CN109457024A - Shr gene polymorphism fluorescent PCR solubility curve detection kit and application - Google Patents
Shr gene polymorphism fluorescent PCR solubility curve detection kit and application Download PDFInfo
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Abstract
The present invention relates to a kind of shr gene polymorphism fluorescent PCR solubility curve detection kits, and its application and a kind of multiplex PCR melting curve detection method for detecting hypertension medication gene.The kit mainly includes primer combination, probe combinations, and the PCR reaction reagent such as thermal starting polymerase, PCR reaction buffer, dNTPs, the 7 kinds of genetic polymorphism detections of correlation of medication containing hypertension, it is CYP2D6*10, CYP2C9*3, ADRB1 (1165G > C), AGTR1 (1166A > C), CYP3A5*3, NPPA (T2238C), ACE (I/D) respectively, PCR amplification and liquation are carried out by 3 reaction solutions, the case where judging 7 gene-correlation polymorphisms, to instruct the personalized application of 5 major class drug of Clinical Hypertension.The present invention is easily accomplished detection process by 3 reaction tubes, while detection process is not necessarily to nucleic acid extraction process, simplifies operating procedure, and interpretation method is simple.
Description
(1) technical field
The present invention relates to a kind of shr gene polymorphism fluorescent PCR solubility curve detection kits, and its application, and
A kind of multiplex PCR melting curve detection method detecting hypertension medication gene.
(2) background technique
Hypertension is higher as a kind of Chronic Non-Communicable Diseases and China's illness rate, disability rate is higher and disease is negative
Carry on a shoulder pole heavier chronic disease.The data of the publication of national health State Family Planning Commission in 2016 are shown: China 18 years old and the above adult hypertension
Illness rate is 25.2%.Although hypertension awareness, treatment rate, the control rate of population of China have improvement in recent years, place
In reduced levels.Global disease burden is shown: Chinese population because caused by hypertension Disability adjusted life years (DALY) it is high
Up to 37,940,000 man-years, the 12.0% of the total DALY of Zhan, account for the 63.5% of the total DALY of cardiovascular disease；Wherein disability Years of life lost
It (YLD) is 35,570,000 man-years, early death Years of life lost (YLL) is 236.5 ten thousand man-years, accounts for cardiovascular disease YLD and YLL
50.1% and 64.5%, it is the first risk factor of cardiovascular disease burden.The whole nation every year because blood pressure increase caused by premature death
Number is up to more than 200 ten thousand, and annual direct medical cost is up to 36,600,000,000, the reach mark blood pressure rate of the hypertensive patient after China's treatment
29.6%.For hypertension as the most important risk factor of cardiovascular and cerebrovascular diseases, epidemic status is serious, major complications such as stroke,
The lethality height of disabling of myocardial infarction, heart failure and chronic kidney disease etc., serious consumption medical treatment and social resources, to family and
Society causes heavy burden, it has also become the important public health problem in one, China.Although the diagnosis of hypertension in recent years and controlling
Treatment makes significant progress, and hypertension therapeutic drug also emerges one after another, but condition of medicine treatment for hypertension also exist it is many it is unreasonable it
Thus place also affects compliance, duration and the controlling of blood pressure rate of patient's treatment.
The individual difference of drug response is the principal element that clinically current controlling of blood pressure rate is low, generates this species diversity
Reason has very much, for example many factors including raising, gene, gender, weight, disease condition can lead to Different Individual for medicine
The difference of the reaction of object, however inherent cause is wherein the most fundamental factor, the metabolism of drug, transhipment, receptor gene polymorphic
Property directly determine its coding protein function difference, thus be drug metabolism absorb generate difference, lead to people's blood concentration
It is dramatically different.Current clinically common 5 major class drug and corresponding oligogene polymorphism and function effect (table as follows
1):
Table 1: the clinical meaning of hypertension drug associated genotype
Above-described gene mutation have it is larger may be related to reaction of the hypertension drug in Different Individual, it is identical
The drug of dosage generates notable difference in Different Individual, and effectively, some individuals are absolutely void or even somebody produces for some individuals
Raw drug response.Therefore, genetic test is carried out for hypertensive patient using effectively genetic polymorphism detection technology, according to inspection
The types of medicines or dosage for surveying the adjustable patient of result, can make drug play the best use, while can prevent drug
Toxic side effect.This provides optimal scientific practice argument for Personalized medicine in precisely medical treatment, to meet wider individuation
The arriving in medical epoch keeps punching.
Have much for the technology of genetic polymorphism detection at present, it is most common to have polymerase chain reaction-restricted
The technologies such as segment length polymorphism analysis (PCR-RFLP method), sequence-specific PCR, generation sequencing, the sequencing of two generations, but these skills
Art has respective applied defect, and what is had is cumbersome, and detection cycle is long, can not carry out high-throughput multidigit point while detect, so that
It is clinically still undesirable for polymorphism detection technique application at present, it is unable to satisfy clinical detection demand always.
Multiple fluorescence PCR combination high-resolution sonde method melting curve method is a kind of novel snp analysis technology, is from HRM
It is improved on the basis of (high-resolution solubility curve) technology, the saturable dye in HRM is substituted with probe, and probe is set
The position of meter just in SNP site, keeps testing result more direct, and more specifically, and maximum advantage is can to carry out parting
Detection, a probe distinguish all polymorphic implementations of a SNP site, detect in conjunction with multicolor fluorescence, make to detect flux significantly
It is promoted, and one fluorescence channel of conventional fluorescent PCR method can only detect a kind of genotype, if using sonde method melting curve method one
A fluorescence channel can detecte 3 kinds of genotype or even can be more, and flux is made to amplify 3 times, and what it is with greater need for explanation is using conventional
Fluorescence PCR method faces the interference between hypotype, so that the method that interpretation majority uses Ct value difference, makes testing result become unstable
It is fixed.
(3) summary of the invention
In order to overcome the deficiencies of the prior art, the object of the present invention is to provide hypertension individuation medication guide genetic tests
Kit and its application, including detect respectively CYP2C9*3 gene, CYP2D6 gene, CYP3A5*3 gene, ADRB1 gene,
The reagent of AGTR1 gene, ACE gene and NPPA gene pleiomorphism.
