CN109897896A - A kind of TaqMan-MGB probe technique detection influences the development of methodology of fat-reducing medicament curative effect gene - Google Patents
A kind of TaqMan-MGB probe technique detection influences the development of methodology of fat-reducing medicament 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 fat-reducing medicament curative effect gene, detecting influences fat-reducing medicament curative effect gene polynorphisms in sample to be tested, the present invention is the same as six 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 is by detecting SLCO1B1 c.521T > C (rs4149056), SLCO1B1 c.388A > G (rs2302683), SLCO1B1 c.-910G > A (rs4149015), ApoE c.388T > C (rs429358 ), ApoE c.526C these functional meanings of > T (rs7412) and NPC1L1 c.-18C > A (rs41279633) mutation can adjuvant clinical doctor select suitable drug and dosage, or avoid the generation of drug interaction, patient is set to obtain 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 fat-reducing medicament curative effect gene SLCO1B1 is c.521T
> C, SLCO1B1 c.388A > G, SLCO1B1 c.-910G > A, ApoE c.388T > C, ApoE c.526C > T and
The detection method and its primer special, probe of each genotype of NPC1L1 c.-18C > A are led to using TaqMan/MGB probe technique
It crosses Real-TimePCR analysis system and quickly carries out genomic DNA amplification and fluorescence signal detection.
Background technique
According to statistics, hyperlipidemia number of patients in China's has been up to 1.6 hundred million, has 25,000,000 people in 35 years old or more crowd while suffering from
There are hypertension and hyperlipidemia.Although dyslipidemia non-evident sympton, once morbidity may cause disability or death.But China
Resident is also not nearly enough to the attention degree of dyslipidemia, does not recognize the harmfulness of dyslipidemia, largely with dyslipidemia
People fails to be found in time, and the blood lipid control of most patients diagnosed is unsatisfactory.According to solid to the metropolitan high gallbladder in 12, China
The investigation of alcoholemia patient shows that their blood lipid level controls up to standard only 26.5%, wherein patients with coronary heart disease control reaches
Target only has 16.6%.
Fat-reducing medicament can effectively prevent cardiovascular disease, but individual difference leads to some patient's unsatisfactory curative effects.About China,
The extensive Bloodlipid-lowering of Asia and European ethnic group is the study found that there is patient's LDL (low-density lipoprotein of 38.5%-59.5%
Cholesterol) fail to reach therapeutic purpose.There are many factor for influencing Bloodlipid-lowering, and inherent cause is one of Different therapeutical effect between individual
Main cause, participate in the albumen of lipid-metabolism, drug transporters and drug targets genetic polymorphism have to Bloodlipid-lowering it is important
It influences, such as SLCO1B1, ApoE, NPC1L1.
Statins are the drugs of a kind for the treatment of hypercholesterolemia, belong to 3- hydroxyl 3- methyl glutaryl coenzyme A
(HMG-CoA) reductase inhibitor, in the treatment guidelines of current abnormalities of sugar/lipid metabolism and atherosclerosis disease by
Recommend extensively, is the foundation stone of atherosclerotic cardiovascular disease primary prevention and secondary prevention.The whole world has more than 200,000,000
People takes such drug.Common statins have Pitavastatin (Pitavastatin), Pravastatin
(Pravastatin), Atorvastatin (Atorvastatin), Lovastatin (Lovastatin), Simvastatin
(Simvastatin), Fluvastatin (Fluvastatin) etc..Although statins are to cardiovascular disease and hyperlipemic patients
There is good curative effect, but in clinical application in more than 20 years, for statins there is also individual difference, some people will appear malicious pair
Effect, mainly includes hepatotoxicity wind agitation, muscle poison and neurotoxicity etc., incidence is respectively 1%~3%, 1.5%~3%,
0.045%, wherein muscle poison is especially prominent, serious to cause rhabdomyolysis (Rhabdomyolysis) even dead.
In drug combination, drug interaction can occur, serious muscle poison incidence can be increased.
Ezetimibe (Ezetimibe) is a kind of non-statins that can reduce enteral cholesterol absorption.Increase statin
Drug, Ezetimibe can preferably play the ability of persistently progressive reduction LDL cholesterol level, and can improve painstaking effort
It runs affairs the prognosis of part.In addition, reducing LDL cholesterol to than previous set lower level, more preferably effect can get.
