CN106367479B - Instruct detection combination object and application, the kit and detection method of hypertension medication - Google Patents

Instruct detection combination object and application, the kit and detection method of hypertension medication Download PDF

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CN106367479B
CN106367479B CN201610732578.5A CN201610732578A CN106367479B CN 106367479 B CN106367479 B CN 106367479B CN 201610732578 A CN201610732578 A CN 201610732578A CN 106367479 B CN106367479 B CN 106367479B
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林源吉
丁佳女
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Hangzhou Hundred Biological Ltd By Share Ltd
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Abstract

The present invention relates to the detection combination object, kit and the detection methods that instruct hypertension medication.Including detection CYP2C9*3 (rs1057910), CYP2D6 (rs1065852), CYP3A5*3 (rs776746), ADRB1 (rs1801253), AGTR1 (rs5186), ACE (rs4646994), NPPA (rs5065), genotype.The genotype in above-mentioned 7 sites is detected using the present invention, method is simple and practicable, rapidly and efficiently, of low cost, and the new way of a simple and direct judgement is provided for the clinical application of hypertension.

Description

Instruct detection combination object and application, the kit and detection method of hypertension medication
Technical field
The present invention relates to molecular biology and medical domain, and in particular to instructs hypertension medication correlation SNP site Detection combination object, kit and detection method.
Background technology
With the change of the development and resident living mode of social economy, Chronic Non-Communicable Diseases (abbreviation chronic disease) is As influencing the great public health problem of China or even global residents ' health, and hypertension be the higher chronic disease of illness rate it One and the most important risk factor of cardiovascular and cerebrovascular disease.It is shown according to the World Health Organization (WHO) statistics, 2012 complete Centre of sphere angiosis death toll is 17,000,000, accounts for the 46% of chronic disease death toll, wherein hypertension complication dead 9,400,000, Having become influences the primary risk factor of global disease burden.The World Bank in 2011《Create healthy harmonious life containment China Slow disease is popular》Report is pointed out：Chronic disease has become the No.1 health threat of China.It is led in about 10,300,000 every year different reasons In the Died Patients of cause, chronic disease proportion is more than 80%, and wherein cardiovascular and cerebrovascular disease death occupies chronic disease cause of the death first place, The generation of 50%~75% palsy and 40%~50% myocardial infarction is related with blood pressure raising.2010~the year two thousand forty, every year If cardiovascular death rate can be made to reduce by 1%, it is equivalent to the annual economic receipts for creating GDP 15% in 2010 Beneficial (2.34 trillion dollars), and if cardiovascular death rate declines 3%, annual economic well-being of workers and staff is up to domestic production in 2010 34% (5.4 trillion dollars) of total value.On the contrary, if chronic disease cannot be successfully managed, these diseases certainly will will aggravate can be pre- The aging of population seen and workforce population reduce caused economic and social impacts.
Since founding of New, carry out respectively 4 times of the China of nineteen fifty-nine, 1979,1991,2002 for 15 years old and The investigation of the above Hypertension among Residents in Urban epidemic status, Chinese residents nutrition and health survey in 2002,2004~2013 years 4 field investigations and 2010~2012 years Chinese residents nutrition surveys etc. of Chinese chronic disease and its risk factor monitoring obtain Obtained a large amount of Hypertensions and control data.These data show that China's adult hypertension illness rate constantly increases, by The 5.11% of nineteen fifty-nine rises to 17.65% in 2002, newest publication《Chinese residents nourishment and chronic disease status report (2015)》It has been shown that, China in 2012 18 years old and the above prevalence of hypertension rate are 25.2%, and male is higher than women, and city is high In rural area, estimate that current China's adult hypertension patient is about 2.6 hundred million；Compared with 2002, Prevalence of Hypertension obviously rises, Rural area increases rapider.But China's adult hypertension illness awareness is only 46.5%, treatment rate 41.1%, control Rate is 13.8%.At the same time, (such as smoking, excessive consumption of alcohol, the intake of with high salt and high lipid food, activity are or not Risk Factors of Hypertension Sufficient, overweight and fat and total cholesterol raising etc.) generally existing in crowd, and constantly increase or remain high, become high The potential threat of the cardiovascular and cerebrovascular diseases such as blood pressure, myocardial infarction and palsy.And 2011~2012 years hypertension awarenesses in the U.S., Treatment rate and control rate have respectively reached 82.7%, 75.6% and 51.8%.Compared with developed countries, the hypertension of China resident Number of patients is more, high still in reduced levels although hypertension awareness, treatment rate and control rate increase in recent years Controlling of blood pressure rate regional disparity is larger, and great challenge is brought for chronic diseases in China prevention and control situation.