A kind of shr gene polymorphism fluorescent PCR solubility curve detection kit, it is main include three groups of primer pairs and
PCR reaction reagent, which is characterized in that the primer sequence is as follows:
Primed probe group one is CYP3A5*3 and NPPA primer:
CYP3A5*3 primer probe sequence is as follows:
Upstream primer: 5 '-TAATGAAGTATTTTAAACATATA-3 '
Downstream primer: 5 '-GTTCTAGTTCATTAGGGTGTGACAC-3 '
Fluorescence probe: FAM-TTTGTCTTTCAATATCTC-BQ1；
NPPA primer probe sequence is as follows:
Upstream primer: 5 '-GATGGGATGATCACAAC-3 '
Downstream primer: 5 '-CCAGGTCACCAAGCCAGATA-3 '
Fluorescence probe: HEX-TGTTATCTTCAGTAC-BQ2；
Primed probe group two is AGTR1, CYP2C9*3 and ACE (I/D) primer:
AGTR1 primer probe sequence is as follows:
Upstream primer: 5 '-TTCTAAAATATATTCCCC-3 '
Downstream primer: 5 '-GAAAAGTCGGTTCAGTCCACATAAT-3 '
Fluorescence probe: FAM-CCAAATGAGCATTAGCT-BQ1；
CYP2C9*3 primer probe sequence is as follows:
Upstream primer: 5 '-AGAGATTGAACGTGTGATTGGCA-3 '
Downstream primer: 5 '-TGATACTATGAATTTGGGGACTTCG-3 '
Fluorescence probe: HEX-GAGATACATTGACC-BQ2；
ACE (I/D) primer probe sequence is as follows:
Upstream primer: 5 '-TCCCATCCTTTCTCCCATTTCTCTA-3 '
Downstream primer: 5 '-CCCTTAGCTCACCTCTGCTTGTA-3 '
Fluorescence probe: CY5-GCTGCCTATACAGTCACT-BQ2；
Primed probe group three is ADRB1 and CYP2D6*10 primer:
ADRB1 primer probe sequence is as follows:
Upstream primer: 5 '-CCCATCATCTACTGCCGCAGC-3 '
Downstream primer: 5 '-TCTCCGTGGGTCGCGTG-3 '
Fluorescence probe: FAM-CTTCCAGGGACTGCTCT-BQ1；
CYP2D6*10 primer probe sequence is as follows:
Upstream primer: 5 '-CCCATTTGGTAGTGAGGCAGGT-3 '
Downstream primer: 5 '-GACCTGATGCACCGGCG-3 '
Fluorescence probe: HEX-GCACGCTACCCACCAGG-BQ2.
When specific detection, PCR reaction solution 1 is prepared with primed probe group one and PCR reaction reagent, with two He of primed probe group
PCR reaction reagent prepares PCR reaction solution 2, prepares PCR reaction solution 3 with primed probe group three and PCR reaction reagent, passes through 3
Reaction solution detects the results of 7 gene pleiomorphisms respectively.Each reaction solution includes at least two fluorescence channel, detects equipotential base
Because of type, to instruct clinical application.
What above-mentioned primer specifically detected is the site rs1057910, the CYP2D6 gene of CYP2C9*3 gene
The site rs1065852, the site rs776746 of CYP3A5*3 gene, the site rs1801253 of ADRB1 gene, AGTR1 gene
The site rs5186, ACE gene the site rs4646994 and NPPA gene the site rs5065 polymorphism.
Reaction solution 1 include thermal starting polymerase, PCR buffer, dNTPs, CYP3A5*3 primer, CYP3A5*3 probe,
NPPA primer, NPPA probe.
Reaction solution 2 includes: thermal starting polymerase, PCR buffer, dNTPs, AGTR1 primer, AGTR1 probe, CYP2C9*3
Primer, CYP2C9*3 probe, ACE (I/D) primer, ACE (I/D) probe.
Reaction solution 3 includes: thermal starting polymerase, PCR buffer, dNTPs, ADRB1 primer, ADRB1 probe, CYP2D6*
10 primers, CYP2D6*10 probe.
The kit also typically includes 7 gene pleiomorphism plasmid standards as positive control, the standard items sequence
It arranges as follows:
CYP3A5*3 standard items sequence:
CYP3A5*3 saltant type:
5’-CTTTTTGATAATGAAGTATTTTAAACATATAAAACATTATGGAGAGTGGCA TAGGAGATACCCA
CGTATGTACCACCCAGCTTAACGAATGCTCTACTGTCATTTC TAACCATAATCTCTTTAAAGAGCTCTTTTGTCT
TTCAGTATCTCTTCCCTGTTTGG ACCACATTACCCTTCATCATATGAAGCCTTGGGTGGCTCCTGTGTGAGACTC
TTG CTGTGTGTCACACCCTAATGAACTAGAACCTAAGGTTGCTGTGTGTCGTACAACT AGGGGTAT-3'；
CYP3A5*3 wild type:
5’-CTTTTTGATAATGAAGTATTTTAAACATATAAAACATTATGGAGAGTGGCA TAGGAGATACCCA
CGTATGTACCACCCAGCTTAACGAATGCTCTACTGTCATTTC TAACCATAATCTCTTTAAAGAGCTCTTTTGTCT
TTCAATATCTCTTCCCTGTTTGG ACCACATTACCCTTCATCATATGAAGCCTTGGGTGGCTCCTGTGTGAGACTC
TTG CTGTGTGTCACACCCTAATGAACTAGAACCTAAGGTTGCTGTGTGTCGTACAACT AGGGGTAT-3'；
NPPA standard items sequence:
NPPA saltant type:
5’-GCTTAGATGGGATGATCACAACTCCATGGCAACAAGATGACACAAATGC AGCAGAGACCCCAGG
GGACAGGAGCCTCTTGCAGTCTGTCCCTAGGCCCAGCC CTGCTTGTCCTCCCTGGCTGTTATCTTCGGTACTGCA
AAGAGAACACAGACATAT CTGGCTTGGTGACCTGGCTGTCCTGGAAAAGTCAGCTTCATGTATGAGTGTGCC CA
TCCTCTGAACTTGATTACTGACCACCTGCTTCCCACCGGCCCCCACCCCAGCC TGATGACCCTCTGA-3'；
NPPA wild type:
5’-GCTTAGATGGGATGATCACAACTCCATGGCAACAAGATGACACAAATGC AGCAGAGACCCCAGG
GGACAGGAGCCTCTTGCAGTCTGTCCCTAGGCCCAGCC CTGCTTGTCCTCCCTGGCTGTTATCTTCAGTACTGCA
AAGAGAACACAGACATAT CTGGCTTGGTGACCTGGCTGTCCTGGAAAAGTCAGCTTCATGTATGAGTGTGCC CA
TCCTCTGAACTTGATTACTGACCACCTGCTTCCCACCGGCCCCCACCCCAGCC TGATGACCCTCTGA-3'；
AGTR1 standard items sequence:
AGTR1 saltant type:
5’-CTCCAGCTTCTAAAATATATTCCCCCAAAAGCCAAATCCCACTCAAACCT TTCAACAAAAATGA
GCACGCTTTCCTACCGCCCCTCAGATAATGTAAGCTCATCC ACCAAGAAGCCTGCACCATGTTTTGAGGTTGAGT