Medical field wishes to confirm that it is suitble to be treated and can be reached with which kind of lipid-lowering medicine by the detection to some Patient genotype
To best therapeutic effect, and the generation of side effect is reduced, but to reach this " personalized medicine " target, also to be permitted
More researchs.This detection kit can assist doctor to carry out the selection of a few class fat-reducing medicaments, dosage, toxic reaction and curative effect
Prediction, can not only improve treatment curative effect, moreover it is possible to reduce the generation of adverse events.
Up to the present, the detection method of domestic only a few detection SLCO1B1 and ApoE gene pleiomorphism.This hair
It is bright for the c.521T > C of SLCO1B1 in qualitative detection human whole blood sample (EDTA or citric acid anticoagulated whole blood)
(rs4149056), SLCO1B1 c.388A > G (rs2302683), SLCO1B1 c.-910G > A (rs4149015), ApoE
C.388T > C (rs429358), ApoE c.526C > T (rs7412) and NPC1L1 c.-18C > A (rs41279633) six
The polymorphism in site.Testing result of the present invention only represent to mankind SLCO1B1 c.521T > C, SLCO1B1 c.388A > G,
The detection of SLCO1B1 c.-910G > A, ApoE c.388T > C, ApoE c.526C > T and NPC1L1 c.-18C > A gene
As a result, helping doctor to design personalized medicine scheme for patient provides reference.
Summary of the invention
The present invention, which provides a kind of detection, influences fat-reducing medicament curative effect gene SLCO1B1 c.521T > C, SLCO1B1
C.388A > G, SLCO1B1 c.-910G > A, ApoE c.388T > C, ApoE c.526C > T and NPC1L1 c.-18C > A
The detection method of each genotype, the present invention also provides for the SNP site progress on above-mentioned influence fat-reducing medicament related gene
The primer and TaqMan/MGB probe of real-time fluorescence PCR detection.
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 SLCO1B1 gene T521C SNP site:
Upstream primer: 5 '-AAAGGAATCTGGGTCATAC-3 '
Downstream primer: 5 '-CCTATTCCACGAAGCATA-3 '
Wild probe: 5 '-FAM-CCATGAACACATATA-MGB-3 '
Mutant probe: 5 '-HEX-CATGAACGCATATA-MGB-3 '
2) primer and TaqMan/MGB probe for the detection of SLCO1B1 Gene A 388G SNP site:
Upstream primer: 5 '-TTGATTAATTAAACAAGT-3 '
Downstream primer: 5 '-TCAGTGATGTTCTTACAG-3 '
Wild probe: 5 '-FAM-ATGAATTGATATTAG-MGB-3 '
Mutant probe: 5 '-HEX-ATGAATCGATATTAG-MGB-3 '
3) primer and TaqMan/MGB probe for the detection of SLCO1B1 gene G910A SNP site:
Upstream primer: 5 '-CTCATATATGCATCCTC-3 '
Downstream primer: 5 '-TATATACACACAAACAT-3 '
Wild probe: 5 '-FAM-TGTATACAGGTAAA-MGB-3 '
Mutant probe: 5 '-HEX-TGTATACAGGTAAA-MGB-3 '
4) primer and TaqMan/MGB probe for the detection of ApoE gene T388C SNP site:
Upstream primer: 5 '-CTGTCCAAGGAGCTG-3 '
Downstream primer: 5 '-CCGCGCACGTCCT-3 '
Wild probe: 5 '-FAM-CCGCACACGTCCT-MGB-3 '
Mutant probe: 5 '-HEX-CCGCGCACGTCCT-MGB-3 '
5) primer and TaqMan/MGB probe for the detection of ApoE gene C 526T SNP site:
Upstream primer: 5 '-CCTCGCCTCCCACC-3 '
Downstream primer: 5 '-CCCGGCCTGGTAC-3 '
Wild probe: 5 '-FAM-TGCCAGGCGCTTC-MGB-3 '
Mutant probe: 5 '-HEX-TGCCAGGCACTTC-MGB-3 '
6) primer and TaqMan/MGB probe for the detection of NPC1L1 gene C 18A SNP site:
Upstream primer: 5 '-TCACCAAGCGCAGGAG-3 '
Downstream primer: 5 '-CTGCCTTAATGTGTGTTCA-3 '
Wild probe: 5 '-FAM-CGCTGAGCCCTTC-MGB-3 '
Mutant probe: 5 '-HEX-CGCTGATCCCTTC-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 SLCO1B1 Gene A 521G 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) is extracted into the PCR reaction solution that obtained DNA is added to mankind's SLCO1B1 Gene A 388G 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；
4) rapid (1) is extracted into the PCR reaction solution that obtained DNA is added to mankind's SLCO1B1 gene G910A 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；
