ l, the amount of rTaq enzyme (5U/ul) used is 0.1 μ l, the amount of upstream primer (10uM) used is 0.5 μ l, the amount of downstream primer (10uM) used is 0.5 μ l, the amount of probe 1 or 3 or 5 or 7 or 9 or 11 or 13(10uM) used is 0.5 μ l, the amount of probe 2 or 4 or 6 or 8 or 10 or 12 or 14(10uM) (10uM) used is 0.5 μ l, the amount of DMSO used is 0.5 μ l, and the amount of ROX used is 0.1 μ l.
In a fluorescent quantitative PCR detection system, the reagent in the detection composition for guiding the hypertension drug comprises the primer and the probe and is used for detecting the polymorphism of CYP2C9 gene, CYP2D6 gene, CYP3A5 gene, ADPB1 gene, AGTR1 gene, ACE gene and NPPA gene. Furthermore, other reagents required by the PCR amplification reaction system can be included.
Example 2 fluorescent quantitative PCR detection method
The fluorescent quantitative PCR detection method for detecting the 7 polymorphic sites comprises the following steps:
(a) extracting genomic DNA in a sample;
(b) amplifying and detecting the gene polymorphism of a region containing the rs1057910 site in the CYP2C9 x 3 gene, the gene polymorphism of a region containing the rs1065852 site in the CYP2D6 gene, the gene polymorphism of a region containing the rs776746 site in the CYP3A5 x 3 gene, the gene polymorphism of a region containing the rs1801253 site in the ADRB1 gene, the gene polymorphism of a region containing the rs5186 site in the AGTR1 gene, the gene polymorphism of a region containing the rs 46994 site in the ACE gene, and the gene polymorphism of a region containing the rs5065 site in the NPPA gene.
The method for extracting the genome DNA comprises the following steps: traditional phenol chloroform extraction, a commercialized silica gel membrane extraction kit and a commercialized magnetic bead extraction kit. The extracted genomic DNA can satisfy the following conditions: OD260/280 is 1.7-2.0; the DNA concentration is less than or equal to 5 ng/mul and less than or equal to 100 ng/mul.
Wherein, the reaction conditions of the fluorescent quantitative PCR amplification are shown in the table 2:
TABLE 2
In example 1 and example 2, a two-probe detection system without reference gene detection is adopted, and primers and probes for reference gene detection are not included. The operation in the experimental process is reduced, and experimental errors caused by excessive operation are avoided; meanwhile, the system without internal reference also reduces the experiment cost.
In another embodiment, the amplification primers and probes described in example 1 are also suitable for use in a detection system comprising an internal reference gene.
Example 3 analysis of clinical samples Using fluorescent quantitative PCR
150 clinical saliva samples and 150 clinical blood samples were collected, genomic DNA was extracted using a commercially available magnetic bead extraction kit, and 7 genes were then typed and tested using the methods of examples 1 and 2. Meanwhile, the first-generation sequencing method is used for carrying out genotyping detection on the extracted DNA, and the genotyping result of the method is compared with the genotyping result of the first-generation sequencing. Wherein, the comparative test results of CYP2C9 x 3(rs1057910) are shown in table 3; the results of comparative testing of CYP2D6(rs1065852) are shown in table 4; comparative test results for CYP3a5 x 3(rs776746) are shown in table 5; comparative test results for ADRB1(rs1801253) are shown in table 6; comparative test results for AGTR1(rs5186) are shown in table 7; comparative test results for ACE (rs4646994) are shown in table 8; the results of comparative testing of NPPA (rs5065) are shown in table 9.
Table 3: comparative test results for CYP2C9 x 3(rs1057910)
Table 4: comparative test results for CYP2D6(rs1065852)
Table 5: comparative test results for CYP3a5 x 3(rs776746)
Table 6: comparative test results for ADRB1(rs1801253)
Table 7: comparative test results for AGTR1(rs5186)
Table 8: results of comparative tests for ACE (rs4646994) (I for insertion and D for deletion)
Table 9: comparative test results for NPPA (rs5065)
Referring to FIGS. 1 to 6, the results of typing and first-generation sequencing of ADRB1(rs1801253) gene measured by the method of the present invention were completely identical by comparing the detection profiles of the two methods. Wherein the comparison of ADRB1(rs1801253) gene wild type (GG) is shown in FIG. 1 and FIG. 2; the results of comparison of ADRB1(rs1801253) gene mutation heterozygotes (GC) are shown in FIGS. 3 and 4; the results of mutation homozygote (CC) of ADRB1(rs1801253) gene are shown in fig. 5 and 6. Similarly, the typing test patterns of CYP2C9 x 3(rs1057910), CYP2D6(rs1065852), CYP3A5 x 3(rs776746), AGTR1(rs5186), ACE (rs4646994) and NPPA (rs5065) genes determined by the method of the present invention are compared with the first-generation sequencing test pattern, and the results are also completely consistent.