In order to effectively control chronic disease, WHO is promulgated within 2013《Global non-communicable diseases prevention and control action plan (2013~2020)》, 15 ministries and commissions of China combine and promulgate《Chinese prevention and treatment of chronic diseases work planning (2012~2015)》, it is intended to By multi-field, multi-sector cooperation, control chronic disease danger factor increases, and contains or reduce chronic disease incidence and the death rate, Disease Spectrum caused by reducing chronic disease.Therefore, in order to reinforce the preventing and controlling of China's Hypertension among Residents in Urban, multidisciplinary participation is answered to make Determine relevant policies, such as formulates the price for reducing Cardia Salt, the limitation of food addition salinity, increases physical training facility and improve ring Border etc. advocates fitness-for-all life style, reduces the prevalence of Risk Factors of Hypertension；It is advocated energetically through medical institutions head It examines measuring blood pressure and the screening of Hypertension is reinforced in residents ' health physical examination, prevalence of hypertension awareness is improved, so as to early hair Existing, early diagnosis, early treatment；Large hospital should be given full play to the directive function of basic medical unit, specification in drug treatment The same of primary health institution professional technician quantity and technical merit is being continuously improved in condition of medicine treatment for hypertension flow When, the health control of hypertensive patient and Canonical management in basic public health service are further strengthened, it is reasonable to improve hypertension Drug level and hypertension control rate.
Current clinically used drug for hypertension has 5 major class：Diuretics, angiotensin converting enzyme inhibitors, blood vessel Angiotensin receptor blocker, beta-blocker, calcium ion antagonist.Drug arrives a variety of drugs involved in metabolic process in vivo Metabolic enzyme, transporter and receptor, their existing gene pleiomorphisms have eventually led to patient and have taken same drug in the same manner When, therapeutic effect, the bad development of generation and its there are apparent individual differences to tolerance of drug etc..
Currently, the hypertension research based on pharmacogenetic group is primarily upon the diversity and drug efficacy of antihypertensive treatment of gene Relationship, influence of the gene difference to drug efficacy of antihypertensive treatment caused by different ethnic groups, age, gender and diet and living environment Deng, less focus on gene mutation to drug be depressured after remote effect.The following hypertension drug genetics group research should be it is multidisciplinary, Including the system research engineering that molecular biology, clinical medicine, science of heredity, mathematics, sociology etc. cooperate jointly, base is not only paid close attention to Cause and the relationship between physiology, pathology will also pay close attention to the relationship between gene and environment, humanity, society, be final hypertension etc. Disease prevention and treatment provide foundation.
It is mutual due to various factors such as the hereditary difference of Different Individual, ethnic group, age, gender and diet and living environments Effect, makes Different Individual and group for the neurological susceptibility of hypertension and the reaction of antihypertensive drugs in sex, race and in terms of the age Also different effects is showed.Should be treatment and prevention for the treatment of hypertension.As gene prevents the arriving in epoch, pass through base Because of test, the early prevention of Different Individual is carried out for different Susceptible population, by the assessment of genetic test and composite factor, Suitable individualized treatment is provided for it, make its medication reach safely, effectively, it is economical.
Invention content
In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a kind of detection groups instructing hypertension medication Object is closed, including detects CYP2C9*3 genes, CYP2D6 genes, CYP3A5*3 genes, ADRB1 genes, AGTR1 genes, ACE respectively The reagent of gene and NPPA gene pleiomorphisms.
Further, detection be the sites rs1057910 of CYP2C9*3 genes, CYP2D6 genes rs1065852 Site, the sites rs776746 of CYP3A5*3 genes, the sites rs1801253 of ADRB1 genes, AGTR1 genes the positions rs5186 The polymorphism in the sites rs5065 of point, the sites rs4646994 of ACE genes and NPPA genes.
Detecting the reagent of said gene polymorphism can make according to the different testing principles reagent different with method choice For detection combination object.The reagent of the detection combination object of one of which type includes being designed based on said gene polymorphic site Amplimer and probe, probe are two probes of the wild type and saltant type that are directed to gene polymorphism sites design respectively.
The detection combination object is applied to instruct the detection kit of hypertension medication, wherein composition includes detection CYP2C9*3 genes, CYP2D6 genes, CYP3A5*3 genes, ADRB1 genes, AGTR1 genes, ACE genes and NPPA genes are more The reagent of state property.
The detection kit of hypertension medication is instructed the present invention also provides simple operation, including is detected respectively CYP2C9*3 genes, CYP2D6 genes, CYP3A5*3 genes, ADRB1 genes, AGTR1 genes, ACE genes and NPPA genes are more The reagent of state property.
