CN109897895A - A kind of TaqMan-MGB probe technique detection influences the development of methodology of antihypertensive drugs curative effect gene - Google Patents
A kind of TaqMan-MGB probe technique detection influences the development of methodology of antihypertensive drugs curative effect gene Download PDFInfo
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Abstract
The invention discloses a kind of methods that TaqMan-MGB probe for real-time fluorescence quantitative PCR technique joint-detection influences antihypertensive drugs curative effect gene, detecting influences antihypertensive drugs curative effect gene polynorphisms in sample to be tested, the present invention is the same as five SNP sites of detection when reaching, the primer and TaqMan-MGB probe specificity used is strong, PCR product pollution, which can be reduced, causes the risk of false positive to be grasped, it is easy to operate, as a result accurate and reliable, the present invention passes through detection NEDD4L-326G > A (rs4149601), AGTR1 1186A > C (rs5186), ACE I/D (rs1799752), ADRB1 1165G > C (rs1801253) and CYP3A5*3 (rs77674 6) mutation of these functional meanings can adjuvant clinical doctor select suitable drug and dosage, or avoid the generation of drug interaction, so that patient is obtained optimal therapeutic effect, to achieve the purpose that real " drug usage individuation ".
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of influence antihypertensive drugs curative effect gene NEDD4L c.-326G
> A, AGTR1c.*86A > C, ACE Indel, ADRB1 c.1165G the detection method of each genotype of > C and CYP3A5*3 and
Its primer special, probe quickly carry out genome by Real-TimePCR analysis system using TaqMan/MGB probe technique
DNA cloning and fluorescence signal detection.
Background technique
Hypertension is one of most independent, most important risk factor in many risk factors of cardiovascular and cerebrovascular disease.With high blood
Pressure, cerebral apoplexy and coronary heart disease are the cardiovascular and cerebrovascular disease of representative, seriously endanger human health.China is a hypertensive populations
Big country.Newest Chinese population cohort study that the periodical of JAMA in 2016 Internal Medicine is delivered (queue number:
500223) show that China's adult's Prevalence of Hypertension is 32.5%, diagnosis 30.5%, overall controlling of blood pressure rate is no more than
5%, hypertension prevention and control situation is extremely severe.
Figure one, all ages and classes, the average systolic (SBP) of gender and diastolic pressure (DBP)
The distribution of figure two, all ages and classes, gender hypertension.(four groups: having controlled, treated but do not controlled, diagnosed and do not controlled
It treats, diagnose)
Multiple factors affect the control rate of hypertension, such as: diagnosis is low, treatment rate is low, depressor is efficient low.With
People's health consciousness improve, diagnosis, treatment rate etc. can gradually take on a new look, and the effective percentage of depressor becomes pendulum in doctor and patient
And the problem huge together between family and society.The effective percentage of five big hypotensors be respectively thiazide diuretic class (~
50%), angiotensin-ii receptor retarding agent class (~50%), angiotensin converting enzyme inhibitors class (50-70%), β by
Body retarding agent class (50-60%), calcium ion antagonist class (50-60%).Clinic, which selects medicine, at present is administered by the method for trial and error,
I.e. empirical medication, best decompression drug effect also only has 20-70% according to statistics, and clinical medicine side reaction is difficult to avoid that, this
Kind invalid and unreasonable medication increases the financial burden of patient and delays the treatment of disease, therefore, according to individual with
Different therapeutic schemes is formulated in the hereditary variation of the relevant metabolic enzyme of drug therapy, transporter and receptor, realizes drug therapy
Body is not only the developing direction of current pharmacogenetics and clinical drug therapy, and has highly important social effect
And economic significance.
CYP2C9 and CYP2D6 is two kinds of main drug metabolic enzymes in human body, they participate in respectively metabolism Losartan etc. by
1 Adrenergic receptor blocker of the β such as body blocking agent and metoprolol；1 adrenocepter of β (ADRB1) is the kidneys such as metoprolol
The action target spot of upper parathyrine receptor blocker；Angiotensin-ii receptor (AGTR1) is the work of the AT1 receptor antagonist such as Losartan
Use target spot；Angiotensin converting enzyme (related gene ACE) is the effect of the angiotensin converting enzyme inhibitors such as benazepil
Target spot.Corresponding hypertension therapeutic drug will be significantly impacted by encoding the functional meaning mutation that the genes of these albumen is occurred
Individual reaction, this kit by detection NEDD4L -326G > A (rs4149601), AGTR1 1186A > C (rs5186),
ACE I/D (rs1799752), ADRB1 1165G > C (rs1801253) and CYP3A5*3 (rs776746) these functional meanings
The mutation of justice carrys out adjuvant clinical doctor and selects suitable drug and dosage, or avoids the generation of drug interaction, makes patient
Optimal therapeutic effect is obtained, to achieve the purpose that real " drug usage individuation ".