For the fluorescent quantitative PCR typing experiment, an internal reference gene is generally adopted to indicate the normality of a reaction system, so that the typing result is ensured to be credible. Namely: the PCR typing result is effective only under the premise that the internal reference gene amplification is normal. Therefore, the use of reference genes is an indispensable design for general PCR typing. The invention adopts the design of a double-probe system, not only can complete all typing tests in one reaction tube, but also avoids the use of internal reference genes, and can judge whether the PCR typing result is effective or not by utilizing the detection genes. The judging method comprises the following steps: when only the fluorescence signal of the wild type is present, the sample is indicated to be wild type. When only the fluorescence signal of the mutant type appears, the sample is indicated as the homozygous mutant type. When the wild type and mutant signals are simultaneously present, the sample is indicated to be heterozygous mutant. When no fluorescence signal is present, this detection fails. Therefore, the method not only can reduce experiment operation, but also can reduce experiment cost.
EXAMPLE 4 specificity of fluorescent quantitative PCR Dual Probe System
The key to the success of the two-probe system is the specificity of the probes. In the invention, a large number of target gene sequences are compared, a region including an rs region and having higher conservatism is selected for primer design, and then specific bases are selected to respectively design TaqMan probes aiming at a wild type and a mutant type and respectively mark fluorescent groups, such as a wild type marked FAM fluorescent group and a mutant type marked VIC fluorescent group. The probe designed by the invention has extremely high specificity, not only can play a good role in typing in a double-probe system, but also can control the Ct value within 33. In the current commercial products, for the reagent for fluorescent quantitative PCR typing, not only a single probe system with complex operation and high cost is used, but also the Ct value is generally controlled within 35. Therefore, compared with the existing detection system, the double-probe system has good specificity and can realize high-efficiency typing.
Example 5 high sensitivity of fluorescent quantitative PCR detection System
The sensitivity of the PCR detection system is mainly characterized by the requirement of the system on the amplification starting template amount, and the higher the sensitivity is, the PCR system can obtain an amplification signal at a lower template (genomic DNA in the invention). Generally, commercial products require that the initial amount of genomic DNA be controlled to 10ng or more, whereas the present invention can reduce the initial amount of genomic DNA to 5 ng. The method of examples 1 and 2 was used to detect genomic DNA with a template amount of 5ng, and as a control, the method was used to detect genomic DNA from the same sample by one-generation sequencing, and comparative experiments showed that when the template amount of genomic DNA was 5ng, the detection result was consistent with the one-generation sequencing result. Therefore, the primers, the probes and the detection method are very beneficial to detection in some trace samples (such as serum/plasma DNA).
Example 6 accurate medication guidance for five major classes of hypertension drugs
After analyzing the precise medication guidance of hypertension, the current commercial products mainly guide the medication of three major drugs through five sites, wherein the three major drugs comprise angiotensin II receptor blockers (such as losartan and irbesartan), beta 1 adrenergic receptor blockers (such as metoprolol and carvedilol), and angiotensin converting enzyme inhibitors (such as enalapril and fosinopril). However, in the guidelines for hypertension issued by the national institute of health and computing committee, two main classes of antihypertensive drugs, in addition to the three classes of antihypertensive drugs, are the mainstream drugs, namely diuretics and calcium ion antagonists. Through various researches and analyses, the invention creatively designs the medicine which can guide the accurate medication of five main classes of hypertension main medicines through the detection and analysis of 7 sites (including CYP2C9 x 3(rs1057910), CYP2D6(rs1065852), CYP3A5 x 3(rs776746), ADRB1(rs1801253), AGTR1(rs5186), ACE (rs4646994) and NPPA (rs 5065)). The medicine not only can comprehensively cover five main medicines of hypertension, but also can play a guiding role in the combined medication of hypertension. The details of the 7-site directed five major classes of hypertensive drugs of the present invention are shown in tables 10 to 14:
(1) diuretic table 10
(2) Angiotensin inhibitor invertase table 11
(3) Angiotensin II receptor blockers table 12
(4) Beta 1 adrenoceptor blockers table 13
(5) Calcium ion antagonists table 14
Example 7 other methods for testing the accurate administration of five major classes of hypertension drugs
Through experimental analysis, the detection composition for guiding the hypertension medication is respectively prepared by aiming at a fluorescent quantitative PCR non-double probe system, a first-generation sequencing method, pyrosequencing, PCR-HRM, arms-PCR, Restriction Fragment Length Polymorphism (RFLP), In Situ Hybridization (ISH), a gene chip or high-throughput sequencing method and the like, and reagents in the composition comprise reagents required by the corresponding methods. The reagents are used for analyzing the polymorphism of CYP2C9 x 3 gene, CYP2D6 gene, CYP3A5 x 3 gene, ADRB1 gene, AGTR1 gene, ACE gene and NPPA gene, and the obtained detection result can provide reference information for the medication guidance of hypertension.