Amplimer and probe mentioned above, is respectively selected from：
Expand the primer and probe sequence of CYP2C9*3 (rs1057910)：
CYP2C9*3 (rs1057910) sense primer：ATTTAATGTCACAGGTCACTGC(SEQ NO1)
CYP2C9*3 (rs1057910) downstream primer：CACATGCCCTACACAGAT(SEQ NO2)
Probe 1：AGATACATTGACCTT(SEQ NO16)
Probe 2：AGATACCTTGACCTT(SEQ NO17)
Expand the primer and probe sequence of CYP2D6 (rs1065852)：
CYP2D6 (rs1065852) sense primer：TGCTCCTGGTGGACCTGA(SEQ NO3)
CYP2D6 (rs1065852) downstream primer：AGTCCACATGCAGCAGGT(SEQ NO4)
Probe 3：CGCTACTCACCAG(SEQ NO18)
Probe 4：CGCTACCCACCAG(SEQ NO19)
Expand the primer and probe sequence of CYP3A5*3 (rs776746)：
CYP3A5*3 (rs776746) sense primer：AGCTTAACGAATGCTCTAC(SEQ NO5)
CYP3A5*3 (rs776746) downstream primer：AGCAAGAGTCTCACACAGG(SEQ NO6)
Probe 5：CTTTCAATATCTCTT(SEQ NO20)
Probe 6：CTTTCAGTATCTCTT(SEQ NO21)
Expand the primer and probe sequence of ADRB1 (rs1801253)：
ADRB1 (rs1801253) sense primer：CCTTCAACCCCATCATCT(SEQ NO7)
ADRB1 (rs1801253) downstream primer：GGTCTCCGTGGGTCGCG(SEQ NO8)
Probe 7：TTCCAGGGACTGC(SEQ NO22)
Probe 8：TTCCAGCGACTGC(SEQ NO23)
Expand the primer and probe sequence of AGTR1 (rs5186)：
AGTR1 (rs5186) sense primer：CATTCCTCTGCAGCACTTCACT(SEQ NO9)
AGTR1 (rs5186) downstream primer：CGGTTCAGTCCACATAATGCAT(SEQ NO10)
Probe 9：AATGAGCATTAGCTAC(SEQ NO24)
Probe 10：AATGAGCCTTAGCTAC(SEQ NO25)
Expand the primer and probe sequence of ACE (rs4646994)：
ACE (rs4646994) sense primer：TTTCTCCCATTTCTCTAGACCT(SEQ NO11)
ACE (rs4646994) downstream primer 1：ATCCCGCCACTGCACT(SEQ NO12)
ACE (rs4646994) downstream primer 2：TCAGAGAATTTCAGAGCTG(SEQ NO13)
Probe 11：CGCTCTGTCGCCCAGGC(SEQ NO26)
Probe 12：CTATACAGTCACTTTTATGTGGTTT(SEQ NO27)
Expand the primer and probe sequence of NPPA (rs5065)：
NPPA (rs5065) sense primer：CCAGCCCTGCTTGT(SEQ NO14)
NPPA (rs5065) downstream primer：AGGATGGGCACACTCAT(SEQNO15).
Probe 13：TTATCTTCAGTACTG(SEQ NO28)
Probe 14：TTATCTTCGGTACTG(SEQ NO29).
The present invention also provides a kind of detection methods instructing hypertension medication, include the following steps：
(a) genomic DNA in sample is extracted；
(b) it expands and detects the polymorphism in the sites CYP2C9*3 gene rs1057910, CYP2D6 genes rs1065852 Point polymorphism, the polymorphism in the sites CYP3A5*3 gene rs776746, the polymorphism in the sites ADRB1 gene rs1801253, The polymorphism in the sites AGTR1 gene rs5186, the polymorphism in the sites ACE gene rs4646994, NPPA gene rs5065 sites Polymorphism.
Hypertension medication of the present invention includes diuretics, angiotensin converting enzyme inhibitors, angiotensins Receptor blocker, beta-blocker and calcium ion antagonist.
Technical scheme of the present invention has the following advantages：The present invention enumerates and the five relevant genes of major class hypertension drug The analysis of polymorphic site, by the detection and analysis of 7 loci polymorphisms, capable of effectively instructing mainstream drug for hypertension, (five is big Class drug is diuretics, angiotensin converting enzyme inhibitors, angiotensin receptor blocker, beta-blocker, calcium respectively Antagonism of ions agent) Clinical practice, the type of similar product more on the market, covering is wider.And using specificity when detecting Double probes of high wild-type probe and saltant type probe design, and avoid the internal reference needed using current fluorescence quantitative PCR detection Primer and probe.This not only enhances the specificity of detection, also reduces operating error and reduces testing cost.It is visited using Taqman Not only significant increase detection is specific after needle, and detection time is effectively also shorten to 1 hour left side by original 2.5-3 hours The right side, to substantially increase the accuracy and detection efficiency of testing result.
Description of the drawings
The generation sequencing result of Fig. 1 ADRB1 (rs1801253) wild type (GG).
The genotyping result of the embodiment of the present invention of Fig. 2 ADRB1 (rs1801253) wild type (GG).
The generation sequencing result of Fig. 3 ADRB1 (rs1801253) heterozygous mutation (GC).
The genotyping result of the embodiment of the present invention of Fig. 4 ADRB1 (rs1801253) heterozygous mutation (GC).
The generation sequencing result of Fig. 5 ADRB1 (rs1801253) no mutant homozygote (CC).
The genotyping result of the embodiment of the present invention of Fig. 6 ADRB1 (rs1801253) no mutant homozygote (CC).
Specific implementation mode
Inventor by analyze clinically hypertensive patient's common drug use and be metabolized and the individual difference of drug effect, sieve 7 polymorphic sites with the five closely related genes of major class drug for hypertension are selected, and prepares and detects 7 polymorphic positions The composition of point and the detection kit for using the composition, and accurate detection method is established to five major class anti-hypertensions Drug realizes effective medication guide.The five major class drugs for hypertension are diuretics, angiotensin converting enzyme inhibition Agent, angiotensin receptor blocker, beta-blocker, calcium ion antagonist.Described is directed to five major class drugs for hypertension 7 polymorphic sites (specific No. RS), respectively CYP2C9*3 (rs1057910), CYP2D6 (rs1065852), CYP3A5* 3 (rs776746), ADRB1 (rs1801253), AGTR1 (rs5186), ACE (rs4646994) and NPPA (rs5065).Described 7 Its hypertension medication curative effect of the mutated individual of loci gene type and risk are different from general population.