5) rapid (1) obtained DNA is extracted to be added in the PCR reaction solution of mankind's ApoE gene T388C 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 is bent
1. the channel FAM without amplification curve, VIC channel C t≤32 is judged as saltant type to line, and 2. the channel VIC is without amplification curve, the channel FAM
Ct≤32 are judged as wild type, and 3. FAM, VIC channel C t≤32, then be judged as heterozygous；
6) rapid (1) obtained DNA is extracted to be added in the PCR reaction solution of mankind's ApoE gene C 526T polymorphic site
PCR amplification is carried out, the Ct value in wild-type probe (channel FAM), the Ct value of saltant type probe (channel VIC) and the channel ROX are measured
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 expansion
Increase curve, FAM channel C t≤32 are judged as wild type, and 3. FAM, VIC channel C t≤32, then be judged as heterozygous；
7) rapid (1) obtained DNA is extracted to be added in the PCR reaction solution of mankind's NPC1L1 gene C 18A polymorphic site
PCR amplification is carried out, the Ct value in wild-type probe (channel FAM), the Ct value of saltant type probe (channel VIC) and the channel ROX are measured
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 expansion
Increase curve, FAM channel C t≤32 are 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 fat-reducing medicament curative effect gene, 25 μ L reaction
System specifically influences the PCR reaction solution of antihypertensive drugs curative effect gene mutation including detection, it is anti-that reaction solution contains PCR
Answer necessary buffer (buffer raw material: Tris, glycine betaine, potassium chloride, glycerol, Tween-20, magnesium chloride, dNTP),
Outside the substances such as Taq archaeal dna polymerase (being purchased from Fei Peng Biological Co., Ltd., article No.: MD001), H2O (distillation and separation method), also
Corresponding includes 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 fat-reducing medicament 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. expanding, 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 fat-reducing medicament 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 for influencing antihypertensive drugs curative effect related gene simultaneously, as a result
It is easy interpretation.This hair using MGB probe there are 3 ' end quenching groups not shine, closer in the position in space with reporter group,
The stability of hybridization is increased, keeps the result of experiment more accurate, 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 36 is quantitative using influence fat-reducing medicament 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: for influencing the TaqMan/MGB probe for real-time fluorescence PCR detection system of fat-reducing medicament curative effect gene
Primer and the screening of 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.
According to influence fat-reducing medicament curative effect related gene SLCO1B1 c.521T > C, SLCO1B1 c.388A > G,
C.388T c.526C the conserved region of > T and NPC1L1 c.-18C > A are set by > C, ApoE by SLCO1B1 c.-910G > A, ApoE
It counts primer and TaqMan/MGB probe, particular sequence is as follows:
1) primer and TaqMan/MGB probe for the detection of SLCO1B1 gene T521C SNP site:
Upstream primer: 5 '-AAAGGAATCTGGGTCATAC-3 '
Downstream primer: 5 '-CCTATTCCACGAAGCATA-3 '
Wild probe: 5 '-FAM-CCATGAACACATATA-MGB-3 '
Mutant probe: 5 '-HEX-CATGAACGCATATA-MGB-3 '
2) primer and TaqMan/MGB probe for the detection of SLCO1B1 Gene A 388G SNP site:
Upstream primer: 5 '-TTGATTAATTAAACAAGT-3 '
Downstream primer: 5 '-TCAGTGATGTTCTTACAG-3 '
Wild probe: 5 '-FAM-ATGAATTGATATTAG-MGB-3 '
Mutant probe: 5 '-HEX-ATGAATCGATATTAG-MGB-3 '
3) primer and TaqMan/MGB probe for the detection of SLCO1B1 gene G910A SNP site:
Upstream primer: 5 '-CTCATATATGCATCCTC-3 '
Downstream primer: 5 '-TATATACACACAAACAT-3 '
Wild probe: 5 '-FAM-TGTATACAGGTAAA-MGB-3 '
Mutant probe: 5 '-HEX-TGTATACAGGTAAA-MGB-3 '
4) primer and TaqMan/MGB probe for the detection of ApoE gene T388C SNP site:
Upstream primer: 5 '-CTGTCCAAGGAGCTG-3 '
Downstream primer: 5 '-CCGCGCACGTCCT-3 '
Wild probe: 5 '-FAM-CCGCACACGTCCT-MGB-3 '
Mutant probe: 5 '-HEX-CCGCGCACGTCCT-MGB-3 '
5) primer and TaqMan/MGB probe for the detection of ApoE gene C 526T SNP site:
Upstream primer: 5 '-CCTCGCCTCCCACC-3 '
Downstream primer: 5 '-CCCGGCCTGGTAC-3 '
Wild probe: 5 '-FAM-TGCCAGGCGCTTC-MGB-3 '
Mutant probe: 5 '-HEX-TGCCAGGCACTTC-MGB-3 '
6) primer and TaqMan/MGB probe for the detection of NPC1L1 gene C 18A SNP site:
Upstream primer: 5 '-TCACCAAGCGCAGGAG-3 '
Downstream primer: 5 '-CTGCCTTAATGTGTGTTCA-3 '
Wild probe: 5 '-FAM-CGCTGAGCCCTTC-MGB-3 '
Mutant probe: 5 '-HEX-CGCTGATCCCTTC-MGB-3 '.
Embodiment 2: the TaqMan/MGB probe for real-time fluorescence PCR detection of fat-reducing medicament 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 includes 6 kinds of reaction solutions: c.521T c.388A > G reacts SLCO1B1 by > C reaction solution, SLCO1B1
Liquid, SLCO1B1 c.-910G > A reaction solution, ApoE c.388T > C reaction solution, ApoE c.526C > T reaction solution and
NPC1L1 c.-18C > A reaction solution, each reaction solution include its corresponding primer, TaqMan/MGB probe, PCR reaction buffering
Liquid (Tris, glycine betaine, potassium chloride, glycerol, Tween-20, magnesium chloride, dNTP), Taq DNA polymerase are (purchased from luxuriant and rich with fragrance roc biology stock
Part Co., Ltd, article No.: MD001), the substances such as H2O (distillation and separation method).
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 Taq enzyme, 10
μM each 1.0 μ L of 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 amplification, 95 DEG C of 3min；3. expanding, 95 DEG C of 10sec, 61 DEG C of 40sec, totally 35 circulation, terminates in the extension of each circulation
The detection of Shi Jinhang fluorescence signal.Detection pattern is triple channel sonde method, 25 μ L of reaction volume.Name saves file in desktop,
Run 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 that c.521T (Fig. 1 is the site > C fluorescent quantitative PCR curve graph 3 sample SLCO1B1
The SLCO1B1 c.521T wild amplification curve of > C, internal reference amplification curve diagram；Fig. 2 is that c.521T > C mutation amplification is bent by SLCO1B1
Line, internal reference amplification curve diagram；Fig. 3 is SLCO1B1 c.521T > C heterozygosis amplification curve, internal reference amplification curve diagram；) Fig. 4~6 are
The corresponding 3 sample SLCO1B1 c.521T site > C sequencer map, (Fig. 4 is that sequencing result is that TT is wild, Fig. 5 result is CC prominent
Become, Fig. 6 result is TC 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 | 26.44 | NoCt | 23.91 |
| 2 | NoCt | 25.81 | 23.88 |
| 3 | 24.03 | 24.10 | 23.10 |
Fig. 7~9 are that c.388A (Fig. 7 is the site > G fluorescent quantitative PCR curve graph 3 sample SLCO1B1
The SLCO1B1 c.388A wild amplification curve of > G, internal reference amplification curve diagram；Fig. 8 is that c.388A > G mutation amplification is bent by SLCO1B1
Line, internal reference amplification curve diagram；Fig. 9 is SLCO1B1 c.388A > G heterozygosis amplification curve, internal reference amplification curve diagram；) Figure 10~12
For the corresponding 3 sample SLCO1B1 c.388A site > G sequencer map, (Figure 10 is that sequencing result is that AA is wild, Figure 11 result is
GG mutation, Figure 12 result are AG heterozygosis), consistent with real time fluorescent quantitative PCE testing result, following table is each sample amplification pair
Answer Ct value.