Claims (10)

1. A test composition for hypertension-guiding drugs, comprising a reagent for detecting a CYP2C9 x 3 gene polymorphism, a CYP2D6 gene polymorphism, a CYP3A5 x 3 gene polymorphism, an ADRB1 gene polymorphism, an AGTR1 gene polymorphism, an ACE gene polymorphism and an NPPA gene polymorphism.
2. The assay composition of claim 1, wherein the polymorphisms are detected at the rs1057910 site of the CYP2C9 x 3 gene, the rs1065852 site of the CYP2D6 gene, the rs776746 site of the CYP3a5 x 3 gene, the rs1801253 site of the ADRB1 gene, the rs5186 site of the AGTR1 gene, the rs4646994 site of the ACE gene, and the rs5065 site of the NPPA gene.
3. The detection composition according to any one of claims 1 to 2, wherein the reagents comprise amplification primers, and two probes of a wild type and a mutant type designed for the polymorphic sites of the gene, respectively.
4. The detection composition according to claim 3,
the probe sequence for detecting CYP2C9 x 3(rs1057910) polymorphism comprises:
1, probe 1: AGATACATTGACCTT (SEQ NO16)
And (3) probe 2: AGATACCTTGACCTT (SEQ NO17)
The probe sequence for detecting CYP2D6(rs1065852) polymorphism comprises:
and 3, probe 3: CGCTACTCACCAG (SEQ NO18)
And 4, probe 4: CGCTACCCACCAG (SEQ NO19)
The probe sequence for detecting the CYP3A5 x 3(rs776746) polymorphism comprises:
and 5, probe: CTTTCAATATCTCTT (SEQ NO20)
And 6, probe 6: CTTTCAGTATCTCTT (SEQ NO21)
The probe sequence for detecting ADRB1(rs1801253) polymorphism comprises:
and (7) probe: TTCCAGGGACTGC (SEQ NO22)
And (3) probe 8: TTCCAGCGACTGC (SEQ NO23)
The probe sequence for detecting AGTR1(rs5186) polymorphism comprises:
and (3) probe 9: AATGAGCATTAGCTAC (SEQ NO24)
A probe 10: AATGAGCCTTAGCTAC (SEQ NO25)
The probe sequence for detecting ACE (rs4646994) polymorphism comprises:
a probe 11: CGCTCTGTCGCCCAGGC (SEQ NO26)
The probe 12: CTATACAGTCACTTTTATGTGGTTT (SEQ NO27)
The probe sequence for detecting NPPA (rs5065) polymorphism comprises:
and (3) probe 13: TTATCTTCAGTACTG (SEQ NO28)
The probe 14: TTATCTTCGGTACTG (SEQ NO 29).
5. The assay composition of claim 1,
primer sequences for amplifying CYP2C9 x 3(rs 1057910):
CYP2C9 x 3(rs1057910) upstream primer: ATTTAATGTCACAGGTCACTGC (SEQ NO1)
CYP2C9 x 3(rs1057910) downstream primer: CACATGCCCTACACAGAT (SEQ NO2)
Primer sequences for amplifying CYP2D6(rs 1065852):
CYP2D6(rs1065852) upstream primer: TGCTCCTGGTGGACCTGA (SEQ NO3)
CYP2D6(rs1065852) downstream primer: AGTCCACATGCAGCAGGT (SEQ NO4)
Primer sequences for amplifying CYP3a5 x 3(rs 776746):
CYP3a5 x 3(rs776746) upstream primer: AGCTTAACGAATGCTCTAC (SEQ NO5)
CYP3a5 × 3(rs776746) downstream primer: AGCAAGAGTCTCACACAGG (SEQ NO6)
Primer sequence for amplification of ADRB1(rs 1801253):
ADRB1(rs1801253) upstream primer: CCTTCAACCCCATCATCT (SEQ NO7)
ADRB1(rs1801253) downstream primer: GGTCTCCGTGGGTCGCG (SEQ NO8)
Primer sequence for amplification of AGTR1(rs 5186):
AGTR1(rs5186) upstream primer: CATTCCTCTGCAGCACTTCACT (SEQ NO9)
AGTR1(rs5186) downstream primer: CGGTTCAGTCCACATAATGCAT (SEQ NO10)
Primer sequence for amplifying ACE (rs 4646994):
ACE (rs4646994) upstream primer: TTTCTCCCATTTCTCTAGACCT (SEQ NO11)
ACE (rs4646994) downstream primer 1: ATCCCGCCACTGCACT (SEQ NO12)
ACE (rs4646994) downstream primer 2: TCAGAGAATTTCAGAGCTG (SEQ NO13)
Primer sequence for amplification of NPPA (rs 5065):
NPPA (rs5065) upstream primer: CCAGCCCTGCTTGT (SEQ NO14)
NPPA (rs5065) downstream primer: AGGATGGGCACACTCAT (SEQ NO 15).
6. Use of the test composition according to any one of claims 1 to 5 in a test kit for the administration of a medicament for the treatment of hypertension.