The sites rs1057910 of CYP2C9*3 genes, the sites rs1065852 of CYP2D6 genes, CYP3A5*3 genes The sites rs776746, the sites rs1801253 of ADRB1 genes, the sites rs5186 of AGTR1 genes, ACE genes The sites rs4646994 and the sites rs5065 of NPPA genes can be expressed as CYP2C9*3 (rs1057910), CYP2D6 respectively (rs1065852)、CYP3A5*3(rs776746)、ADRB1(rs1801253)、AGTR1(rs5186)、ACE(rs4646994) With NPPA (rs5065).
Embodiment 1 utilizes the detection kit of fluorescence quantitative PCR detection system
Detection kit includes amplimer, probe, and carries out other matched reagents of fluorescence quantitative PCR detection.
Amplimer and probe are for CYP2C9*3 (rs1057910), CYP2D6 (rs1065852), CYP3A5*3 (rs776746), ADRB1 (rs1801253), AGTR1 (rs5186), ACE (rs4646994) and NPPA (rs5065) 7 are polymorphic Property site design.
One, the design and synthesis of amplimer
Sites rs1065852 in region, CYP2D6 genes based on the sites rs1057910 in CYP2C9*3 genes The region in the sites rs776746 in region, CYP3A5*3 genes, the sites rs1801253 in ADRB1 genes region, In the region in the sites rs5186 in AGTR1 genes, the region in the middle sites rs4646994 of ACE genes, NPPA genes The region in the sites rs5065, designs amplimer, wherein
Expand the primer sequence of CYP2C9*3 (rs1057910)：
CYP2C9*3 (rs1057910) sense primer：ATTTAATGTCACAGGTCACTGC(SEQ NO1)
CYP2C9*3 (rs1057910) downstream primer：CACATGCCCTACACAGAT(SEQ NO2)
Expand the primer sequence of CYP2D6 (rs1065852)：
CYP2D6 (rs1065852) sense primer：TGCTCCTGGTGGACCTGA(SEQ NO3)
CYP2D6 (rs1065852) downstream primer：AGTCCACATGCAGCAGGT(SEQ NO4)
Expand the primer sequence of CYP3A5*3 (rs776746)：
CYP3A5*3 (rs776746) sense primer：AGCTTAACGAATGCTCTAC(SEQ NO5)
CYP3A5*3 (rs776746) downstream primer：AGCAAGAGTCTCACACAGG(SEQ NO6)
Expand the primer sequence of ADRB1 (rs1801253)：
ADRB1 (rs1801253) sense primer：CCTTCAACCCCATCATCT(SEQ NO7)
ADRB1 (rs1801253) downstream primer：GGTCTCCGTGGGTCGCG(SEQ NO8)
Expand the primer sequence of AGTR1 (rs5186)：
AGTR1 (rs5186) sense primer：CATTCCTCTGCAGCACTTCACT(SEQ NO9)
AGTR1 (rs5186) downstream primer：CGGTTCAGTCCACATAATGCAT(SEQ NO10)
Expand the primer sequence of ACE (rs4646994)：
ACE (rs4646994) sense primer：TTTCTCCCATTTCTCTAGACCT(SEQ NO11)
ACE (rs4646994) downstream primer 1：ATCCCGCCACTGCACT(SEQ NO12)
ACE (rs4646994) downstream primer 2：TCAGAGAATTTCAGAGCTG(SEQ NO13)
Expand the primer sequence of NPPA (rs5065)：
NPPA (rs5065) sense primer：CCAGCCCTGCTTGT(SEQ NO14)
NPPA (rs5065) downstream primer：AGGATGGGCACACTCAT(SEQ NO15)
Since the polymorphism of ACE (rs4646994) is non-simple point mutation, but insertion/deletion is mutated, in order to ensure to expand Validity, the present invention devises a sense primer and two downstream primers.Experiment shows drawing using SEQ NO.11 to 13 Object and SEQ NO.26 can detect the polymorphism of all ACE (rs4646994) to 27 probes, and amplification efficiency is high, there is good spy Anisotropic and accuracy.
Two, the design and synthesis of probe
Based on CYP2C9*3 (rs1057910), CYP2D6 (rs1065852), CYP3A5*3 (rs776746), ADRB1 (rs1801253), AGTR1 (rs5186), ACE (rs4646994) and the characteristics of NPPA (rs5065) single nucleotide polymorphisms, if The high specific probe analyzed for quantitative fluorescent PCR is counted out, and the probe is to be set respectively for gene polymorphism sites The wild type of meter and the two of saltant type probes.Wherein,
Detection CYP2C9*3 (rs1057910) polymorphism probe sequence include：
Probe 1：AGATACATTGACCTT(SEQ NO16)
Probe 2：AGATACCTTGACCTT(SEQ NO17)
Detection CYP2D6 (rs1065852) polymorphism probe sequence include：
Probe 3：CGCTACTCACCAG(SEQ NO18)
Probe 4：CGCTACCCACCAG(SEQ NO19)
Detection CYP3A5*3 (rs776746) polymorphism probe sequence include：
Probe 5：CTTTCAATATCTCTT(SEQ NO20)
Probe 6：CTTTCAGTATCTCTT(SEQ NO21)
Detection ADRB1 (rs1801253) polymorphism probe sequence include：
Probe 7：TTCCAGGGACTGC(SEQ NO22)
Probe 8：TTCCAGCGACTGC(SEQ NO23)
Detection AGTR1 (rs5186) polymorphism probe sequence include：
Probe 9：AATGAGCATTAGCTAC(SEQ NO24)
Probe 10：AATGAGCCTTAGCTAC(SEQ NO25)
Detection ACE (rs4646994) polymorphism probe sequence include：
Probe 11：CGCTCTGTCGCCCAGGC(SEQ NO26)
Probe 12：CTATACAGTCACTTTTATGTGGTTT(SEQ NO27)
Detection NPPA (rs5065) polymorphism probe sequence include：
Probe 13：TTATCTTCAGTACTG(SEQ NO28)
Probe 14：TTATCTTCGGTACTG(SEQ NO29).