Summary of the invention
The present invention provides a kind of influence antihypertensive drugs curative effect related gene, NEDD4L c.-326G > A, AGTR1c.*86A
The detection method of > C, ACE Indel, ADRB1 c.1165G each genotype of > C and CYP3A5*3, the present invention also provides with
The primer and TaqMan/MGB probe of real-time fluorescence PCR detection are carried out in the SNP site to above-mentioned antihypertensive drugs related gene.
The purpose of the present invention is what is be achieved through the following technical solutions:
1. gene library searching, sequence alignment screen distinguished sequence；
2. designing probe and primer；
3. optimizing reaction condition；
The concentration of various reagents is prepared in 4.PCR reaction system；
5. pair TaqMan/MGB probe PCR technology established is verified, the method for sanger sequencing detects each genotype.
The nucleotide sequence of TaqMan/MGB probe and primer provided by the invention is as follows:
1) primer and TaqMan/MGB probe for the detection of NEDD4L gene G326A SNP site:
Upstream primer: 5 '-AGAACTTGCTCTGCCCTT-3 '
Downstream primer: 5 '-ATGTGGAGAGCTTCAGAAT-3 '
Wild probe: 5 '-FAM-CGAGTGTTACCTG-MGB-3 '
Mutant probe: 5 '-HEX-CGAGTGTTACTTGC-MGB-3 '
2) primer and TaqMan/MGB probe for the detection of AGTR1 Gene A 86C SNP site:
Upstream primer: 5 '-GAAGGAGCAAGAGAAC-3 '
Downstream primer: 5 '-GTCGGTTCAGTCCACAT-3 '
Wild probe: 5 '-FAM-AAATGAGCATTAG-MGB-3 '
Mutant probe: 5 '-HEX-AAATGAGCCTTAG-MGB-3 '
3) primer and TaqMan/MGB probe for the detection of ACE gene In/del SNP site:
Upstream primer: 5 '-AGCCACTCCCATCCTTTCTC-3 '
Downstream primer: 5 '-CCCTTAGCTCACCTCTGC-3 '
Wild probe: 5 '-FAM-ATAAAAGTGACTGTATCACG-MGB-3 '
Mutant probe: 5 '-HEX-ATAAAAGTGACTGTATAGG-MGB-3 '
4) primer and TaqMan/MGB probe for the detection of ADRB1 gene G1165C SNP site:
Upstream primer: 5 '-CTTCAACCCCATCATCTAC-3 '
Downstream primer: 5 '-GTCTCCGTGGGTCGC-3 '
Wild probe: 5 '-FAM-CAGAGCAGTCCCT-MGB-3 '
Mutant probe: 5 '-HEX-CAGAGCAGTCGCT-MGB-3 '
5) primer and TaqMan/MGB probe for the detection of CYP3A5 gene * 1/*3 SNP site:
Upstream primer: 5 '-ACTGTCATTTCTAACCATAATC-3 '
Downstream primer: 5 '-ACAGCAAGAGTCTCACAC-3 '
Wild probe: 5 '-FAM-TTTGTCTTTCAATAT-MGB-3 '
Mutant probe: 5 '-HEX-TTTTGTCTTTCAGTAT-MGB-3 '.