7. A detection kit for guiding hypertension medication comprises reagents for detecting CYP2C9 x 3 gene polymorphism, CYP2D6 gene polymorphism, CYP3A5 x 3 gene polymorphism, ADRB1 gene polymorphism, AGTR1 gene polymorphism, ACE gene polymorphism and NPPA gene polymorphism.
8. The test kit of claim 7, wherein the reagents comprise amplification primers and probes, wherein
Primer and probe sequences for amplifying CYP2C9 x 3(rs 1057910):
CYP2C9 x 3(rs1057910) upstream primer: ATTTAATGTCACAGGTCACTGC (SEQ NO1)
CYP2C9 x 3(rs1057910) downstream primer: CACATGCCCTACACAGAT (SEQ NO2)
1, probe 1: AGATACATTGACCTT (SEQ NO16)
And (3) probe 2: AGATACCTTGACCTT (SEQ NO17)
Primer and probe sequences for amplifying CYP2D6(rs 1065852):
CYP2D6(rs1065852) upstream primer: TGCTCCTGGTGGACCTGA (SEQ NO3)
CYP2D6(rs1065852) downstream primer: AGTCCACATGCAGCAGGT (SEQ NO4)
And 3, probe 3: CGCTACTCACCAG (SEQ NO18)
And 4, probe 4: CGCTACCCACCAG (SEQ NO19)
Primer and probe sequences for amplifying CYP3a5 x 3(rs 776746):
CYP3a5 x 3(rs776746) upstream primer: AGCTTAACGAATGCTCTAC (SEQ NO5)
CYP3a5 × 3(rs776746) downstream primer: AGCAAGAGTCTCACACAGG (SEQ NO6)
And 5, probe: CTTTCAATATCTCTT (SEQ NO20)
And 6, probe 6: CTTTCAGTATCTCTT (SEQ NO21)
Primer and probe sequences for amplification of ADRB1(rs 1801253):
ADRB1(rs1801253) upstream primer: CCTTCAACCCCATCATCT (SEQ NO7)
ADRB1(rs1801253) downstream primer: GGTCTCCGTGGGTCGCG (SEQ NO8)
And (7) probe: TTCCAGGGACTGC (SEQ NO22)
And (3) probe 8: TTCCAGCGACTGC (SEQ NO23)
Primer and probe sequences for amplification of AGTR1(rs 5186):
AGTR1(rs5186) upstream primer: CATTCCTCTGCAGCACTTCACT (SEQ NO9)
AGTR1(rs5186) downstream primer: CGGTTCAGTCCACATAATGCAT (SEQ NO10)
And (3) probe 9: AATGAGCATTAGCTAC (SEQ NO24)
A probe 10: AATGAGCCTTAGCTAC (SEQ NO25)
Primer and probe sequences for amplifying ACE (rs 4646994):
ACE (rs4646994) upstream primer: TTTCTCCCATTTCTCTAGACCT (SEQ NO11)
ACE (rs4646994) downstream primer 1: ATCCCGCCACTGCACT (SEQ NO12)
ACE (rs4646994) downstream primer 2: TCAGAGAATTTCAGAGCTG (SEQ NO13)
A probe 11: CGCTCTGTCGCCCAGGC (SEQ NO26)
The probe 12: CTATACAGTCACTTTTATGTGGTTT (SEQ NO27)
Primer and probe sequences for amplification of NPPA (rs 5065):
NPPA (rs5065) upstream primer: CCAGCCCTGCTTGT (SEQ NO14)
NPPA (rs5065) downstream primer: AGGATGGGCACACTCAT (SEQ NO 15).
And (3) probe 13: TTATCTTCAGTACTG (SEQ NO28)
The probe 14: TTATCTTCGGTACTG (SEQ NO 29).
9. The detection method for guiding the hypertension medication comprises the following steps:
(a) extracting genomic DNA in a sample;
(b) amplifying and detecting polymorphism of rs1057910 site of CYP2C9 x 3 gene, polymorphism of rs1065852 site of CYP2D6 gene, polymorphism of rs776746 site of CYP3A5 x 3 gene, polymorphism of rs1801253 site of ADRB1 gene, polymorphism of rs5186 site of AGTR1 gene, polymorphism of rs4646994 site of ACE gene and polymorphism of rs5065 site of NPPA gene.
10. The assay of claim 9 wherein said hypertensive agent comprises a diuretic, an angiotensin converting enzyme inhibitor, an angiotensin receptor blocker, a beta receptor blocker, and a calcium antagonist.
CN201610732578.5A 2016-08-25 2016-08-25 Instruct detection combination object and application, the kit and detection method of hypertension medication Active CN106367479B (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN201811169467.3A CN109706240A (en) 2016-08-25 2016-08-25 It detects the oligonucleotides of NPPA gene pleiomorphism and is instructing the application in hypertension medication
CN201610732578.5A CN106367479B (en) 2016-08-25 2016-08-25 Instruct detection combination object and application, the kit and detection method of hypertension medication
CN201810301466.3A CN108690876A (en) 2016-08-25 2016-08-25 Detect primer, probe and application, kit and the detection method of ACE gene pleiomorphisms