GACATGTTCGAAACCTGTCC ATAAAGTAATTTTGTGAAAGAAGGAGCAAGAGAACATTCCTCTGCAGCACTTCA
CTACCAAATGAGCCTTAGCTACTTTTCAGAATTGAAGGAGAAAATGCATTATGTG GACTGAACCGACTTTTCTAA
AGCTCTGAACAAAAGCTTTTCTTTCCTTTTGCAA CAAGACAAAGCAAAGCCACATTTTGCATTAGACAGATGACG
GCTGCTCGAAGA ACAATGTCAGAAACTCGATGAATGTGTTGATTTGAGAAATTTTACTGACAGAAAT
GCAATCTCCCTAGCCTGCTT-3'；
AGTR1 wild type:
5’-CTCCAGCTTCTAAAATATATTCCCCCAAAAGCCAAATCCCACTCAAACCT TTCAACAAAAATGA
GCACGCTTTCCTACCGCCCCTCAGATAATGTAAGCTCATCC ACCAAGAAGCCTGCACCATGTTTTGAGGTTGAGT
GACATGTTCGAAACCTGTCC ATAAAGTAATTTTGTGAAAGAAGGAGCAAGAGAACATTCCTCTGCAGCACTTCA
CTACCAAATGAGCATTAGCTACTTTTCAGAATTGAAGGAGAAAATGCATTATGTG GACTGAACCGACTTTTCTAA
AGCTCTGAACAAAAGCTTTTCTTTCCTTTTGCAA CAAGACAAAGCAAAGCCACATTTTGCATTAGACAGATGACG
GCTGCTCGAAGA ACAATGTCAGAAACTCGATGAATGTGTTGATTTGAGAAATTTTACTGACAGAAAT
GCAATCTCCCTAGCCTGCTT-3'；
CYP2C9*3 standard items sequence:
CYP2C9*3 saltant type:
5’-TCTCCTTTTCCATCAGTTTTTACTTGTGTCTTATCAGCTAAAGTCCAGGA AGAGATTGAACGTG
TGATTGGCAGAAACCGGAGCCCCTGCATGCAAGACAGGA GCCACATGCCCTACACAGATGCTGTGGTGCACGAGG
TCCAGAGATACCTTGACC TTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGACATTAAATTCAGAAACTA T
CTCATTCCCAAGGTAAGTTTGTTTCTCCTACACTGCAACTCCATGTTTTCGAAG TCCCCAAATTCATAGTATCAT
TTTTAAACCTCT-3'；
CYP2C9*3 wild type:
5’-AGAGATTGAACGTGTGATTGGCAGAAACCGGAGCCCCTGCATGCAAGA CAGGAGCCACATGCCC
TACACAGATGCTGTGGTGCACGAGGTCCAGAGATACAT TGACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACC
TGTGACATTAAATTCAG AAACTATCTCATTCCCAAGGTAAGTTTGTTTCTCCTACACTGCAACTCCATGTTTT C
GAAGTCCCCAAATTCATAGTATCATTTTTAAACCTCT-3’
ACE (I/D) standard items sequence:
ACE-D plasmid:
5’-AGACTCAAGCACGCCCCTCACAGGACTGCTGAGGCCCTGCAGGTGTCT GCAGCATGTGGCCCCA
GGCCGGGGACTCTGTAAGCCACTGCTGGAGAGCCACT CCCATCCTTTCTCCCATTTCTCTAGACCTGCTGCCTAT
ACAGTCACTTTTATGTGG TTTCGCCAATTTTATTCCAGCTCTGAAATTCTCTGAGCTCCCCTTACAAGCAGAG G
TGAGCTAAGGGCTGGAGCTCAAGGCATTCAAACCCCTACCAGATCTGACGAAT GTGATGGCCACGTCCCGGAAAT
ATGAAGACCTGTTATGGGCATGGGAGGGCTGG CGAGACAAGGCGGGGAGAGCCATCCTCCAGTTTTACCCGAAAT
ACGTGGAACT CATCAACCAGGCTGCCCGGCTCAATG-3'；
ACE-I plasmid:
5’-AGACTCAAGCACGCCCCTCACAGGACTGCTGAGGCCCTGCAGGTGTCT GCAGCATGTGGCCCCA
GGCCGGGGACTCTGTAAGCCACTGCTGGAGAGCCACT CCCATCCTTTCTCCCATTTCTCTAGACCTGCTGCCTAT
ACAGTCACTTTTTTTTTTT TTTTGAGACGGAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCGGGATCT C
GGCTCACTGCAAGCTCCGCCTCCCGGGTTCACGCCATTCTCCTGCCTCAGCCT CCCAAGTAGCTGGGACCACAGG
CGCCCGCCACTACGCCCGGCTAATTTTTTGTA TTTTTAGTAGAGACGGGGTTTCACCGTTTTAGCCGGGATGGTC
TCGATCTCCTGA CCTCGTGATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGATA CAGTCACT
TTTATGTGGTTTCGCCAATTTTATTCCAGCTCTGAAATTCTCTGAGCT CCCCTTACAAGCAGAGGTGAGCTAAGG
GCTGGAGCTCAAGGCATTCAAACCCC TACCAGATCTGACGAATGTGATGGCCACGTCCCGGAAATATGAAGACCT
GTTATG GGCATGGGAGGGCTGGCGAGACAAGGCGGGGAGAGCCATCCTCCAGTTTTACC CGAAATACGTGGAAC
TCATCAACCAGGCTGCCCGGCTCAATG-3'；
ADRB1 standard items sequence:
ADRB1 saltant type:
5’-ATGGGCGTCTTCACGCTCTGCTGGCTGCCCTTCTTCCTGGCCAACGTGG TGAAGGCCTTCCACC
GCGAGCTGGTGCCCGACCGCCTCTTCGTCTTCTTCAACT GGCTGGGCTACGCCAACTCGGCCTTCAACCCCATCA
TCTACTGCCGCAGCCCCG ACTTCCGCAAGGCCTTCCAGCGACTGCTCTGCTGCGCGCGCAGGGCTGCCCGCC GG
CGCCACGCGACCCACGGAGACCGGCCGCGCGCCTCGGGCTGTCTGGCCCGG CCCGGACCCCCGCCATCGCCCGGG
GCCGCCTCGGACGACGACGACGACGATGT CGTCGGGGCCACGCCGCCCGCGCGCCTGCTGGAGCCCTGGGCCGGC
TGCAACG GCGGGGCGGCGGC-3'；
ADRB1 wild type:
5’-ATGGGCGTCTTCACGCTCTGCTGGCTGCCCTTCTTCCTGGCCAACGTGG TGAAGGCCTTCCACC
GCGAGCTGGTGCCCGACCGCCTCTTCGTCTTCTTCAACT GGCTGGGCTACGCCAACTCGGCCTTCAACCCCATCA
TCTACTGCCGCAGCCCCG ACTTCCGCAAGGCCTTCCAGGGACTGCTCTGCTGCGCGCGCAGGGCTGCCCGC CGG
CGCCACGCGACCCACGGAGACCGGCCGCGCGCCTCGGGCTGTCTGGCCCG GCCCGGACCCCCGCCATCGCCCGGG
GCCGCCTCGGACGACGACGACGACGATG TCGTCGGGGCCACGCCGCCCGCGCGCCTGCTGGAGCCCTGGGCCGGC
TGCAAC GGCGGGGCGGCGGC-3'；
CYP2D6*10 standard items sequence:
CYP2D6*10 saltant type:
5’-GTGCTGAGAGTGTCCTGCCTGGTCCTCTGTGCCTGGTGGGGTGGGGGT GCCAGGTGTGTCCAGA
GGAGCCCATTTGGTAGTGAGGCAGGTATGGGGCTAGA AGCACTGGTGCCCCTGGCCGTGATAGTGGCCATCTTCC
TGCTCCTGGTGGACCT GATGCACCGGCGCCAACGCTGGGCTGCACGCTACTCACCAGGCCCCCTGCCACT GCCC
GGGCTGGGCAACCTGCTGCATGTGGACTTCCAGAACACACCATACTGCTT CGACCAGGTGAGGGAGGAGGTCCTG
GAGGGCGGCAGAGGTGCTGAGGCTCCC CTACCAGAAGCAAACATGGATGGTGGGTGAAACCACAGGCTGGACCAG
AAGCC AGGCTGAGAAGGGGA-3'；
CYP2D6*10 wild type:
5’-GTGCTGAGAGTGTCCTGCCTGGTCCTCTGTGCCTGGTGGGGTGGGGGT GCCAGGTGTGTCCAGA
GGAGCCCATTTGGTAGTGAGGCAGGTATGGGGCTAGA AGCACTGGTGCCCCTGGCCGTGATAGTGGCCATCTTCC
TGCTCCTGGTGGACCT GATGCACCGGCGCCAACGCTGGGCTGCACGCTACCCACCAGGCCCCCTGCCAC TGCCC
GGGCTGGGCAACCTGCTGCATGTGGACTTCCAGAACACACCATACTGCT TCGACCAGGTGAGGGAGGAGGTCCTG
GAGGGCGGCAGAGGTGCTGAGGCTCC CCTACCAGAAGCAAACATGGATGGTGGGTGAAACCACAGGCTGGACCAG
AAGC CAGGCTGAGAAGGGGA-3’。
The invention further relates to application of the kit in detection hypertension medication gene.
The invention further relates to a kind of multiplex PCR melting curve detections using mentioned reagent box detection hypertension medication gene
Method, the method are as follows:
(1) clinical blood sample is acquired, is directly used after being diluted by Sample dilution, extension rate is between 5-40 times；
(2) PCR reaction solution 1 is prepared with primed probe group one and PCR reaction reagent, is reacted with primed probe group two and PCR
Preparation of reagents PCR reaction solution 2 prepares PCR reaction solution 3 with primed probe group three and PCR reaction reagent, and the blood sample after dilution is
Template is directly added separately to PCR reaction solution 1, PCR reaction solution 2, in PCR reaction solution 3, and amplification cumulative volume is 20 microlitres, wherein
Sample volume is 2 microlitres；
(3) PCR amplification program is set and melts program；
(4) result interpretation corresponds to different polymorphic sites by different Tm value melting peakss and directly carries out interpretation.
The Sample dilution is one of following:
(1) sodium hydroxide solution, concentration 0.01M~0.1M；
(2) DMSO, sodium hydroxide solution, wherein DMSO solution concentration is 1%~10%, concentration of sodium hydroxide solution
0.01M~0.1M；
(3) glycine betaine, sodium hydroxide solution, wherein beet alkali concentration is 1M~5M, concentration of sodium hydroxide solution 0.01M
~0.1M；
(4) mannitol, sodium hydroxide solution, wherein mannitol concentration is 1%~5%, concentration of sodium hydroxide solution
0.01M~0.1M.
The PCR reaction solution composition is as follows:
PCR reaction solution 1:Tris (PH8.5) 20mmol/L, KCL 50mmol/L, MgCL2 2mmol/L、 dNTPs
2mmol/L, polymerase 1U, CYP3A5*3 upstream primer 250nmol/L, CYP3A5*3 downstream primer 50nmol/L, CYP3A5*3
Fluorescence probe 100nmol/L, NPPA upstream primer 250nmol/L, NPPA downstream primer 50nmol/L, NPPA fluorescence probe
100nmol/L；
PCR reaction solution 2:Tris (PH8.5) 20mmol/L, KCL 50mmol/L, MgCL2 2mmol/L、 dNTPs
2mmol/L, polymerase 1U, CYP2C9 upstream primer 100nmol/L, CYP2C9 downstream primer 500nmol/L, CYP2C9 fluorescence
Probe 500nmol/L, AGTR1 upstream primer 100nmol/L, AGTR1 downstream primer 500nmol/L, AGTR1 fluorescence probe
500nmol/L, ACE upstream primer 100nmol/L, ACE downstream primer 500nmol/L, ACE fluorescence probe 500nmol/L；
PCR reaction solution 3:Tris (PH8.5) 20mmol/L, KCL 50mmol/L, MgCL2 2mmol/L、 dNTPs
2mmol/L, polymerase 1U, DMSO 2%, ADRB1 upstream primer 100nmol/L, ADRB1 downstream primer 500nmol/L,
ADRB1 fluorescence probe 500nmol/L, CYP2D6 upstream primer 100nmol/L, CYP2D6 downstream primer 500nmol/L,
CYP2D6 fluorescence probe 500nmol/L.
The present invention is different by the Tm value difference that the difference of different bases generates, and can easily distinguish very much different mutation types.From
The present invention can be seen that 7 kinds of genes in total and be related to 21 hypotypes, and the present invention is easily accomplished detection process by 3 reaction tubes,
Detection process is not necessarily to nucleic acid extraction process simultaneously, simplifies operating procedure, and interpretation method is simple.
The beneficial effects are mainly reflected as follows:
1) simple and quick, it is not necessarily to nucleic acid purification process, saves the time.
2) high sensitivity can detect the nucleic acid down to 0.5ng.
3) one-time detection flux is big: not only covering 21 kinds of hypotypes of 7 allele, and required reaction liquid pipe number is few.
4) interpretation is intuitive: different subtype, instrument automatic interpretation can be distinguished according to the difference of Tm value.
(4) Detailed description of the invention
Fig. 1 is 1# sample results figure；
Fig. 2 is 2# sample results figure；
Fig. 3 is 3# sample results figure；
Fig. 4 is 4# sample results figure；
Fig. 5 is 6# sample results figure
(5) specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
Embodiment 1:
1. reaction solution is prepared
1) reaction solution 1 is prepared
Take 2 milliliters of cryopreservation tubes, be separately added into 200 μ l of Tris (PH8.5) of 0.2mol/L, 200 μ l of KCL of 0.5mol/L,
The MgCL of 20mmol/L2200 μ l, 40 μ l of dNTPs of 100mmol/L, polymerase 100U, 100 μM of the upstream CYP3A5*3 are drawn
5 μ l of object, 100 μM of 1 μ l of CYP3A5*3 downstream primer, 100 μM of 2 μ l of CYP3A5*3 fluorescence probe, 100 μM of the upstream NPPA
5 μ l of primer, 100 μM of 1 μ l of NPPA downstream primer, 100 μM of 2 μ l of NPPA fluorescence probe.Volume is supplied to 1.8ml with purified water,
It is spare to mix well rear stored frozen.
2) reaction solution 2 is prepared
Take 2 milliliters of cryopreservation tubes, be separately added into 200 μ l of Tris (PH8.5) of 0.2mol/L, 200 μ l of KCL of 0.5mol/L,
The MgCL of 20mmol/L2200 μ l, 40 μ l of dNTPs of 100mmol/L, polymerase 100U, 100 μM of CYP2C9 upstream primer
2 μ l, 100 μM of 10 μ l of CYP2C9 downstream primer, 100 μM of 10 μ l of CYP2C9 fluorescence probe, 100 μM of AGTR1 upstream primer
2 μ l, 100 μM of 10 μ l of AGTR1 downstream primer, 100 μM of 10 μ l of AGTR1 fluorescence probe, 100 μM of 2 μ l of ACE upstream primer,
100 μM of 10 μ l of ACE downstream primer, 100 μM of 1 μ l of ACE fluorescence probe.Volume is supplied to 1.8ml with purified water, is mixed well
Stored frozen is spare afterwards.
3) reaction solution 3 is prepared
Take 2 milliliters of cryopreservation tubes, be separately added into 200 μ l of Tris (PH8.5) of 0.2mol/L, 200 μ l of KCL of 0.5mol/L,
The MgCL of 20mmol/L2200 μ l, 40 μ l of dNTPs of 100mmol/L, polymerase 100U, 100 μM of ADRB1 upstream primer
2.5 μ l, 100 μM of 15 μ l of ADRB1 downstream primer, 100 μM of 10 μ l of ADRB1 fluorescence probe, 100 μM of the upstream CYP2D6 are drawn
2 μ l of object, 100 μM of 10 μ l of CYP2D6 downstream primer, 100 μM of 10 μ l of CYP2D6 fluorescence probe.Volume is supplied extremely with purified water
It is spare to mix well rear stored frozen by 1.8ml.
4) Sample dilution is prepared
Sodium hydroxide solution is prepared, concentration 0.04M is divided in 2 milliliters of cryopreservation tubes, and room temperature preservation is spare.
5) negative controls are prepared
Using purified water, it is divided in 2 milliliters of cryopreservation tubes according to 0.5ml, room temperature preservation is spare.
6) positive reference substance is prepared
Take 2 milliliters of cryopreservation tubes, be separately added into 10 6 power concentration CYP2D6*10, CYP2C9*3, ADRB1 (1165G >
C), AGTR1 (1166A > C), CYP3A5*3, NPPA (T2238C), ACE (I/D) plasmid, each gene include wild type and dash forward
Modification.It is spare to mix well rear stored frozen.
2. Sample pretreatment
10 μ l whole blood samples (room temperature is melted and mixed well) are taken, 290 μ l Sample dilutions is added, mixes well standby
With extraction product can also be used.
3. sample-adding
Single reaction total volume is 20 μ l, therefore is divided in order into the PCR thin-wall tube that above-mentioned packing has 18 μ l reaction solutions
2 μ l positive controls, 2 μ l negative controls, 2 μ l specimen samples are not added, 8 union pipe lids are covered tightly, after then mixing gently 8 unions
Micro- centrifugation is finally transferred to PCR detection zone.Sample-adding can refer to 96 orifice plate suggestions layout (table 2).
96 orifice plate suggestion of table 2:PCR instrument layout
4.PCR amplification and fluorescence detection
Cycling condition setting is as follows
Table 3: response procedures
Instrument channel selects: reaction solution 1 and reaction solution 3 are FAM and HEX, and reaction solution 2 is FAM/HEX/CY5.
5. interpretation of result
1) negative control should may be contaminated or operate for reagent without obvious melting peakss, if there are melting peakss after analysis
Pollution in the process is detected again behind the source that please decontaminate.
2) positive control after melting curve analysis should every each channel of pipe have 2 melting peakss, if positive control pipe without
Only there is 1 melting peaks in melting peakss, and whether still before the deadline reagent used in PLSCONFM, determines whether operation strictly presses
Book carries out as directed.
3) sample interpretation method carries out interpretation according to following table standard
Table 4: target gene and positive control melt Tm value range
According to testing result, the corresponding different melting peakss in different channels, represent different hypotypes.
Embodiment 2:
1. reagent susceptibility test
1) experiment sample, each gene hypotype clinical sample, extraction product dilution to kit minimum detection limit 0.5ng,
Retest 3 times, negative or positive findings are judged by last liquation.
2) loading is tested
2 μ l of susceptibility sample, each sample retest 3 times are separately added into reaction solution 1, reaction solution 2, reaction solution 3
Post analysis result.
3) testing result
Three reaction solution detection sensitivity sample results are the positive, and reproducible results is consistent.
Table 5: susceptibility pattern detection result
2. specific test sample
1) experiment sample is verified using specificity of the specific sample to reagent, wherein 2 parts are Escherichia coli, 2
Part be BCG vaccine, 2 parts be soybean genome.
2) loading is tested
Specific 2 μ l of sample is separately added into reaction solution 1, reaction solution 2, reaction solution 3, while it is right that the negative and positive is arranged
According to product, 1 post analysis result of each sample retest.
3) testing result
It is feminine gender that three reaction solutions, which detect specific sample results, illustrates that kit specificity is functional.
Table 6: specific pattern detection result
| Susceptibility sample | Reaction solution 1 | Reaction solution 2 | Reaction solution 3 |
| Escherichia coli 1 | It is negative | It is negative | It is negative |
| Escherichia coli 2 | It is negative | It is negative | It is negative |
| BCG vaccine 1 | It is negative | It is negative | It is negative |
| BCG vaccine 2 | It is negative | It is negative | It is negative |
| Soybean gene 1 | It is negative | It is negative | It is negative |
| Soybean gene 2 | It is negative | It is negative | It is negative |
Embodiment 3: detection clinical sample 20
1) experiment sample, clinical sample derives from Chifeng hospital, by Sample dilution in kit, according to
20 samples are carried out pre-treatment by method in embodiment 1.
2) loading is tested
2 μ l of clinical sample after being separately added into dilution in reaction solution 1, reaction solution 2, reaction solution 3, each sample repeat to survey
Try 1 post analysis result.
3) testing result
Three reaction solution detection sensitivity sample results are the positive, and testing result is as follows:
Table 7: clinical sample testing result
The method of the present invention that the above embodiments are only used to help understand and its core concept.It should be pointed out that for
For those skilled in the art, without departing from the principle of the present invention, if can also be carried out to the present invention
Dry improvement and modification, these improvement and modification are also fallen into the claims in the present invention protection scope.
Claims (6)
1. a kind of shr gene polymorphism fluorescent PCR solubility curve detection kit mainly includes three groups of primer pairs and PCR anti-
Answer reagent, which is characterized in that the primer sequence is as follows:
Primed probe group one is CYP3A5*3 and NPPA primer:
CYP3A5*3 primer probe sequence is as follows:
Upstream primer: 5 '-TAATGAAGTATTTTAAACATATA-3 '
Downstream primer: 5 '-GTTCTAGTTCATTAGGGTGTGACAC-3 '
Fluorescence probe: FAM-TTTGTCTTTCAATATCTC-BQ1；
NPPA primer probe sequence is as follows:
Upstream primer: 5 '-GATGGGATGATCACAAC-3 '
Downstream primer: 5 '-CCAGGTCACCAAGCCAGATA-3 '
Fluorescence probe: HEX-TGTTATCTTCAGTAC-BQ2；
Primed probe group two is AGTR1, CYP2C9*3 and ACE (I/D) primer:
AGTR1 primer probe sequence is as follows:
Upstream primer: 5 '-TTCTAAAATATATTCCCC-3 '
Downstream primer: 5 '-GAAAAGTCGGTTCAGTCCACATAAT-3 '
Fluorescence probe: FAM-CCAAATGAGCATTAGCT-BQ1；
CYP2C9*3 primer probe sequence is as follows:
Upstream primer: 5 '-AGAGATTGAACGTGTGATTGGCA-3 '
Downstream primer: 5 '-TGATACTATGAATTTGGGGACTTCG-3 '
Fluorescence probe: HEX-GAGATACATTGACC-BQ2；
ACE (I/D) primer probe sequence is as follows:
Upstream primer: 5 '-TCCCATCCTTTCTCCCATTTCTCTA-3 '
Downstream primer: 5 '-CCCTTAGCTCACCTCTGCTTGTA-3 '
Fluorescence probe: CY5-GCTGCCTATACAGTCACT-BQ2；
Primed probe group three is ADRB1 and CYP2D6*10 primer:
ADRB1 primer probe sequence is as follows:
Upstream primer: 5 '-CCCATCATCTACTGCCGCAGC-3 '
Downstream primer: 5 '-TCTCCGTGGGTCGCGTG-3 '
Fluorescence probe: FAM-CTTCCAGGGACTGCTCT-BQ1；
CYP2D6*10 primer probe sequence is as follows:
Upstream primer: 5 '-CCCATTTGGTAGTGAGGCAGGT-3 '
Downstream primer: 5 '-GACCTGATGCACCGGCG-3 '
Fluorescence probe: HEX-GCACGCTACCCACCAGG-BQ2.
2. kit as described in claim 1, it is characterised in that the kit further includes 7 gene pleiomorphism plasmid controls
Product, the standard items sequence are as follows:
CYP3A5*3 standard items sequence:
CYP3A5*3 saltant type:
5’-CTTTTTGATAATGAAGTATTTTAAACATATAAAACATTATGGAGAGTGGCATAGGAGATACCCACGTAT
GTACCACCCAGCTTAACGAATGCTCTACTGTCATTTCTAACCATAATCTCTTTAAAGAGCTCTTTTGTCTTTCAGT
ATCTCTTCCCTGTTTGGACCACATTACCCTTCATCATATGAAGCCTTGGGTGGCTCCTGTGTGAGACTCTTGCTGT
GTGTCACACCCTAATGAACTAGAACCTAAGGTTGCTGTGTGTCGTACAACTAGGGGTAT-3'；
CYP3A5*3 wild type:
5’-CTTTTTGATAATGAAGTATTTTAAACATATAAAACATTATGGAGAGTGGCATAGGAGATACCCACGTAT
GTACCACCCAGCTTAACGAATGCTCTACTGTCATTTCTAACCATAATCTCTTTAAAGAGCTCTTTTGTCTTTCAAT
ATCTCTTCCCTGTTTGGACCACATTACCCTTCATCATATGAAGCCTTGGGTGGCTCCTGTGTGAGACTCTTGCTGT
GTGTCACACCCTAATGAACTAGAACCTAAGGTTGCTGTGTGTCGTACAACTAGGGGTAT-3'；
NPPA standard items sequence:
NPPA saltant type:
5’-GCTTAGATGGGATGATCACAACTCCATGGCAACAAGATGACACAAATGCAGCAGAGACCCCAGGGGACA
GGAGCCTCTTGCAGTCTGTCCCTAGGCCCAGCCCTGCTTGTCCTCCCTGGCTGTTATCTTCGGTACTGCAAAGAGA
ACACAGACATATCTGGCTTGGTGACCTGGCTGTCCTGGAAAAGTCAGCTTCATGTATGAGTGTGCCCATCCTCTGA
ACTTGATTACTGACCACCTGCTTCCCACCGGCCCCCACCCCAGCCTGATGACCCTCTGA-3'；
NPPA wild type:
5’-GCTTAGATGGGATGATCACAACTCCATGGCAACAAGATGACACAAATGCAGCAGAGACCCCAGGGGACA
GGAGCCTCTTGCAGTCTGTCCCTAGGCCCAGCCCTGCTTGTCCTCCCTGGCTGTTATCTTCAGTACTGCAAAGAGA
ACACAGACATATCTGGCTTGGTGACCTGGCTGTCCTGGAAAAGTCAGCTTCATGTATGAGTGTGCCCATCCTCTGA
ACTTGATTACTGACCACCTGCTTCCCACCGGCCCCCACCCCAGCCTGATGACCCTCTGA-3'；
AGTR1 standard items sequence:
AGTR1 saltant type:
5’-CTCCAGCTTCTAAAATATATTCCCCCAAAAGCCAAATCCCACTCAAACCTTTCAACAAAAATGAGCACG
CTTTCCTACCGCCCCTCAGATAATGTAAGCTCATCCACCAAGAAGCCTGCACCATGTTTTGAGGTTGAGTGACATG
TTCGAAACCTGTCCATAAAGTAATTTTGTGAAAGAAGGAGCAAGAGAACATTCCTCTGCAGCACTTCACTACCAAA
TGAGCCTTAGCTACTTTTCAGAATTGAAGGAGAAAATGCATTATGTGGACTGAACCGACTTTTCTAAAGCTCTGAA
CAAAAGCTTTTCTTTCCTTTTGCAACAAGACAAAGCAAAGCCACATTTTGCATTAGACAGATGACGGCTGCTCGAA
GAACAATGTCAGAAACTCGATGAATGTGTTGATTTGAGAAATTTTACTGACAGAAATGCAATCTCCCTAGCCTGC
TT-3'；
AGTR1 wild type:
5’-CTCCAGCTTCTAAAATATATTCCCCCAAAAGCCAAATCCCACTCAAACCTTTCAACAAAAATGAGCACG
CTTTCCTACCGCCCCTCAGATAATGTAAGCTCATCCACCAAGAAGCCTGCACCATGTTTTGAGGTTGAGTGACATG
TTCGAAACCTGTCCATAAAGTAATTTTGTGAAAGAAGGAGCAAGAGAACATTCCTCTGCAGCACTTCACTACCAAA
TGAGCATTAGCTACTTTTCAGAATTGAAGGAGAAAATGCATTATGTGGACTGAACCGACTTTTCTAAAGCTCTGAA
CAAAAGCTTTTCTTTCCTTTTGCAACAAGACAAAGCAAAGCCACATTTTGCATTAGACAGATGACGGCTGCTCGAA
GAACAATGTCAGAAACTCGATGAATGTGTTGATTTGAGAAATTTTACTGACAGAAATGCAATCTCCCTAGCCTGC
TT-3'；
CYP2C9*3 standard items sequence:
CYP2C9*3 saltant type:
5’-TCTCCTTTTCCATCAGTTTTTACTTGTGTCTTATCAGCTAAAGTCCAGGAAGAGATTGAACGTGTGATT
GGCAGAAACCGGAGCCCCTGCATGCAAGACAGGAGCCACATGCCCTACACAGATGCTGTGGTGCACGAGGTCCAGA
GATACCTTGACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGACATTAAATTCAGAAACTATCTCATTCC
CAAGGTAAGTTTGTTTCTCCTACACTGCAACTCCATGTTTTCGAAGTCCCCAAATTCATAGTATCATTTTTAAACC
TCT-3'；
CYP2C9*3 wild type:
5’-AGAGATTGAACGTGTGATTGGCAGAAACCGGAGCCCCTGCATGCAAGACAGGAGCCACATGCCCTACAC
AGATGCTGTGGTGCACGAGGTCCAGAGATACATTGACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGAC
ATTAAATTCAGAAACTATCTCATTCCCAAGGTAAGTTTGTTTCTCCTACACTGCAACTCCATGTTTTCGAAGTCCC
CAAATTCATAGTATCATTTTTAAACCTCT-3’
ACE (I/D) standard items sequence:
ACE-D plasmid:
5’-AGACTCAAGCACGCCCCTCACAGGACTGCTGAGGCCCTGCAGGTGTCTGCAGCATGTGGCCCCAGGCCG
GGGACTCTGTAAGCCACTGCTGGAGAGCCACTCCCATCCTTTCTCCCATTTCTCTAGACCTGCTGCCTATACAGTC
ACTTTTATGTGGTTTCGCCAATTTTATTCCAGCTCTGAAATTCTCTGAGCTCCCCTTACAAGCAGAGGTGAGCTAA
GGGCTGGAGCTCAAGGCATTCAAACCCCTACCAGATCTGACGAATGTGATGGCCACGTCCCGGAAATATGAAGACC
TGTTATGGGCATGGGAGGGCTGGCGAGACAAGGCGGGGAGAGCCATCCTCCAGTTTTACCCGAAATACGTGGAACT
CATCAACCAGGCTGCCCGGCTCAATG-3'；
ACE-I plasmid:
5’-AGACTCAAGCACGCCCCTCACAGGACTGCTGAGGCCCTGCAGGTGTCTGCAGCATGTGGCCCCAGGCCG
GGGACTCTGTAAGCCACTGCTGGAGAGCCACTCCCATCCTTTCTCCCATTTCTCTAGACCTGCTGCCTATACAGTC
ACTTTTTTTTTTTTTTTGAGACGGAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCGGGATCTCGGCTCACT
GCAAGCTCCGCCTCCCGGGTTCACGCCATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGACCACAGGCGCCCGCCA
CTACGCCCGGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTTTTAGCCGGGATGGTCTCGATCTCCT
GACCTCGTGATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGATACAGTCACTTTTATGTGGTTT
CGCCAATTTTATTCCAGCTCTGAAATTCTCTGAGCTCCCCTTACAAGCAGAGGTGAGCTAAGGGCTGGAGCTCAAG
GCATTCAAACCCCTACCAGATCTGACGAATGTGATGGCCACGTCCCGGAAATATGAAGACCTGTTATGGGCATGGG
AGGGCTGGCGAGACAAGGCGGGGAGAGCCATCCTCCAGTTTTACCCGAAATACGTGGAACTCATCAACCAGGCTGC
CCGGCTCAATG-3'；
ADRB1 standard items sequence:
ADRB1 saltant type:
5’-ATGGGCGTCTTCACGCTCTGCTGGCTGCCCTTCTTCCTGGCCAACGTGGTGAAGGCCTTCCACCGCGAG
CTGGTGCCCGACCGCCTCTTCGTCTTCTTCAACTGGCTGGGCTACGCCAACTCGGCCTTCAACCCCATCATCTACT
GCCGCAGCCCCGACTTCCGCAAGGCCTTCCAGCGACTGCTCTGCTGCGCGCGCAGGGCTGCCCGCCGGCGCCACGC
GACCCACGGAGACCGGCCGCGCGCCTCGGGCTGTCTGGCCCGGCCCGGACCCCCGCCATCGCCCGGGGCCGCCTCG
GACGACGACGACGACGATGTCGTCGGGGCCACGCCGCCCGCGCGCCTGCTGGAGCCCTGGGCCGGCTGCAACGGCG
GGGCGGCGGC-3'；
ADRB1 wild type:
5’-ATGGGCGTCTTCACGCTCTGCTGGCTGCCCTTCTTCCTGGCCAACGTGGTGAAGGCCTTCCACCGCGAG
CTGGTGCCCGACCGCCTCTTCGTCTTCTTCAACTGGCTGGGCTACGCCAACTCGGCCTTCAACCCCATCATCTACT
GCCGCAGCCCCGACTTCCGCAAGGCCTTCCAGGGACTGCTCTGCTGCGCGCGCAGGGCTGCCCGCCGGCGCCACGC
GACCCACGGAGACCGGCCGCGCGCCTCGGGCTGTCTGGCCCGGCCCGGACCCCCGCCATCGCCCGGGGCCGCCTCG
GACGACGACGACGACGATGTCGTCGGGGCCACGCCGCCCGCGCGCCTGCTGGAGCCCTGGGCCGGCTGCAACGGCG
GGGCGGCGGC-3'；
CYP2D6*10 standard items sequence:
CYP2D6*10 saltant type:
5’-GTGCTGAGAGTGTCCTGCCTGGTCCTCTGTGCCTGGTGGGGTGGGGGTGCCAGGTGTGTCCAGAGGAGC
CCATTTGGTAGTGAGGCAGGTATGGGGCTAGAAGCACTGGTGCCCCTGGCCGTGATAGTGGCCATCTTCCTGCTCC
TGGTGGACCTGATGCACCGGCGCCAACGCTGGGCTGCACGCTACTCACCAGGCCCCCTGCCACTGCCCGGGCTGGG
CAACCTGCTGCATGTGGACTTCCAGAACACACCATACTGCTTCGACCAGGTGAGGGAGGAGGTCCTGGAGGGCGGC
AGAGGTGCTGAGGCTCCCCTACCAGAAGCAAACATGGATGGTGGGTGAAACCACAGGCTGGACCAGAAGCCAGGCT
GAGAAGGGGA-3'；
CYP2D6*10 wild type:
5’-GTGCTGAGAGTGTCCTGCCTGGTCCTCTGTGCCTGGTGGGGTGGGGGTGCCAGGTGTGTCCAGAGGAGC
CCATTTGGTAGTGAGGCAGGTATGGGGCTAGAAGCACTGGTGCCCCTGGCCGTGATAGTGGCCATCTTCCTGCTCC
TGGTGGACCTGATGCACCGGCGCCAACGCTGGGCTGCACGCTACCCACCAGGCCCCCTGCCACTGCCCGGGCTGGG
CAACCTGCTGCATGTGGACTTCCAGAACACACCATACTGCTTCGACCAGGTGAGGGAGGAGGTCCTGGAGGGCGGC
AGAGGTGCTGAGGCTCCCCTACCAGAAGCAAACATGGATGGTGGGTGAAACCACAGGCTGGACCAGAAGCCAGGCT
GAGAAGGGGA-3’。
3. application of the kit of any of claims 1 or 2 in detection hypertension medication gene.
4. a kind of multiplex PCR melting curve detection method using the detection hypertension medication gene of kit described in claim 1,
The method is as follows:
(1) clinical blood sample is acquired, is directly used after being diluted by Sample dilution, extension rate is between 5-40 times；
(2) PCR reaction solution 1 is prepared with primed probe group one and PCR reaction reagent, with primed probe group two and PCR reaction reagent
PCR reaction solution 2 is prepared, PCR reaction solution 3 is prepared with primed probe group three and PCR reaction reagent, the blood sample after dilution is that template is straight
It connects and is added separately to PCR reaction solution 1, PCR reaction solution 2, in PCR reaction solution 3, amplification cumulative volume is 20 microlitres, wherein sample body
Product is 2 microlitres；
(3) PCR amplification program is set and melts program；
(4) result interpretation corresponds to different polymorphic sites by different Tm value melting peakss and directly carries out interpretation.
5. method as claimed in claim 4, it is characterised in that the Sample dilution is one of following:
(1) sodium hydroxide solution, concentration 0.01M~0.1M；
(2) DMSO, sodium hydroxide solution, wherein DMSO solution concentration is 1%~10%, concentration of sodium hydroxide solution 0.01M~
0.1M；
(3) glycine betaine, sodium hydroxide solution, wherein beet alkali concentration be 1M~5M, concentration of sodium hydroxide solution 0.01M~
0.1M；
(4) mannitol, sodium hydroxide solution, wherein mannitol concentration is 1%~5%, concentration of sodium hydroxide solution 0.01M~
0.1M。
6. method as claimed in claim 4, it is characterised in that the PCR reaction solution composition is as follows:
PCR reaction solution 1:Tris (PH8.5) 20mmol/L, KCL 50mmol/L, MgCL22mmol/L, dNTPs 2mmol/L, gather
Synthase 1U, CYP3A5*3 upstream primer 250nmol/L, CYP3A5*3 downstream primer 50nmol/L, CYP3A5*3 fluorescence probe
100nmol/L, NPPA upstream primer 250nmol/L, NPPA downstream primer 50nmol/L, NPPA fluorescence probe 100nmol/L；
PCR reaction solution 2:Tris (PH8.5) 20mmol/L, KCL 50mmol/L, MgCL22mmol/L, dNTPs 2mmol/L, gather
Synthase 1U, CYP2C9 upstream primer 100nmol/L, CYP2C9 downstream primer 500nmol/L, CYP2C9 fluorescence probe 500nmol/
L, on AGTR1 upstream primer 100nmol/L, AGTR1 downstream primer 500nmol/L, AGTR1 fluorescence probe 500nmol/L, ACE
Swim primer 100nmol/L, ACE downstream primer 500nmol/L, ACE fluorescence probe 500nmol/L；
PCR reaction solution 3:Tris (PH8.5) 20mmol/L, KCL 50mmol/L, MgCL22mmol/L, dNTPs 2mmol/L, gather
Synthase 1U, DMSO 2%, ADRB1 upstream primer 100nmol/L, ADRB1 downstream primer 500nmol/L, ADRB1 fluorescence probe
500nmol/L, CYP2D6 upstream primer 100nmol/L, CYP2D6 downstream primer 500nmol/L, CYP2D6 fluorescence probe
500nmol/L。
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| CN109929925A (en) * | 2019-03-22 | 2019-06-25 | 踏石生物科技（苏州）有限公司 | Using 65 site CC type of people ADBR1 gene 11 as the positive reference product of template |
| CN109943629A (en) * | 2019-03-22 | 2019-06-28 | 踏石生物科技（苏州）有限公司 | A kind of ADRB1 G1165C genotype detection kit and its detection method |
| CN110257506A (en) * | 2019-07-09 | 2019-09-20 | 杭州千基生物科技有限公司 | Polymorphism detection kit and method for hypertension accurate medication related gene |
| CN110863042A (en) * | 2019-10-11 | 2020-03-06 | 济南和合医学检验有限公司 | Method for detecting hypertension related gene by using multiple PCR technology |
| CN111100929A (en) * | 2020-01-17 | 2020-05-05 | 郑州安图生物工程股份有限公司 | Kit for detecting polymorphism of drug-related gene for hypertension |
| CN111334573A (en) * | 2020-04-28 | 2020-06-26 | 重庆浦洛通基因医学研究院有限公司 | Gene detection kit for hypertension medication and use method |
| CN111793676A (en) * | 2020-07-21 | 2020-10-20 | 北京安智因生物技术有限公司 | Method and kit for detecting gene polymorphism and application thereof |
| CN112695083A (en) * | 2021-02-05 | 2021-04-23 | 北京核子华曦医学检验实验室有限公司 | Nucleic acid composition and kit for detecting gene polymorphism of medicine for hypertension |
| CN114480594A (en) * | 2021-12-23 | 2022-05-13 | 郑州华之源医学检验实验室有限公司 | Method and kit for detecting multiple single nucleotide polymorphisms and application thereof |
| CN116814777A (en) * | 2023-08-25 | 2023-09-29 | 北京宏微特斯生物科技有限公司 | Kit for guiding related gene polymorphic sites by using hypertension and application method of kit |
| CN116814777B (en) * | 2023-08-25 | 2023-12-12 | 北京宏微特斯生物科技有限公司 | Kit for guiding related gene polymorphic sites by using hypertension and application method of kit |
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