| Sample number | The channel FAM | The channel VIC | The channel ROX |
| 4 | 22.53 | NoCt | 22.27 |
| 5 | NoCt | 23.87 | 22.91 |
| 6 | 23.69 | 24.47 | 22.20 |
Figure 13~15 are that (Figure 13 is for 3 site sample SLCO1B1 c.-910G > A fluorescent quantitative PCR curve graphs
The wild amplification curve of SLCO1B1 c.-910G > A, internal reference amplification curve diagram；Figure 14 is that SLCO1B1 c.-910G > A mutation is expanded
Increase curve, internal reference amplification curve diagram；Figure 15 is SLCO1B1 c.-910G > A heterozygosis amplification curve, internal reference amplification curve diagram；) figure
16~19 be corresponding 3 site sample SLCO1B1 c.-910G > A sequencer maps, and (Figure 16 is that sequencing result is that GG is wild, Figure 17
As a result it is that AA is mutated, Figure 18 result is GA heterozygosis), consistent with real time fluorescent quantitative PCE testing result, following table is the amplification of each sample
As a result Ct value is corresponded to.
| Sample number | The channel FAM | The channel VIC | The channel ROX |
| 7 | 22.55 | NoCt | 21.82 |
| 8 | NoCt | 23.33 | 22.44 |
| 9 | 22.18 | 22.87 | 22.58 |
Figure 19~21 are the 3 sample ApoE c.388T site > C fluorescent quantitative PCR curve graph (Figure 19 ApoE
C.388T the wild amplification curve of > C, internal reference amplification curve diagram；Figure 20 is that c.388T > C is mutated amplification curve to ApoE, internal reference expands
Increase curve graph；Figure 21 is ApoE c.388T > C heterozygosis amplification curve, internal reference amplification curve diagram；) Figure 22~24 are corresponding 3
The sample ApoE c.388T site > C sequencer map, (Figure 22 is that sequencing result is that TT is wild, Figure 23 result is CC mutation, Tu24Jie
Fruit is TC 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 |
| 10 | 24.81 | NoCt | 21.16 |
| 11 | NoCt | 26.02 | 22.71 |
| 12 | 25.94 | 24.18 | 23.59 |
Figure 25~27 are the 3 sample ApoE c.526C site > T fluorescent quantitative PCR curve graph (Figure 25 ApoE
C.526C the wild amplification curve of > T, internal reference amplification curve diagram；Figure 26 is that c.526C > T is mutated amplification curve to ApoE, internal reference expands
Increase curve graph；Figure 27 is ApoE c.526C > T heterozygosis amplification curve, internal reference amplification curve diagram；) Figure 28~30 are corresponding 3
The sample ApoE c.526C site > T sequencer map, (Figure 28 is that sequencing result is that CC is wild, Figure 29 result is TT mutation, Tu30Jie
Fruit is CT 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 |
| 13 | 25.31 | NoCt | 23.13 |
| 14 | NoCt | 25.28 | 22.91 |
| 15 | 23.60 | 25.44 | 24.22 |
Figure 31~33 are that (Figure 31 is for 3 site sample NPC1L1 c.-18C > A fluorescent quantitative PCR curve graphs
The wild amplification curve of NPC1L1 c.-18C > A, internal reference amplification curve diagram；Figure 32 is that NPC1L1 c.-18C > A mutation amplification is bent
Line, internal reference amplification curve diagram；Figure 33 is NPC1L1 c.-18C > A heterozygosis amplification curve, internal reference amplification curve diagram；) Figure 34~36
For corresponding 3 site sample NPC1L1 c.-18C > A sequencer maps, (Figure 34 is that sequencing result is that CC is wild, Figure 35 result is AA
Mutation, Figure 36 result are CA heterozygosis), consistent with real time fluorescent quantitative PCE testing result, following table is each sample amplification pair
Answer Ct value.