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610732578.5A CN106367479B (en) 2016-08-25 2016-08-25 Instruct detection combination object and application, the kit and detection method of hypertension medication

Related Child Applications (2)

Application Number Title Priority Date Filing Date
CN201810301466.3A Division CN108690876A (en) 2016-08-25 2016-08-25 Detect primer, probe and application, kit and the detection method of ACE gene pleiomorphisms
CN201811169467.3A Division CN109706240A (en) 2016-08-25 2016-08-25 It detects the oligonucleotides of NPPA gene pleiomorphism and is instructing the application in hypertension medication

Publications (2)

Publication Number Publication Date
CN106367479A true CN106367479A (en) 2017-02-01
CN106367479B CN106367479B (en) 2018-10-30

Family

ID=57903203

Family Applications (3)

Application Number Title Priority Date Filing Date
CN201610732578.5A Active CN106367479B (en) 2016-08-25 2016-08-25 Instruct detection combination object and application, the kit and detection method of hypertension medication
CN201811169467.3A Pending CN109706240A (en) 2016-08-25 2016-08-25 It detects the oligonucleotides of NPPA gene pleiomorphism and is instructing the application in hypertension medication
CN201810301466.3A Pending CN108690876A (en) 2016-08-25 2016-08-25 Detect primer, probe and application, kit and the detection method of ACE gene pleiomorphisms

Family Applications After (2)

Application Number Title Priority Date Filing Date
CN201811169467.3A Pending CN109706240A (en) 2016-08-25 2016-08-25 It detects the oligonucleotides of NPPA gene pleiomorphism and is instructing the application in hypertension medication
CN201810301466.3A Pending CN108690876A (en) 2016-08-25 2016-08-25 Detect primer, probe and application, kit and the detection method of ACE gene pleiomorphisms

Country Status (1)

Country Link
CN (3) CN106367479B (en)