The present invention provides a kind of result accurately and reliably TaqMan/MGB probe for real-time fluorescence quantitative detecting method, specific to wrap
Include that steps are as follows:
1) extraction of human genome DNA to be measured；
2) rapid (1) is extracted into the PCR reaction solution that obtained DNA is added to mankind's NEDD4L gene G326A polymorphic site
Middle carry out PCR amplification measures the Ct value of wild-type probe (channel FAM) and the Ct value of saltant type probe (channel VIC), if amplification
1. the channel FAM without amplification curve, VIC channel C t≤32 is judged as saltant type to curve, and 2. without amplification curve, FAM is logical in the channel VIC
Road Ct≤32 are judged as wild type, and 3. FAM, VIC channel C t≤32, then be judged as heterozygous；
3) rapid (1) obtained DNA is extracted to be added in the PCR reaction solution of human AGT R1 Gene A 86C polymorphic site
PCR amplification is carried out, the Ct value of wild-type probe (channel FAM) and the Ct value of saltant type probe (channel VIC) are measured, if amplification
1. the channel FAM without amplification curve, VIC channel C t≤32 is judged as saltant type to curve, and 2. without amplification curve, FAM is logical in the channel VIC
Road Ct≤32 are judged as wild type, and 3. FAM, VIC channel C t≤32, then be judged as heterozygous；
4) rapid (1) obtained DNA is extracted to be added in the PCR reaction solution of mankind's ACE gene In/del polymorphic site
PCR amplification is carried out, the Ct value of wild-type probe (channel FAM) and the Ct value of saltant type probe (channel VIC) are measured, if amplification
1. the channel FAM without amplification curve, VIC channel C t≤32 is judged as saltant type to curve, and 2. without amplification curve, FAM is logical in the channel VIC
Road Ct≤32 are judged as wild type, and 3. FAM, VIC channel C t≤32, then be judged as heterozygous；
5) rapid (1) is extracted into the PCR reaction solution that obtained DNA is added to mankind's ADRB1 gene G1165C polymorphic site
Middle carry out PCR amplification measures the Ct value of wild-type probe (channel FAM) and the Ct value of saltant type probe (channel VIC), if amplification
1. the channel FAM without amplification curve, VIC channel C t≤32 is judged as saltant type to curve, and 2. without amplification curve, FAM is logical in the channel VIC
Road Ct≤32 are judged as wild type, and 3. FAM, VIC channel C t≤32, then be judged as heterozygous；
6) rapid (1) is extracted into the PCR reaction solution that obtained DNA is added to mankind's CYP3A5 gene * 1/*3 polymorphic site
It is logical to measure the Ct value in wild-type probe (channel FAM), the Ct value of saltant type probe (channel VIC) and ROX for middle carry out PCR amplification
The Ct value in road, if 1. the channel FAM without amplification curve, VIC channel C t≤32 is judged as saltant type to amplification curve, 2. the channel VIC without
Amplification curve, FAM channel C t≤32, is judged as wild type, and 3. FAM, VIC channel C t≤32, then be judged as heterozygous；
The TaqMan/MGB probe, 5 ' ends are marked with reporter group (Reporter, R), such as FAM, HEX, VIC or
The quenching group of ROX etc., 3 ' end labels use non-fluorescence quenching group (NFQ), itself does not generate fluorescence, can substantially reduce
The intensity of background signal, while MGB modification group is also connected on probe, primer Tm is worth low 10 DEG C or so than probe Tm, visits
The label at needle sequence both ends is ' FAM-3 ' MGB, 5 ' HEX-3 ' MGB, 5 ' ROX-3 ' BHQ2.
The TaqMan/MGB probe for real-time fluorescence PCR detection system of above-mentioned influence antihypertensive drugs curative effect gene, 25 μ L reaction
System, specifically, the PCR reaction solution including detecting the gene mutation of antihypertensive drugs curative effect, reaction solution contains PCR reaction must
Buffer (buffer raw material: Tris, glycine betaine, potassium chloride, glycerol, Tween-20, magnesium chloride, dNTP), the Taq DNA of palpus
It is also corresponding outside the substances such as polymerase (being purchased from Fei Peng Biological Co., Ltd., article No.: MD001), H2O (distillation and separation method)
Include above-mentioned detection primer and probe.The kit that assembling is formed saves under the conditions of -20 DEG C.
The TaqMan/MGB probe for real-time fluorescence PCR detection system of above-mentioned influence antihypertensive drugs curative effect gene, reaction condition
For 1. UNG enzymatic treatment, 37 DEG C of 3min；2. initial denaturation expands, 95 DEG C of 3min；3. it expands, 95 DEG C of 10sec, 61 DEG C of 40sec,
Totally 35 circulation carries out the detection of fluorescence signal at the end of the extension of each circulation.
For the accuracy of verification result, the TaqMan/MGB probe of above-mentioned influence antihypertensive drugs curative effect genotype is glimmering in real time
Further include the steps that detecting RNP reference gene in light PCR detection system.
The establishment of above-mentioned TaqMan/MGB probe for real-time fluorescence PCR detection architecture is in the full-automatic fluorescent PCR instrument of ABI7500
Upper progress, the optimization of screening, reaction system, reaction condition including primer and TaqMan/MGB probe.
Compared with prior art, the present invention at least have by above scheme it is following the utility model has the advantages that
1) present invention establishes the TaqMan/MGB probe for real-time fluorescence PCR detection for influencing antihypertensive drugs curative effect gene for the first time
Method, the present invention can quickly detect five SNP sites of antihypertensive drugs curative effect related gene simultaneously, as a result be easy
Interpretation.This hair using MGB probe there are 3 ' end quenching groups not shine, closer in the position in space with reporter group, increase
The stability of hybridization keeps the result of experiment more accurate, and resolution ratio is higher；
2) the detection primer in technical solution of the present invention and TaqMan/MGB probe price are lower, and testing cost is lower,
One polymorphic site only needs pipe qPCR detection can be completed, easy to operate.And the method for the present invention does not need to carry out PCR production
The subsequent analysis of object is not required to PCR product purifying, electrophoresis, digestion, hybridization etc., while saving testing cost, greatly shortens
Detection cycle, improves the efficiency of detection, in addition reduces the risk that PCR product pollution causes false positive.