| Sample number | The channel FAM | The channel VIC | The channel ROX |
| 16 | 22.86 | NoCt | 22.62 |
| 17 | NoCt | 24.56 | 22.89 |
| 18 | 24.40 | 24.93 | 23.21 |
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 fat-reducing medicament 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 SLCO1B1 gene T521C SNP site:
Upstream primer: 5 '-AAAGGAATCTGGGTCATAC-3 ',
Downstream primer: 5 '-CCTATTCCACGAAGCATA-3 ',
Wild probe: 5 '-FAM-CCATGAACACATATA-MGB-3 ',
Mutant probe: 5 '-HEX-CATGAACGCATATA-MGB-3 '；
2) primer and TaqMan/MGB probe for the detection of SLCO1B1 Gene A 388G SNP site:
Upstream primer: 5 '-TTGATTAATTAAACAAGT-3 ',
Downstream primer: 5 '-TCAGTGATGTTCTTACAG-3 ',
Wild probe: 5 '-FAM-ATGAATTGATATTAG-MGB-3 ',
Mutant probe: 5 '-HEX-ATGAATCGATATTAG-MGB-3 '；
3) primer and TaqMan/MGB probe for the detection of SLCO1B1 gene G910A SNP site:
Upstream primer: 5 '-CTCATATATGCATCCTC-3 ',
Downstream primer: 5 '-TATATACACACAAACAT-3 ',
Wild probe: 5 '-FAM-TGTATACAGGTAAA-MGB-3 ',
Mutant probe: 5 '-HEX-TGTATACAGGTAAA-MGB-3 '；
4) primer and TaqMan/MGB probe for the detection of ApoE gene T388C SNP site:
Upstream primer: 5 '-CTGTCCAAGGAGCTG-3 ',
Downstream primer: 5 '-CCGCGCACGTCCT-3 ',
Wild probe: 5 '-FAM-CCGCACACGTCCT-MGB-3 ',
Mutant probe: 5 '-HEX-CCGCGCACGTCCT-MGB-3 '；
5) primer and TaqMan/MGB probe for the detection of ApoE gene C 526T SNP site:
Upstream primer: 5 '-CCTCGCCTCCCACC-3 ',
Downstream primer: 5 '-CCCGGCCTGGTAC-3 ',
Wild probe: 5 '-FAM-TGCCAGGCGCTTC-MGB-3 ',
Mutant probe: 5 '-HEX-TGCCAGGCACTTC-MGB-3 '；
6) primer and TaqMan/MGB probe for the detection of NPC1L1 gene C 18A SNP site:
Upstream primer: 5 '-TCACCAAGCGCAGGAG-3 ',
Downstream primer: 5 '-CTGCCTTAATGTGTGTTCA-3 ',
Wild probe: 5 '-FAM-CGCTGAGCCCTTC-MGB-3 ',
Mutant probe: 5 '-HEX-CGCTGATCCCTTC-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 lipid-lowering medicine 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 SLCO1B1 Gene A 521G 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) by rapid (1) extract obtained DNA be added in the PCR reaction solution of mankind's SLCO1B1 Gene A 388G 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；
4) by rapid (1) extract obtained DNA be added in the PCR reaction solution of mankind's SLCO1B1 gene G910A 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；
5) DNA that rapid (1) extraction obtains is added in the PCR reaction solution of mankind's ApoE gene T388C 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；
6) DNA that rapid (1) extraction obtains is added in the PCR reaction solution of mankind's ApoE gene C 526T polymorphic site and is carried out
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 expansion
Increase curve, FAM channel C t≤32 are judged as wild type, and 3. FAM, VIC channel C t≤32, then be judged as heterozygous；
7) DNA that rapid (1) extraction obtains is added in the PCR reaction solution of mankind's NPC1L1 gene C 18A polymorphic site and is carried out
PCR amplification measures the Ct value, the Ct value of saltant type probe (channel VIC) and the Ct in the channel ROX in wild-type probe (channel FAM)
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 bent without amplification
Line, 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 fat-reducing medicament 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. the TaqMan/MGB probe for real-time fluorescence PCR detection system of fat-reducing medicament curative effect genotype according to claim 3
In further include the steps that RNP reference gene detect.
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