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106755560A (en) * 2017-03-30 2017-05-31 德必碁生物科技（厦门）有限公司 A kind of multiple fluorescence PCR method detects the kit of hypertension medication gene pleiomorphism
CN107058583A (en) * 2017-06-07 2017-08-18 上海龙鼎医药科技有限公司 Carry out the genotyping detection method of ADD1 gene rs4961 loci polymorphisms
CN107435076A (en) * 2017-09-08 2017-12-05 银川安龙基因科技有限公司 A kind of hypertension medication genetic test Solid phase PCR kit
CN107988356A (en) * 2017-12-22 2018-05-04 上海派森诺医学检验所有限公司 A kind of primer sets and detection kit for detecting hypertension relative gene polymorphism
CN108070646A (en) * 2017-06-23 2018-05-25 安徽安龙基因医学检验所有限公司 A kind of accurate medication genetic test Solid phase PCR kit of cardiovascular and cerebrovascular disease
CN108179180A (en) * 2017-10-25 2018-06-19 广州和康医疗技术有限公司 A kind of method and kit that genotype detection is carried out to CYP3A5*3 sites
CN108531578A (en) * 2018-04-26 2018-09-14 宁波美丽人生医学检验所有限公司 A kind of Primer composition and kit for detecting hypertension medication related gene
CN108690876A (en) * 2016-08-25 2018-10-23 杭州百迈生物股份有限公司 Detect primer, probe and application, kit and the detection method of ACE gene pleiomorphisms
CN108977531A (en) * 2018-09-05 2018-12-11 武汉康录生物技术股份有限公司 A kind of human hypertension's risk genes polymorphic detection kit and its preparation method and application
CN109082464A (en) * 2017-06-14 2018-12-25 合肥中科普瑞昇生物医药科技有限公司 A kind of primer sets and kit detecting hypertension drug metabolism related gene
CN109182509A (en) * 2018-10-29 2019-01-11 广州金域医学检验集团股份有限公司 Primer group, kit and method for detecting polymorphic sites of hypertension-related drug genes
CN109355368A (en) * 2018-10-22 2019-02-19 江苏美因康生物科技有限公司 A kind of kit and method of quick detection hypertension individuation medication gene pleiomorphism
CN109457024A (en) * 2018-03-23 2019-03-12 杭州泰领生物技术有限公司 Shr gene polymorphism fluorescent PCR solubility curve detection kit and application
CN109837339A (en) * 2019-03-27 2019-06-04 西安奥蓝泰生物科技有限公司 Primer sets, probe groups, kit and method for the detection of children's safety medication related gene
CN109897895A (en) * 2019-01-11 2019-06-18 江苏百世诺医疗科技有限公司 A kind of TaqMan-MGB probe technique detection influences the development of methodology of antihypertensive drugs curative effect gene
CN110129430A (en) * 2019-05-27 2019-08-16 广州海思医疗科技有限公司 A kind of detection kit and detection method for hypertensive patient's medication
CN110129427A (en) * 2019-04-15 2019-08-16 江苏医药职业学院 ACE insertion mutation type probe, ACE deficient mutant probe, kit and its application
CN110257506A (en) * 2019-07-09 2019-09-20 杭州千基生物科技有限公司 Polymorphism detection kit and method for hypertension accurate medication related gene
CN110511989A (en) * 2019-08-01 2019-11-29 杭州和壹基因科技有限公司 A kind of high-flux sequence method and its application of hypertension therapeutic pharmaceutical relevant gene
CN110863042A (en) * 2019-10-11 2020-03-06 济南和合医学检验有限公司 Method for detecting hypertension related gene by using multiple PCR technology
CN111100929A (en) * 2020-01-17 2020-05-05 郑州安图生物工程股份有限公司 Kit for detecting polymorphism of drug-related gene for hypertension
CN112608989A (en) * 2020-12-18 2021-04-06 成都和合医学检验所有限公司 Primer group, kit and method for detecting gene polymorphism
CN113549686A (en) * 2021-06-09 2021-10-26 湖南菲思特精准医疗科技有限公司 Detection kit for calcium ion antagonist metabolic marker, detection method and application thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113373213B (en) * 2021-06-17 2023-05-12 苏州大学 DNA methylation marker and application thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101240320A (en) * 2007-02-06 2008-08-13 上海主健生物工程有限公司 Kit for detecting cardiovascular disease incidence inheritance risk
WO2008157834A1 (en) * 2007-06-21 2008-12-24 University Of South Florida Materials and methods for diagnosis of asthma
CN101760528A (en) * 2008-12-26 2010-06-30 上海基康生物技术有限公司 Medicine metabolic relevant loci detection method
CN102876786A (en) * 2012-09-13 2013-01-16 周宏灏 Kit for detecting natriuretic peptide precursor A (NPPA) gene polymorphism by pyro-sequencing method and method
CN105018338A (en) * 2014-04-28 2015-11-04 郑州大学 Gene mutation detection chip for predicting curative effect and safety performance of statin drugs
CN105586406A (en) * 2016-01-15 2016-05-18 汪道文 Method for detecting gene polymorphism of ADRB1 and GRK5