In one embodiment, the probe is TaqMan probe, and 5 ' ends of every probe are fluorescent reporter group, 3 ' ends For quenching group.Wherein fluorescent reporter group can be selected from FAM, TET, VIC, JOE, HEX, Cy3, Cy3.5, Cy5, Cy5.5, TAMRA, ROX, Texas Red, LC RED640 and LC RED705 etc.；Quenching group can be selected from MGB, BHQ0, BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse and NFQ etc..
Three, pcr amplification reaction reagent
Pcr amplification reaction reagent includes above-mentioned amplimer and probe, further include pcr amplification reaction system it is required its His matched reagent.
Other described matched reagents include PCR Buffer, Mg2+, dNTP, Taq enzyme.In more optimal solution, other are mating Reagent further includes DMSO and ROX.About the selection of ROX, according to the needs of quantitative fluorescent PCR instrument and experiment that experiment uses Depending on, and not all instrument or experiment is required for that ROX is added.The selection of ROX is for correcting background signal, therefore, in every case The case where being not necessarily to correcting background signal with other background signal calibration functions or reagent or experimental design, can not use ROX carries out background signal correction.
In a specific pcr amplification reaction embodiment, the reagent and dosage progress quantitative fluorescent PCR described in table 1 are used Detection：
Table 1：
Component Dosage
10×buffer(Mg2+) 2μl
MgCl2(25mM) 0-5μl
dNTP(10mM) 0.2-1μl
RTaq enzymes (5U/ul) 0.1-0.5μl
Sense 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 genomic DNAs
ddH2O It is loaded to 20ul
In a preferred embodiment, the MgCl2(25mM) dosage is 3 μ l, and dNTP (10mM) dosage is 0.5 μ l, rTaq enzymes (5U/ul) dosage are 0.1 μ l, and sense primer (10uM) dosage is 0.5 μ l, the downstream primer (10uM) dosage is 0.5 μ l, and the probe 1 or 3 or 5 or 7 or 9 or 11 or 13 (10uM) dosages are 0.5 μ l, the probe 2 or 4 Or 6 or 8 or 10 or 12 or 14 (10uM) (10uM) dosage be 0.5 μ l, the DMSO dosages are 0.5 μ l, and the ROX dosages are 0.1μl。
In fluorescence quantitative PCR detection system, it includes described draw to instruct the reagent in the detection combination object of hypertension medication Object and probe, for detecting CYP2C9*3 genes, CYP2D6 genes, CYP3A5*3 genes, ADPB1 genes, AGTR1 genes, ACE Gene and NPPA gene pleiomorphisms.Further, it can also include other the required mating examinations of pcr amplification reaction system Agent.
2 fluorescent quantitative PCR detection method of embodiment
The fluorescent quantitative PCR detection method for detecting 7 polymorphic sites includes the following steps：
(a) genomic DNA in sample is extracted；
(b) expand and detect gene pleiomorphism, the CYP2D6 in the region comprising the sites rs1057910 in CYP2C9*3 genes Include the area in the sites rs776746 in gene in the gene pleiomorphism, CYP3A5*3 genes in the region comprising the sites rs1065852 Include in the gene pleiomorphism, AGTR1 genes in the region comprising the sites rs1801253 in the gene pleiomorphism in domain, ADRB1 genes The gene pleiomorphism in the region comprising the sites rs4646994 in the gene pleiomorphism in the region in the sites rs5186, ACE genes, The gene pleiomorphism in the region comprising the sites rs5065 in NPPA genes.
The extracting method of genomic DNA includes：Traditional phenol chloroform, the pellosil extracts kit of commercialization, quotient The paramagnetic particle method extracts kit of product.As long as the genomic DNA extracted meets the following conditions：OD260/280 be 1.7~ 2.0；5ng/ μ l≤DNA concentration≤100ng/ μ l.
Wherein, fluorescent quantitative PCR reaction condition such as table 2：
Table 2
Using double probe in detecting systems without reference gene detection in embodiment 1 and embodiment 2, reference gene is not included The primer and probe of detection.The operation in experimentation is reduced, excessive operation is avoided to cause experimental error；Meanwhile without interior The system of ginseng also reduces experimental cost.
In a further embodiment, implement amplimer, probe described in 1 to be also applied for including the detection of reference gene System.
Embodiment 3 analyzes clinical sample using fluorescence quantifying PCR method
150 clinical saliva samples and 150 clinical blood samples are collected, the paramagnetic particle method extracts kit of commercialization is utilized The extraction of genomic DNA is carried out, the parting that 7 genes are then carried out using the method for embodiment 1 and embodiment 2 is detected.Meanwhile Genotyping detection is carried out to the DNA of extraction using generation PCR sequencing PCR, and the genotypic results of this method and a generation are sequenced Genotypic results compared.Wherein, the contrast test result of CYP2C9*3 (rs1057910) such as table 3；CYP2D6 (rs1065852) contrast test result such as table 4；The contrast test result of CYP3A5*3 (rs776746) such as table 5；ADRB1 (rs1801253) contrast test result such as table 6；The contrast test result of AGTR1 (rs5186) such as table 7；ACE (rs4646994) contrast test result such as table 8；The contrast test result of NPPA (rs5065) such as table 9.
Table 3：The contrast test result of CYP2C9*3 (rs1057910)
Table 4：The contrast test result of CYP2D6 (rs1065852)
Table 5：The contrast test result of CYP3A5*3 (rs776746)
Table 6：The contrast test result of ADRB1 (rs1801253)
Table 7：The contrast test result of AGTR1 (rs5186)
Table 8：The contrast test result of ACE (rs4646994) (I indicates to be inserted into, and D indicates missing)
Table 9：The contrast test result of NPPA (rs5065)
If Fig. 1 to 6 is the genotyping result and a generation for measuring ADRB1 (rs1801253) gene using the method for the invention Sequencing as a result, by the test map comparative analyses of two methods, result is completely the same.Wherein ADRB1 (rs1801253) The comparing result of gene wild type (GG) is as depicted in figs. 1 and 2；Pair of ADRB1 (rs1801253) gene mutation heterozygotes (GC) It is more as shown in Figure 3 and Figure 4 than result；The result of ADRB1 (rs1801253) gene mutation homozygotes (CC) is as shown in Figure 5 and Figure 6. Similarly CYP2C9*3 (rs1057910), CYP2D6 (rs1065852), CYP3A5*3 are measured using the method for the invention (rs776746), the parting test map and a generation of AGTR1 (rs5186), ACE (rs4646994) and NPPA (rs5065) gene The test map of sequencing compares, and is as a result also completely the same.
For fluorescence quantitive PCR typing experiment, it can generally show the normality of reaction system using reference gene, So that it is guaranteed that genotyping result is believable.I.e.：Only under the premise of reference gene amplification normally, PCR genotyping results are only effectively 's.Therefore, the use of reference gene is the general essential design of PCR partings.The present invention uses the design of double probe systems, It is not only able to complete whole parting tests in a reaction tube, while also eliminating the use of reference gene, utilize detection base Because itself can determine whether PCR genotyping results are effective.The method of judgement is：When only there is the fluorescence signal of wild type, table Bright sample is wild type.When only there is the fluorescence signal of saltant type, show that sample is homozygous mutant.It is wild when occurring simultaneously When type and mutant signal, show that sample is heterozygous mutant.When no any fluorescence signal occurs, then show the secondary detection Failure.Therefore, this method can not only reduce experimental implementation, also reduce experimental cost.
The specificity of the double probe systems of 4 quantitative fluorescent PCR of embodiment
Double successful keys of probe body system are the specificity of probe.The present invention is by carrying out a large amount of objective gene sequences It compares, selection includes the regions rs while there is the region of higher conservative to carry out design of primers, then specific base is selected to go The TaqMan probe for wild type and saltant type is separately designed, respectively mark fluorescent group, such as the flag F AM of wild type glimmering The label VIC fluorophors of light group and saltant type.Probe designed by the present invention not only can with high specificity It is enough to play the role of good parting in double probe systems, while Ct values can also be controlled within 33.It is commercialized at present In product, for the reagent of fluorescence quantitive PCR typing, Single probe system complicated for operation and expensive is not only used, simultaneously Its Ct value generally can only also control within 35.Therefore, double probe systems of the invention have compared to existing detection architecture Specificity well, can realize efficient parting.
The high sensitivity of 5 fluorescence quantitative PCR detection system of embodiment
The sensitivity of PCR detection architectures, mainly characterizes requirement of the system for amplification starting template amount, and sensitivity is got over Height shows that the PCR system is obtained with amplified signal in lower template (middle finger genomic DNA of the present invention).In general, In the product of commercialization, require control in 10ng or more the initial amount of genomic DNA, and the present invention then can be by base Because the initial amount of group DNA is reduced to 5ng.It is that 5ng genomic DNAs are detected to template quantity using the method for Examples 1 and 2, Simultaneously as a contrast, the genomic DNA of same sample is detected with generation PCR sequencing PCR, contrast experiment shows to work as genome When the template quantity of DNA is 5ng, testing result is consistent with generation sequencing result.Therefore primer, probe and detection of the present invention Method is highly beneficial for the detection in some trace samples (such as serum/plasma DNA).
The accurate medication guide of 6 hypertension of embodiment, five major class drug
It is found after analyzing the accurate medication guide of hypertension, in current commercial prod, mainly passes through five positions It puts to instruct the medication of three categories drug, this three categories drug includes that (such as chlorine is husky for angiotensin II receptor blockers Smooth, Irb etc.), 1 Adrenergic receptor blockers of β (such as metoprolol, Carvedilol), Angiotensin-Converting suppression Preparation (that Puli, fosinopril in such as).However, defending planning commission's promulgation in country《Hypertension medicine guide》In clearly refer to Go out, in addition to above-mentioned three categories antihypertensive beyond the region of objective existence, also two major classes drug for hypertension is mainstream medicine, is diuretics respectively And calcium ion antagonist.By various research and analysis, the present invention creatively design by 7 sites (including CYP2C9*3(rs1057910)、CYP2D6(rs1065852)、CYP3A5*3(rs776746)、ADRB1(rs1801253)、 AGTR1 (rs5186), ACE (rs4646994) and NPPA (rs5065)) detection and analysis, five major class master of hypertension can be instructed Flow the accurate medication of drug.This is not only able to the five major class mainstream medicines for covering hypertension comprehensively, while can also join to hypertension It shares medicine and plays directive function.Instruct the concrete condition such as table 10 of five major class hypertension drugs to table 14 in 7 sites of the present invention：
(1) diuretics table 10
(2) hypertensin inhibitor invertase table 11
(3) angiotensin II receptor blockers table 12
(4) 1 Adrenergic receptor blocker tables 13 of β
(5) calcium ion antagonist table 14
Other detection methods of 7 hypertension of embodiment, the five accurate medication of major class drug
By experimental analysis, respectively be directed to the non-double probe systems of quantitative fluorescent PCR, generation PCR sequencing PCR, pyrosequencing, PCR-HRM, arms-PCR, restriction fragment length polymorphism side, in situ hybridization (ISH), genetic chip or high-flux sequence etc. Method prepares the detection combination object for instructing hypertension medication respectively, and the reagent in composition includes the required examination of corresponding method Agent.These reagents for analyze CYP2C9*3 genes, CYP2D6 genes, CYP3A5*3 genes, ADRB1 genes, AGTR1 genes, The testing result of ACE genes and NPPA gene pleiomorphisms, acquisition can provide reference information for the medication guide of hypertension.

Claims (6)

1. instruct the detection combination object of hypertension medication, including detection CYP2C9*3 gene pleiomorphisms, CYP2D6 gene pleiomorphisms, CYP3A5*3 gene pleiomorphisms, ADRB1 gene pleiomorphisms, AGTR1 gene pleiomorphisms, ACE gene pleiomorphisms and NPPA genes are more The reagent of state property, the reagent include amplimer, the wild type and saltant type for being directed to gene polymorphism sites design respectively Two probes, wherein
Detection CYP2C9*3 (rs1057910) polymorphism primer and probe sequence include：
CYP2C9*3 (rs1057910) sense primer：ATTTAATGTCACAGGTCACTGC(SEQ NO1)
CYP2C9*3 (rs1057910) downstream primer：CACATGCCCTACACAGAT(SEQ NO2)
Probe 1：AGATACATTGACCTT(SEQ NO16)
Probe 2：AGATACCTTGACCTT(SEQ NO17)
Detection CYP2D6 (rs1065852) polymorphism primer and probe sequence include：
CYP2D6 (rs1065852) sense primer：TGCTCCTGGTGGACCTGA(SEQ NO3)
CYP2D6 (rs1065852) downstream primer：AGTCCACATGCAGCAGGT(SEQ NO4)
Probe 3：CGCTACTCACCAG(SEQ NO18)
Probe 4：CGCTACCCACCAG(SEQ NO19)
Detection CYP3A5*3 (rs776746) polymorphism primer and probe sequence include：
CYP3A5*3 (rs776746) sense primer：AGCTTAACGAATGCTCTAC(SEQ NO5)
CYP3A5*3 (rs776746) downstream primer：AGCAAGAGTCTCACACAGG(SEQ NO6)
Probe 5：CTTTCAATATCTCTT(SEQ NO20)
Probe 6：CTTTCAGTATCTCTT(SEQ NO21)
Detection ADRB1 (rs1801253) polymorphism primer and probe sequence include：
ADRB1 (rs1801253) sense primer：CCTTCAACCCCATCATCT(SEQ NO7)
ADRB1 (rs1801253) downstream primer：GGTCTCCGTGGGTCGCG(SEQ NO8)
Probe 7：TTCCAGGGACTGC(SEQ NO22)
Probe 8：TTCCAGCGACTGC(SEQ NO23)
Detection AGTR1 (rs5186) polymorphism primer and probe sequence include：
AGTR1 (rs5186) sense primer：CATTCCTCTGCAGCACTTCACT(SEQ NO9)
AGTR1 (rs5186) downstream primer：CGGTTCAGTCCACATAATGCAT(SEQ NO10)
Probe 9：AATGAGCATTAGCTAC(SEQ NO24)
Probe 10：AATGAGCCTTAGCTAC(SEQ NO25)
Detection ACE (rs4646994) polymorphism primer and probe sequence include：
ACE (rs4646994) sense primer：TTTCTCCCATTTCTCTAGACCT(SEQ NO11)
ACE (rs4646994) downstream primer 1：ATCCCGCCACTGCACT(SEQ NO12)
ACE (rs4646994) downstream primer 2：TCAGAGAATTTCAGAGCTG(SEQ NO13)
Probe 11：CGCTCTGTCGCCCAGGC(SEQ NO26)
Probe 12：CTATACAGTCACTTTTATGTGGTTT(SEQ NO27)
Detection NPPA (rs5065) polymorphism primer and probe sequence include：
NPPA (rs5065) sense primer：CCAGCCCTGCTTGT(SEQ NO14)
NPPA (rs5065) downstream primer：AGGATGGGCACACTCAT(SEQ NO15)
Probe 13：TTATCTTCAGTACTG(SEQ NO28)
Probe 14：TTATCTTCGGTACTG(SEQ NO29).
2. detection combination object according to claim 1, which is characterized in that detection be CYP2C9*3 genes rs1057910 Site, the sites rs1065852 of CYP2D6 genes, the sites rs776746 of CYP3A5*3 genes, ADRB1 genes The sites rs1801253, the sites rs5186 of AGTR1 genes, ACE genes the sites rs4646994 and NPPA genes rs5065 The polymorphism in site.
3. detection combination object according to claim 1, which is characterized in that the probe is TaqMan probe, every probe 5 ' end be fluorescent reporter group, 3 ' end be quenching group, wherein fluorescent reporter group can be selected from FAM, TET, VIC, JOE, HEX, Cy3, Cy3.5, Cy5, Cy5.5, TAMRA, ROX, Texas Red, LC RED640 or LC RED705；Quenching group is optional From MGB, BHQ0, BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse or NFQ.
4. detection combination object according to claim 1, which is characterized in that further include PCR Buffer, Mg2+、dNTP、Taq Enzyme, DMSO and ROX.
5. application of the detection combination object in the detection kit for instructing hypertension medication described in one of Claims 1-4.
6. instruct the detection kit of hypertension medication, including detection CYP2C9*3 gene pleiomorphisms, CYP2D6 gene pleiomorphisms, CYP3A5*3 gene pleiomorphisms, ADRB1 gene pleiomorphisms, AGTR1 gene pleiomorphisms, ACE gene pleiomorphisms and NPPA genes are more Reagent described in the reagent of state property includes amplimer and probe, wherein：
Expand the primer and probe sequence of CYP2C9*3 (rs1057910)：
CYP2C9*3 (rs1057910) sense primer：ATTTAATGTCACAGGTCACTGC(SEQ NO1)
CYP2C9*3 (rs1057910) downstream primer：CACATGCCCTACACAGAT(SEQ NO2)
Probe 1：AGATACATTGACCTT(SEQ NO16)
Probe 2：AGATACCTTGACCTT(SEQ NO17)
Expand the primer and probe sequence of CYP2D6 (rs1065852)：
CYP2D6 (rs1065852) sense primer：TGCTCCTGGTGGACCTGA(SEQ NO3)
CYP2D6 (rs1065852) downstream primer：AGTCCACATGCAGCAGGT(SEQ NO4)
Probe 3：CGCTACTCACCAG(SEQ NO18)
Probe 4：CGCTACCCACCAG(SEQ NO19)
Expand the primer and probe sequence of CYP3A5*3 (rs776746)：
CYP3A5*3 (rs776746) sense primer：AGCTTAACGAATGCTCTAC(SEQ NO5)
CYP3A5*3 (rs776746) downstream primer：AGCAAGAGTCTCACACAGG(SEQ NO6)
Probe 5：CTTTCAATATCTCTT(SEQ NO20)
Probe 6：CTTTCAGTATCTCTT(SEQ NO21)
Expand the primer and probe sequence of ADRB1 (rs1801253)：
ADRB1 (rs1801253) sense primer：CCTTCAACCCCATCATCT(SEQ NO7)
ADRB1 (rs1801253) downstream primer：GGTCTCCGTGGGTCGCG(SEQ NO8)
Probe 7：TTCCAGGGACTGC(SEQ NO22)
Probe 8：TTCCAGCGACTGC(SEQ NO23)
Expand the primer and probe sequence of AGTR1 (rs5186)：
AGTR1 (rs5186) sense primer：CATTCCTCTGCAGCACTTCACT(SEQ NO9)
AGTR1 (rs5186) downstream primer：CGGTTCAGTCCACATAATGCAT(SEQ NO10)
Probe 9：AATGAGCATTAGCTAC(SEQ NO24)
Probe 10：AATGAGCCTTAGCTAC(SEQ NO25)
Expand the primer and probe sequence of ACE (rs4646994)：
ACE (rs4646994) sense primer：TTTCTCCCATTTCTCTAGACCT(SEQ NO11)
ACE (rs4646994) downstream primer 1：ATCCCGCCACTGCACT(SEQ NO12)
ACE (rs4646994) downstream primer 2：TCAGAGAATTTCAGAGCTG(SEQ NO13)
Probe 11：CGCTCTGTCGCCCAGGC(SEQ NO26)
Probe 12：CTATACAGTCACTTTTATGTGGTTT(SEQ NO27)
Expand the primer and probe sequence of NPPA (rs5065)：
NPPA (rs5065) sense primer：CCAGCCCTGCTTGT(SEQ NO14)
NPPA (rs5065) downstream primer：AGGATGGGCACACTCAT(SEQ NO15).
Probe 13：TTATCTTCAGTACTG(SEQ NO28)
Probe 14：TTATCTTCGGTACTG(SEQ NO29).
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