Specific embodiment
Fig. 1-Figure 30 is quantitative using influence antihypertensive drugs curative effect gene TaqMan-MGB probe for real-time fluorescence of the invention
The amplification curve diagram of the method detection clinical sample of PCR detection.
Specific embodiment
The present invention is further discussed in detail with reference to the accompanying drawings and examples, it should be understood that attached drawing and implementation
Example is technical solution of the present invention to be easier to understand for those skilled in the art, and cannot function as the limit of the scope of the present invention
It is fixed.
In following introductions, term " first ", " second " only for descriptive purposes, and should not be understood as instruction or dark
Show relative importance.Following introductions provide multiple embodiments of the invention, can replace or merge between different embodiments
Combination, therefore the present invention is it is also contemplated that all possible combinations comprising documented identical and/or different embodiments.Thus, such as
Fruit one embodiment include feature A, B, C, another embodiment include feature B, D, then the present invention also should be regarded as include containing
A, the every other possible combined embodiment of one or more of B, C, D, although the embodiment may be in the following contents
In have specific literature record.
Embodiment 1: the primer of the TaqMan/MGB probe for real-time fluorescence PCR detection system for antihypertensive drugs curative effect gene
It is screened with TaqMan/MGB probe
Same variant sites allow 2 and 2 or less degeneracy bases in primer and probe design.Alternatively draw extracted
Object meets claimed below screened: 1. probe length (L) is between 19-28bp, and MGB probe length is between 13-20bp；②
Tm value is between 58-61 DEG C；3. GC% is between 25-75%；④poLyN≤4bp；⑤Hairpin≤4bp；6. coverage rate >
90%；7. amplified production length controls between 50-150bp；8. primer Tm is worth low 10 DEG C or so than probe Tm；9. carry out
BLAST screening, L × 0.4 specific score >.Best Tm value is set in 61 DEG C, and best MGB probe length is 13-18bp.Probe
The label at sequence both ends is ' FAM-3 ' MGB, 5 ' HEX-3 ' MGB, 5 ' ROX-3 ' BHQ2.Primer/probe screening principle are as follows:
Select PCR amplification efficiency and the higher primer and probe of probe joint efficiency as candidate drugs and probe in purpose area.
It is set according to the conserved region for influencing antihypertensive drugs curative effect related gene NEDD4L, AGTR1, ACE, ADRB1 and CYP3A5
It counts primer and TaqMan/MGB probe, particular sequence is as follows:
1) primer and TaqMan/MGB probe for the detection of NEDD4L gene G326A SNP site:
Upstream primer: 5 '-AGAACTTGCTCTGCCCTT-3 '
Downstream primer: 5 '-ATGTGGAGAGCTTCAGAAT-3 '
Wild probe: 5 '-FAM-CGAGTGTTACCTG-MGB-3 '
Mutant probe: 5 '-HEX-CGAGTGTTACTTGC-MGB-3 '
2) primer and TaqMan/MGB probe for the detection of AGTR1 Gene A 86C SNP site:
Upstream primer: 5 '-GAAGGAGCAAGAGAAC-3 '
Downstream primer: 5 '-GTCGGTTCAGTCCACAT-3 '
Wild probe: 5 '-FAM-AAATGAGCATTAG-MGB-3 '
Mutant probe: 5 '-HEX-AAATGAGCCTTAG-MGB-3 '
3) primer and TaqMan/MGB probe for the detection of ACE gene In/del SNP site:
Upstream primer: 5 '-AGCCACTCCCATCCTTTCTC-3 '
Downstream primer: 5 '-CCCTTAGCTCACCTCTGC-3 '
Wild probe: 5 '-FAM-ATAAAAGTGACTGTATCACG-MGB-3 '
Mutant probe: 5 '-HEX-ATAAAAGTGACTGTATAGG-MGB-3 '
4) primer and TaqMan/MGB probe for the detection of ADRB1 gene G1165C SNP site:
Upstream primer: 5 '-CTTCAACCCCATCATCTAC-3 '
Downstream primer: 5 '-GTCTCCGTGGGTCGC-3 '
Wild probe: 5 '-FAM-CAGAGCAGTCCCT-MGB-3 '
Mutant probe: 5 '-HEX-CAGAGCAGTCGCT-MGB-3 '
5) primer and TaqMan/MGB probe of CYP3A5 gene * 1/*3SNP site primer are used for:
Upstream primer: 5 '-ACTGTCATTTCTAACCAIAATC-3 '
Downstream primer: 5 '-ACAGCAAGAGTCTCACAC-3 '
Wild probe: 5 '-FAM-TTTGTCTTTCAATAT-MGB-3 '
Mutant probe: 5 '-HEX-TTTTGTCTTTCAGTAT-MGB-3 '.
Embodiment 2: the TaqMan/MGB probe for real-time fluorescence PCR detection of antihypertensive drugs curative effect gene is influenced
1) extraction of human genome DNA
The extraction of human peripheral's blood DNA, using Jiangsu all generations promise medical science and technology, Co., Ltd simply extracts whole blood gene
Group DNA kit carries out extracting genome DNA, and sample DNA for subsequent experimental or is stored in -20 DEG C after extraction.
2) configuration of PCR reaction system
Detection system include 5 kinds of reaction solutions: NEDD4L c.-326G > A reaction solution, AGTR1c.*86A > C reaction solution,
C.1165G > C reaction solution, CYP3A5*3 reaction solution, each reaction solution include that its is corresponding by ACE Indel reaction solution, ADRB1
Primer, TaqMan/MGB probe, PCR reaction buffer (Tris, glycine betaine, potassium chloride, glycerol, Tween-20, magnesium chloride,
DNTP), the objects such as Taq archaeal dna polymerase (being purchased from Fei Peng Biological Co., Ltd., article No.: MD001), H2O (distillation and separation method)
Matter.
The DNA sample extracted using step 1) carries out real-time fluorescence quantitative PCR amplification, using RNP as internal reference base as template
Cause, the reaction system of each PCR amplification are 25ul, include 2 × PCR reaction buffer, 12.5 μ l, 0.5 μ l of 5U/ul nq enzyme, 10 μ
Each 1.0 μ L of M upstream and downstream primer, each 0.5 μ L of 10 μM of probes, H2O supply 20 μ L, each 0.3 μ L of 10 μM of internal control primers, 10 μM of internal references
5 μ l (50ng) of DNA profiling is added in 0.3 μ L of probe, while setting up positive criteria product and negative control.
3) fluorescent PCR detects
ABI7500 fluorescence quantitative PCR instrument carries out each genetic test, reaction condition are as follows: 1. UNG enzymatic treatment, 37 DEG C of 3min；
2. initial denaturation expands, 95 DEG C of 3min；3. expanding, 95 DEG C of 10sec, 61 DEG C of 40sec, totally 35 circulation, in the extension of each circulation
At the end of carry out fluorescence signal detection.Detection pattern is triple channel sonde method, 25 μ L of reaction volume.Name, save file in
Desktop runs program.
4) genotype judges
Detection sample SNP site genotype is judged according to following table:
| Mode 1 | Mode 2 | Mode 3 | |
| The channel FAM | Without Ct | Ct≤32 | Ct≤32 |
| The channel VIC | Ct≤32 | Ct≤32 | Without Ct |
| Judgement | Saltant type | Heterozygous | Wild type |
Embodiment 3: result statistics
Fig. 1~3 are 3 sample NEDD4L c.-326G > A site fluorescent quantitative PCR curve graph (Fig. 1 NEDD4L
The wild amplification curve of c.-326G > A, internal reference amplification curve diagram；Fig. 2 is that NEDD4L c.-326G > A is mutated amplification curve, internal reference
Amplification curve diagram；Fig. 3 is NEDD4L c.-326G > A heterozygosis amplification curve, internal reference amplification curve diagram；) Fig. 4~6 are corresponding 3
The site sample NEDD4L c.-326G > A sequencer map, (Fig. 4 is that sequencing result is that GG is wild, Fig. 5 result is AA mutation, Fig. 6 knot
Fruit is GA heterozygosis), consistent with real time fluorescent quantitative PCE testing result, following table is that each sample amplification corresponds to Ct value.
| Sample number | The channel FAM | The channel VIC | The channel ROX |
| 1 | 23.04 | NoCt | 22.33 |
| 2 | NoCt | 23.44 | 22.12 |
| 3 | 24.06 | 24.99 | 22.83 |
Fig. 7~9 are 3 sample AGTR1c.*86A > C site fluorescent quantitative PCR curve graph (Fig. 7 AGTR1c.*
The wild amplification curve of 86A > C, internal reference amplification curve diagram；Fig. 8 is that AGTR1c.*86A > C is mutated amplification curve, internal reference amplification song
Line chart；Fig. 9 is AGTR1c.*86A > C heterozygosis amplification curve, internal reference amplification curve diagram；) Figure 10~12 are corresponding 3 samples
The site AGTR1c.*86A > C sequencer map, (Figure 10 is that sequencing result is that AA is wild, Figure 11 result is CC mutation, Figure 12 result is
AC heterozygosis), consistent with real time fluorescent quantitative PCE testing result, following table is that each sample amplification corresponds to Ct value.
| Sample number | The channel FAM | The channel VIC | The channel ROX |
| 4 | 23.35 | NoCt | 21.68 |
| 5 | NoCt | 23.16 | 21.82 |
| 6 | 23.71 | 24.60 | 22.89 |
Figure 13~15 are that (Figure 13 is ACE Indel wild to 3 site sample ACE Indel fluorescent quantitative PCR curve graphs
Raw amplification curve, internal reference amplification curve diagram；Figure 14 is that ACE Indel is mutated amplification curve, internal reference amplification curve diagram；Figure 15 is
ACE Indel heterozygosis amplification curve, internal reference amplification curve diagram；) Figure 16~19 are corresponding 3 sample sites ACE Indel sequencing
Figure, (Figure 16 is that sequencing result is that ACE In is wild, Figure 17 result is ACE del mutation, Figure 18,19 results are ACE Indel
Heterozygosis), consistent with real time fluorescent quantitative PCE testing result, following table is that each sample amplification corresponds to Ct value.
| Sample number | The channel FAM | The channel VIC | The channel ROX |
| 7 | 21.80 | NoCt | 22.36 |
| 8 | NoCt | 24.12 | 23.40 |
| 9 | 22.18 | 24.07 | 23.20 |
Figure 20~22 are that c.1165G (Figure 20 is the site > C fluorescent quantitative PCR curve graph 3 sample ADRB1
The ADRB1 c.1165G wild amplification curve of > C, internal reference amplification curve diagram；Figure 21 is that c.1165G > C mutation amplification is bent by ADRB1
Line, internal reference amplification curve diagram；Figure 22 is ADRB1 c.1165G > C heterozygosis amplification curve, internal reference amplification curve diagram；) Figure 23~25
For the corresponding 3 sample ADRB1 c.1165G site > C sequencer map, (Figure 23 is that sequencing result is that GG is wild, Figure 24 result is CC
Mutation, Figure 25 result are GC heterozygosis), consistent with real time fluorescent quantitative PCE testing result, following table is corresponding for each sample amplification
Ct value.
| Sample number | The channel FAM | The channel VIC | The channel ROX |
| 10 | 22.48 | NoCt | 22.07 |
| 11 | NoCt | 21.98 | 21.18 |
| 12 | 23.31 | 22.18 | 21.18 |
Figure 26~28 are that (Figure 26 is that CYP3A5*1 is wild to 3 site sample CYP3A5*3 fluorescent quantitative PCR curve graphs
Amplification curve, internal reference amplification curve diagram；Figure 27 is that CYP3A5*3 is mutated amplification curve, internal reference amplification curve diagram；Figure 28 is
CYP3A5*1/*3 heterozygosis amplification curve, internal reference amplification curve diagram；) Figure 29~31 are corresponding 3 sites sample CYP3A5*3 sequencing
Figure, (Figure 29 is that sequencing result is that AA is wild, Figure 30 result is GG mutation, Figure 31 result is AG heterozygosis), with real time fluorescent quantitative
PCE testing result is consistent, and following table is that each sample amplification corresponds to Ct value.
| Sample number | The channel FAM | The channel VIC | The channel ROX |
| 13 | 24.96 | NoCt | 21.92 |
| 14 | NoCt | 24.47 | 22.00 |
| 15 | 23.60 | 22.50 | 22.13 |
It is described above to be merely a preferred embodiment of the present invention, it is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Each real-time fluorescence PCR detection result passes through the verifying of sanger sequencing identification, and sequencing result and the present invention detect
As a result it compares unanimously, verifying detection method result is accurate and reliable.
Claims (6)
1. primer, TaqMan/MGB probe that a kind of detection influences the gene mutation of antihypertensive drugs curative effect, which is characterized in that primer
The sequence of probe is as follows:
1) primer and TaqMan/MGB probe for the detection of NEDD4L gene G326A SNP site:
Upstream primer: 5 '-AGAACTTGCTCTGCCCTT-3 ',
Downstream primer: 5 '-ATGTGGAGAGCTTCAGAAT-3 ',
Wild probe: 5 '-FAM-CGAGTGTTACCTG-MGB-3 ',
Mutant probe: 5 '-HEX-CGAGTGTTACTTGC-MGB-3 '；
2) primer and TaqMan/MGB probe for the detection of AGTR1 Gene A 86C SNP site:
Upstream primer: 5 '-GAAGGAGCAAGAGAAC-3 ',
Downstream primer: 5 '-GTCGGTTCAGTCCACAT-3 ',
Wild probe: 5 '-FAM-AAATGAGCATTAG-MGB-3 ',
Mutant probe: 5 '-HEX-AAATGAGCCTTAG-MGB-3 '；
3) primer and TaqMan/MGB probe for the detection of ACE gene In/del SNP site:
Upstream primer: 5 '-AGCCACTCCCATCCTTTCTC-3 ',
Downstream primer: 5 '-CCCTTAGCTCACCTCTGC-3 ',
Wild probe: 5 '-FAM-ATAAAAGTGACTGTATCACG-MGB-3 ',
Mutant probe: 5 '-HEX-ATAAAAGTGACTGTATAGG-MGB-3 '；
4) primer and TaqMan/MGB probe for the detection of ADRB1 gene G1165C SNP site:
Upstream primer: 5 '-CTTCAACCCCATCATCTAC-3 ',
Downstream primer: 5 '-GTCTCCGTGGGTCGC-3 ',
Wild probe: 5 '-FAM-CAGAGCAGTCCCT-MGB-3 ',
Mutant probe: 5 '-HEX-CAGAGCAGTCGCT-MGB-3 '；
5) primer and TaqMan/MGB probe of CYP3A5 gene * 1/*3SNP site primer are used for:
Upstream primer: 5 '-ACTGTCATTTCTAACCATAATC-3 ',
Downstream primer: 5 '-ACAGCAAGAGTCTCACAC-3 ',
Wild probe: 5 '-FAM-TTTGTCTTTCAATAT-MGB-3 ',
Mutant probe: 5 '-HEX-TTTTGTCTTTCAGTAT-MGB-3 '.
2. primed probe according to claim 1, it is characterised in that 5 ' ends are marked with reporter group (Reporter, R), such as
The quenching group of FAM, HEX, VIC or ROX etc., 3 ' end labels use non-fluorescence quenching group (NFQ), itself do not generate glimmering
Light can substantially reduce the intensity of background signal, while MGB modification group is also connected on probe, and primer Tm is than probe Tm
It is worth low 10 DEG C or so, the label at probe sequence both ends is ' FAM-3 ' MGB, 5 ' HEX-3 ' MGB, 5 ' ROX-3 ' BHQ2.
3. influencing depressor based on TaqMan-MGB probe for real-time fluorescence quantitative PCR technique joint-detection described in claim 1
The method of object curative effect gene, which is characterized in that specifically include that steps are as follows:
1) extraction of human genome DNA to be measured；
2) by rapid (1) extract obtained DNA be added in the PCR reaction solution of mankind's NEDD4L gene G326A polymorphic site into
Row PCR amplification measures the Ct value of wild-type probe (channel FAM) and the Ct value of saltant type probe (channel VIC), if amplification curve
1. the channel FAM is judged as saltant type without amplification curve, VIC channel C t≤32,2. the channel VIC is without amplification curve, FAM channel C t
≤ 32, it is judged as wild type, 3. FAM, VIC channel C t≤32, then be judged as heterozygous；
3) DNA that rapid (1) extraction obtains is added in the PCR reaction solution of human AGT R1 Gene A 86C polymorphic site and is carried out
PCR amplification measures the Ct value of wild-type probe (channel FAM) and the Ct value of saltant type probe (channel VIC), if amplification curve is 1.
The channel FAM is judged as saltant type without amplification curve, VIC channel C t≤32, and 2. the channel VIC is without amplification curve, FAM channel C t≤
32, it is judged as wild type, 3. FAM, VIC channel C t≤32, then be judged as heterozygous；
4) DNA that rapid (1) extraction obtains is added in the PCR reaction solution of mankind's ACE gene In/del polymorphic site and is carried out
PCR amplification measures the Ct value of wild-type probe (channel FAM) and the Ct value of saltant type probe (channel VIC), if amplification curve is 1.
The channel FAM is judged as saltant type without amplification curve, VIC channel C t≤32, and 2. the channel VIC is without amplification curve, FAM channel C t≤
32, it is judged as wild type, 3. FAM, VIC channel C t≤32, then be judged as heterozygous；
5) by rapid (1) extract obtained DNA be added in the PCR reaction solution of mankind's ADRB1 gene G1165C polymorphic site into
Row PCR amplification measures the Ct value of wild-type probe (channel FAM) and the Ct value of saltant type probe (channel VIC), if amplification curve
1. the channel FAM is judged as saltant type without amplification curve, VIC channel C t≤32,2. the channel VIC is without amplification curve, FAM channel C t
≤ 32, it is judged as wild type, 3. FAM, VIC channel C t≤32, then be judged as heterozygous；
6) by rapid (1) extract obtained DNA be added in the PCR reaction solution of mankind's CYP3A5 gene * 1/*3 polymorphic site into
Row PCR amplification measures the Ct value in wild-type probe (channel FAM), the Ct value of saltant type probe (channel VIC) and the channel ROX
Ct value, if 1. the channel FAM without amplification curve, VIC channel C t≤32 is judged as saltant type to amplification curve, 2. the channel VIC is without amplification
Curve, FAM channel C t≤32, is judged as wild type, and 3. FAM, VIC channel C t≤32, then be judged as heterozygous.
4. detection method according to claim 3, which is characterized in that the reaction system of each PCR amplification is 25ul, includes 2
12.5 μ l of × PCR reaction buffer, 0.5 μ l of 5U/ul Taq enzyme, each 1.0 μ L of 10 μM of upstream and downstream primers, each 0.5 μ L of 10 μM of probes,
10 μM of internal control primers each 0.3 μ L, 10 μM of 0.3 μ L of internal reference probe, H2O supply 20 μ L, and 5 μ l (50ng) of DNA profiling is added.
5. according to claim 3, influencing the TaqMan/MGB probe for real-time fluorescence PCR detection system of antihypertensive drugs curative effect gene
System, reaction condition are 1. UNG enzymatic treatment, 37 DEG C of 3min；2. initial denaturation expands, 95 DEG C of 3min；3. expanding, 95 DEG C of 10sec, 61 DEG C
40sec, totally 35 circulation, carries out the detection of fluorescence signal at the end of the extension of each circulation.
6. influencing the TaqMan/MGB probe for real-time fluorescence PCR detection of antihypertensive drugs curative effect genotype according to claim 3
Further include the steps that detecting RNP reference gene in system.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111172276A (en) * | 2020-03-09 | 2020-05-19 | 东莞博奥木华基因科技有限公司 | Primer for detecting ACE gene polymorphism and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103226667A (en) * | 2013-05-17 | 2013-07-31 | 西安时代基因健康科技有限公司 | Method for acquiring characterization parameters of hypertension and complication |
| CN103757109A (en) * | 2014-01-13 | 2014-04-30 | 宁波海尔施基因科技有限公司 | Primer composition and multiple-gene detection kit for guiding administration of thiazine diuresis drugs and application method thereof |
| US20160153053A1 (en) * | 2010-08-31 | 2016-06-02 | The General Hospital Corporation | Cancer-related biological materials in microvesicles |
| CN106367479A (en) * | 2016-08-25 | 2017-02-01 | 杭州百迈生物股份有限公司 | Detection composition for guiding hypertension medication, applications of detection composition, kit and detection method |
| CN106520979A (en) * | 2016-11-30 | 2017-03-22 | 武汉海吉力生物科技有限公司 | Nucleic acid, kit and method for detecting G1165C polymorphism of human ADRB1 gene |
| 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 |
-
2019
- 2019-01-11 CN CN201910029841.8A patent/CN109897895A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160153053A1 (en) * | 2010-08-31 | 2016-06-02 | The General Hospital Corporation | Cancer-related biological materials in microvesicles |
| CN103226667A (en) * | 2013-05-17 | 2013-07-31 | 西安时代基因健康科技有限公司 | Method for acquiring characterization parameters of hypertension and complication |
| CN103757109A (en) * | 2014-01-13 | 2014-04-30 | 宁波海尔施基因科技有限公司 | Primer composition and multiple-gene detection kit for guiding administration of thiazine diuresis drugs and application method thereof |
| CN106367479A (en) * | 2016-08-25 | 2017-02-01 | 杭州百迈生物股份有限公司 | Detection composition for guiding hypertension medication, applications of detection composition, kit and detection method |
| CN106520979A (en) * | 2016-11-30 | 2017-03-22 | 武汉海吉力生物科技有限公司 | Nucleic acid, kit and method for detecting G1165C polymorphism of human ADRB1 gene |
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| MICHAEL T. EADON AND ARLENE B. CHAPMAN: "A Physiologic Approach to the Pharmacogenomics of Hypertension", 《ADVANCES IN CHRONIC KIDNEY DISEASE》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172276A (en) * | 2020-03-09 | 2020-05-19 | 东莞博奥木华基因科技有限公司 | Primer for detecting ACE gene polymorphism and application thereof |
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