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005295938A (en) * 2004-04-14 2005-10-27 Ebs Kk Method for efficiently typing insertion/deletion polymorphism of angiotensin-converting enzyme gene
CN101343658A (en) * 2007-09-30 2009-01-14 周宏灏 Gene chip for detection of hyperpiesis individual medicine correlated gene mutation and uses thereof
CN104651529A (en) * 2015-03-24 2015-05-27 厦门大学 Method for detecting deletion mutation of nucleic acid molecule
CN106367479B (en) * 2016-08-25 2018-10-30 杭州百迈生物股份有限公司 Instruct detection combination object and application, the kit and detection method of hypertension medication
CN108977531B (en) * 2018-09-05 2019-07-09 武汉康录生物技术股份有限公司 A kind of human hypertension's risk genes polymorphic detection kit and its preparation method and application
CN111718981A (en) * 2019-03-19 2020-09-29 中山大学孙逸仙纪念医院 ACE gene detection primer group, detection kit and detection method

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101240320A (en) * 2007-02-06 2008-08-13 上海主健生物工程有限公司 Kit for detecting cardiovascular disease incidence inheritance risk
WO2008157834A1 (en) * 2007-06-21 2008-12-24 University Of South Florida Materials and methods for diagnosis of asthma
CN101760528A (en) * 2008-12-26 2010-06-30 上海基康生物技术有限公司 Medicine metabolic relevant loci detection method
CN102876786A (en) * 2012-09-13 2013-01-16 周宏灏 Kit for detecting natriuretic peptide precursor A (NPPA) gene polymorphism by pyro-sequencing method and method
CN105018338A (en) * 2014-04-28 2015-11-04 郑州大学 Gene mutation detection chip for predicting curative effect and safety performance of statin drugs
CN105586406A (en) * 2016-01-15 2016-05-18 汪道文 Method for detecting gene polymorphism of ADRB1 and GRK5

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108690876A (en) * 2016-08-25 2018-10-23 杭州百迈生物股份有限公司 Detect primer, probe and application, kit and the detection method of ACE gene pleiomorphisms
CN106755560A (en) * 2017-03-30 2017-05-31 德必碁生物科技（厦门）有限公司 A kind of multiple fluorescence PCR method detects the kit of hypertension medication gene pleiomorphism
CN106755560B (en) * 2017-03-30 2020-03-27 德必碁生物科技（厦门）有限公司 Kit for detecting gene polymorphism of hypertension drug by multiple fluorescence PCR method
CN107058583A (en) * 2017-06-07 2017-08-18 上海龙鼎医药科技有限公司 Carry out the genotyping detection method of ADD1 gene rs4961 loci polymorphisms
CN109082464B (en) * 2017-06-14 2021-12-07 合肥中科普瑞昇生物医药科技有限公司 Primer group and kit for detecting hypertension drug metabolism related genes
CN109082464A (en) * 2017-06-14 2018-12-25 合肥中科普瑞昇生物医药科技有限公司 A kind of primer sets and kit detecting hypertension drug metabolism related gene
CN108070646A (en) * 2017-06-23 2018-05-25 安徽安龙基因医学检验所有限公司 A kind of accurate medication genetic test Solid phase PCR kit of cardiovascular and cerebrovascular disease
CN107435076A (en) * 2017-09-08 2017-12-05 银川安龙基因科技有限公司 A kind of hypertension medication genetic test Solid phase PCR kit
CN108179180A (en) * 2017-10-25 2018-06-19 广州和康医疗技术有限公司 A kind of method and kit that genotype detection is carried out to CYP3A5*3 sites
CN107988356A (en) * 2017-12-22 2018-05-04 上海派森诺医学检验所有限公司 A kind of primer sets and detection kit for detecting hypertension relative gene polymorphism
CN109457024A (en) * 2018-03-23 2019-03-12 杭州泰领生物技术有限公司 Shr gene polymorphism fluorescent PCR solubility curve detection kit and application
CN108531578A (en) * 2018-04-26 2018-09-14 宁波美丽人生医学检验所有限公司 A kind of Primer composition and kit for detecting hypertension medication related gene
CN108977531A (en) * 2018-09-05 2018-12-11 武汉康录生物技术股份有限公司 A kind of human hypertension's risk genes polymorphic detection kit and its preparation method and application
CN109355368A (en) * 2018-10-22 2019-02-19 江苏美因康生物科技有限公司 A kind of kit and method of quick detection hypertension individuation medication gene pleiomorphism
CN109182509B (en) * 2018-10-29 2022-05-06 广州金域医学检验集团股份有限公司 Primer group, kit and method for detecting polymorphic sites of hypertension-related drug genes
CN109182509A (en) * 2018-10-29 2019-01-11 广州金域医学检验集团股份有限公司 Primer group, kit and method for detecting polymorphic sites of hypertension-related drug genes
CN109897895A (en) * 2019-01-11 2019-06-18 江苏百世诺医疗科技有限公司 A kind of TaqMan-MGB probe technique detection influences the development of methodology of antihypertensive drugs curative effect gene
CN109837339A (en) * 2019-03-27 2019-06-04 西安奥蓝泰生物科技有限公司 Primer sets, probe groups, kit and method for the detection of children's safety medication related gene
CN110129427A (en) * 2019-04-15 2019-08-16 江苏医药职业学院 ACE insertion mutation type probe, ACE deficient mutant probe, kit and its application
CN110129430A (en) * 2019-05-27 2019-08-16 广州海思医疗科技有限公司 A kind of detection kit and detection method for hypertensive patient's medication
CN110257506A (en) * 2019-07-09 2019-09-20 杭州千基生物科技有限公司 Polymorphism detection kit and method for hypertension accurate medication related gene
CN110511989A (en) * 2019-08-01 2019-11-29 杭州和壹基因科技有限公司 A kind of high-flux sequence method and its application of hypertension therapeutic pharmaceutical relevant gene
CN110863042A (en) * 2019-10-11 2020-03-06 济南和合医学检验有限公司 Method for detecting hypertension related gene by using multiple PCR technology
CN111100929A (en) * 2020-01-17 2020-05-05 郑州安图生物工程股份有限公司 Kit for detecting polymorphism of drug-related gene for hypertension
CN112608989A (en) * 2020-12-18 2021-04-06 成都和合医学检验所有限公司 Primer group, kit and method for detecting gene polymorphism
CN113549686A (en) * 2021-06-09 2021-10-26 湖南菲思特精准医疗科技有限公司 Detection kit for calcium ion antagonist metabolic marker, detection method and application thereof

Also Published As

Publication number Publication date
CN106367479B (en) 2018-10-30
CN108690876A (en) 2018-10-23
CN109706240A (en) 2019-05-03

Similar Documents

Publication Publication Date Title
CN106367479B (en) Instruct detection combination object and application, the kit and detection method of hypertension medication
Scholtysik et al. Detection of genomic aberrations in molecularly defined Burkitt’s lymphoma by array-based, high resolution, single nucleotide polymorphism analysis
CN112080563B (en) Kit for detecting accurate medication genes of chronic diseases
WO2021036620A1 (en) Application of a group of genes related to ovarian cancer prognosis
CN105803099A (en) Kit for simultaneously detecting SLCO1B1, APOE and LDLR gene multisite mutation
CN110129430A (en) A kind of detection kit and detection method for hypertensive patient's medication
CN107254531B (en) Genetic biomarker for auxiliary diagnosis of early colorectal cancer and application thereof
CN107058538B (en) Primer composition, kit composed of primer composition and application of kit
CN106520950A (en) UGT1A1 gene polymorphism detection primer and probe and kit
CN101962669A (en) Gene combination, primer and probe for detecting alcoholic liver disease susceptibility and application
CN111118138A (en) Kit and method for detecting polymorphism of folate metabolism ability genes MTHFR and MTRR
CN101555528B (en) Method for testing chromosome 22q11.2 microdeletion and microduplication
CN112725431B (en) Primer, probe and kit for detecting polymorphic sites of APOE and SLCO1B1 genes
WO2021239081A1 (en) Reagent capable of being used for detecting npc1l1 mutant genotyping, kit, usage method therfor and application thereof
CN104531881A (en) Fluorescence PCR detection kit for human K-RAS gene mutation
CN1987462A (en) Probe for detecting matrilinear inheritance chondriosome deafness gene A1555G and its use
CN110819709A (en) Method for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR (polymerase chain reaction)
CN107267611B (en) Primer group for detecting p.Pro189Ala locus genotype and detection kit and application thereof
US10731219B1 (en) Method for preventing progression to metabolic syndrome
CN113337593A (en) Primer probe combination, kit and gene detection method for SSRIs and tricyclic antidepressant precise medication
CN104131102B (en) A kind of judge that NSCLC patient is to the test kit of Gefitinib therapeutic response
CN104131101B (en) A kind of reagent and application thereof detecting P53 gene SNP site
CN101586154A (en) Kit for forecasting treatment effect of epidermal growth factor receptor inhibitor
CN111172248A (en) General kit for verifying copy number variation based on fragment analysis technology
CN109182508A (en) The fluorescence in situ hybridization sequencing approach of TCF7L2 gene mononucleotide polymorphism

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant