CN109306377A - SNaPShot serotype specific primer and its detection method - Google Patents

SNaPShot serotype specific primer and its detection method Download PDF

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Publication number
CN109306377A
CN109306377A CN201810486949.5A CN201810486949A CN109306377A CN 109306377 A CN109306377 A CN 109306377A CN 201810486949 A CN201810486949 A CN 201810486949A CN 109306377 A CN109306377 A CN 109306377A
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Prior art keywords
snapshot
specific primer
serotype specific
detection method
primer
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齐军
刘永飞
方罡
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Shanghai Maipu Biotechnology Co Ltd
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Shanghai Maipu Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Abstract

The present invention relates to technical field of biological, specifically a kind of SNaPShot serotype specific primer and its detection method, it is characterised in that: the SNP site for detecting cardiovascular related drugs medication includes: rs776746, rs6720173, rs12041331, rs4244285, rs2072183, rs1065852, rs9923231, rs2306283, rs12248560, rs5186, rs4149056, rs5065, rs662799, rs4986893, rs5443, rs1057910, rs1799853, rs3808607, rs671, rs5918.The present invention compared with the existing technology, using SNAPSHOT typing method carries out parting to gene, and sampling amount is few, the gene loci of the multinomial cardiovascular related drugs medication of a performance detection, and accuracy is high.

Description

SNaPShot serotype specific primer and its detection method
Technical field
The present invention relates to technical field of biological, specifically a kind of SNaPShot serotype specific primer and its detection side Method.
Background technique
Cardiovascular and cerebrovascular disease is that one kind seriously threatens the mankind, the common disease of especially 50 years old or more middle-aged and the old's health, tool There is the characteristics of high illness rate, high disability rate and high mortality, even if the treatment means that application is most advanced, perfect at present, can still have 50% or more cerebrovascular accident survivor life cannot take care of oneself completely, and the number that cardiovascular and cerebrovascular disease is died of in the whole world every year is high Up to 15,000,000 people, it is the first to occupy the various causes of the death.
And cardio-cerebral vascular disease patient includes several genes parting, different genes parting for related drugs sensibility not yet Together.Therefore, it should be understood that the Genotyping of patient, could suit the remedy to the case conducive to doctor before medication.
There are many detection method of the Genotyping of patient at present, and the accuracy of Sanger PCR sequencing PCR is high but at high price； Massarray etc. requires sample size more, and accuracy is low；Chip and other high-throughput method testing costs are higher, and detecting instrument is universal It spends not high.
Summary of the invention
The purpose of the present invention is overcome the deficiencies in the prior art, a small amount of to sample energy using SNaPShot DNA test Genotyping is carried out to cardio-cerebral vascular disease patient, and accuracy is high.
To achieve the above object, a kind of SNaPShot serotype specific primer of cardiovascular drugs medication guide is designed, feature exists In: detect cardiovascular related drugs medication SNP site include: rs776746, rs6720173, rs12041331, rs4244285、rs2072183、rs1065852、rs9923231、rs2306283、rs12248560、rs5186、 rs4149056、rs5065、rs662799、rs4986893、rs5443、rs1057910、rs1799853、rs3808607、 rs671、rs5918。
The angiocarpy related drugs include diuretics NPPA；Angiotensin converting enzyme inhibitors ACE；Beta receptor retardance Agent ADRB1, CYP2D6；Angiotensin II receptor antagonist AGTR1, CYP2C9；Ca2+ overloading CYP3A5, NPPA；The resistance of 1 receptor of α Stagnant dose of GNB3；Statins SLCO1B1；Fibrates APOA5；Ezetimibe NPC1L1；Resinae CYP7A1；Antioxidant ABCG5； Clopidogrel CYP2C19；Aspirin GPIIIa, PEAR1；Warfarin CYP2C9, VKORC1；Nitroglycerin ALDH2.
A kind of detection method of the SNaPShot serotype specific primer, which is characterized in that use following steps:
(1) according to SNAPSHOT typing method, 20 SNP point primers are designed comprising amplimer, extension primer；
(2) DNA is extracted from EDTA anticoagulated blood sample and buccal swab sample；
(3) multiple SNAPSHOT parting: including multiplexed PCR amplification；PCR product purifying；Multiple extension；Extension products Purifying；
(4) Capillary Electrophoresis is detected for the experimental result to multiple SNAPSHOT parting；
(5) data are analyzed.
The present invention compared with the existing technology, using SNAPSHOT typing method carries out parting to gene, and sampling amount is few, once The gene loci of the multinomial cardiovascular related drugs medication of performance detection, and accuracy is high.
Detailed description of the invention
Fig. 1 is the parameter setting table that upper machine mixed liquor is detected in ABI3730x1 sequenator in the embodiment of the present invention.
Fig. 2 is to carry out parting to a upper machine mixed liquor sample using genemapper4.0 software in the embodiment of the present invention Waveform diagram.
Fig. 3 is to be divided using genemapper4.0 software machine mixed liquor sample on another in the embodiment of the present invention The waveform diagram of type.
Specific embodiment
The present invention is further described now in conjunction with accompanying drawings and embodiments.
Embodiment 1
One, design of primers
It is required according to SNAPSHOT typing method design of primers, 20 SNP site primer sequences are as follows:
(1), amplimer
Kong Hao Site Positive (F) Reversely (R)
1 rs6720173 CCTGGGCAGGTTTTCTCAATG GCAGCTCAAATGTTTCTGTGACAAC
1 rs12041331 GGGGTTATCCTATGCTACATGACTT GATTAGAGTTCCTGGTGGACAAGAG
1 rs4244285 CAGAGCTTGGCATATTGTATCTATACC TCAGGAAGCAATCAATAAAGTCCC
1 rs2072183 CAAGGTGACGACGTGGCGA ATGAGGACCAGACTGCCCG
1 rs1065852 CCATTTGGTAGTGAGGCAGGT TCTGGAAGTCCACATGCAGCA
2 rs2306283 ACTATCTCAGGTGATGCTCTATTGAGT AATTTGGGGAAGATAATGGTGC
2 rs12248560 TTCAGAATAACTAATGTTTGGAAGTTGT GCTGAGGTCTTCTGATGCCCA
2 rs5186 AGAACATTCCTCTGCAGCACTTC TTTAGAAAAGTCGGTTCAGTCCACATA
2 rs4149056 GGTTGTTTAAAGGAATCTGGGTCAT AGTAGACAAAGGGAAAGTGATCATACA
2 rs5065 CCAGGGGACAGGAGCCTCTT CCAGGTCACCAAGCCAGATATGT
2 rs9923231 CATTGCCCTGACACCTAGTGG GGAAGTCAAGCAAGAGAAGACCTG
2 rs662799 AGGCAGGGTGAAGATGAGATGG TTTGGGCTTGCTCTCCTCAG
3 rs776746 CGTATGTACCACCCAGCTTAACG ACACAGGAGCCACCCAAGG
3 rs4986893 AGATCAGCAATTTCTTAACTTGATGGA TTGGTCAATATAGAATTTTGGATTTCC
3 rs5443 GGGCAGACCAGGAGCTGATC TGAAGTCGTCGTAGCCAGCG
3 rs1057910 GCAAGACAGGAGCCACATGCC AGAAACAAACTTACCTTGGGAATGAGA
3 rs1799853 GGAATTTTGGGATGGGGAAG CAGTAAGGTCAGTGATATGGAGTAGGG
3 rs3808607 CTTGAACTAAGTCCACAGGTATCAGAA GTTGTCCCCAGGTCCGAATGT
3 rs671 TGGAGCCCAGTCACCCTTTG CAGGTCCCACACTCACAGTTTTC
3 rs5918 GATTGCTGGACTTCTCTTTGGG GCCTCACTCACTGGGAACTCG
(2), extension primer
Two, DNA is extracted
EDTA anticoagulation uses " the poba gene group DNA extraction kit of TIANGEN Biotech (Beijing) Co., Ltd. (0.1-1ml) (DP348) " kit, reagent involved in process are all made of the reagent carried in the kit.
Buccal swab is using " the efficient buccal swab extracting genome DNA of TIANGEN Biotech (Beijing) Co., Ltd. is tried Agent box (centrifugal column type) (DP362) " kit, reagent involved in process are all made of the reagent carried in kit.
DNA extracts the kit that root is all made of TIANGEN Biotech (Beijing) Co., Ltd. in this case, and extracting process is examination The included normal process of agent box.
Three, multiplexed PCR amplification
Hole number configuration amplimer Mix (Primer Mix) is pressed, primer working solution concentration 50uM, ratio is as follows:
(1) it is pre-configured with PCR amplification mixed liquor, every part of every hole 9u1 is dispensed, and PCR mixture system is as follows:
Reagent Dosage (ul)
EXTaq(HS) 10×n
Primer Mix 2×n
2×GC Buffer I 7×n
Wherein, n=detects sample number+1.
(2) it is loaded: 1ul DNA being added in 19ul amplified reaction mixed liquor；
(3) it expands: carrying out PCR amplification program in PCR instrument, amplification program is as follows:
95 ° are denaturalized 2 minutes；Run 12 following circulations later: 95 DEG C are denaturalized 20 seconds, 65 DEG C~59 DEG C every circulations reductions It anneals 40 seconds, 72 DEG C for 0.5 DEG C and extends 1 minute；24 following circulations: 95 DEG C of denaturation 20 seconds, 59 DEG C of annealing 30 seconds, 72 are run later DEG C extend 1 minute；Extend 10 minutes for 72 DEG C later, 4 DEG C of preservations.
Four, PCR product purifies
Be added 5U SAP enzyme and 2U Exonuclease I enzyme in 15 μ l PCR products, 37 DEG C warm bath 1 hour, then 75 DEG C inactivation 15 minutes.
Five, multiple extension
Press hole number configuration extension primer Mix (UEP Mix), primer working solution concentration 50uM.Primer is a kind of in this example The micro and quantitative aliquot of the dry powder-shaped of 1.5ml centrifuge tube dress, is added ddH2Primer working solution is formed after O, and ddH is added2O Amount it is according to the length of primer different, without fixed numbers, the numerical value in following table in dosage refers to concentration being 50uM Primer takes the amount of corresponding volume, and unit is ul:
Hole 1 Dosage (ul) Hole 2 Dosage (ul) Hole 3 Dosage (ul)
rs6720173-UEP 0.5 rs2306283-UEP 3 rs776746-UEP 0.5
rs12041331-UEP 0.5 rs12248560-UEP 3 rs4986893-UEP 3
rs4244285-UEP 3 rs5186-UEP 0.5 rs5443-UEP 1
rs2072183-UEP 3 rs4149056-UEP 3 rs1057910-UEP 1
rs1065852-UEP 3 rs5065-UEP 1 rs1799853-UEP 2
rs662799-UEP 1 rs3808607-UEP 0.5
rs9923231-UEP 3 rs671-UEP 0.5
rs5918-UEP 3
ddH20 15 10.5 13.5
5.1 are pre-configured with extension mixed liquor, and every part of every hole 8ul is dispensed, and PCR mixture system is as follows:
Reagent Dosage (ul)
5xSEQBuffer 2×n
SNaPshot Kit 5×n
UEP Mix 1×n
Wherein, n=detects sample number+5；
5.2 sample-addings: 2ul PCR product after purification is added in 8ul extension mixed liquor；
5.3 extend: PCR is carried out in PCR instrument and extends program, and it is as follows to extend program:
95 DEG C are denaturalized 10 seconds；35 following circulations are run later: being annealed 5 seconds, 60 DEG C within denaturation 10 seconds, 50 DEG C for 95 DEG C and extended 30 Second；Extend 30 seconds for 60 DEG C later, 4 DEG C of preservations.
6. extension products purify: in 10 μ l extension products be added 1U SAP enzyme, 37 DEG C warm bath 1 hour, then go out for 85 DEG C It is 15 minutes living.
7. product carries out capillary electrophoresis detection:
7.1 configure upper machine mixed liquor: taking 9ul to mix after taking ABI GS120-LIZ internal standard and HIDI to mix in 1: 99 ratio PCR product after liquid and lul dilution is mixed and made into machine mixed liquor.
7.2 initial denaturations: by configured upper machine mixed liquor on PCR 95 DEG C after operation 3 minutes in mixture of ice and water it is very fast It is cooling, make denaturation treatment.
7.3 Capillary Electrophoresis: upper machine mixed liquor is detected in ABI3730x1 sequenator, is grouped using G5 color, point Shape parameter setting such as Fig. 1.
The analysis of 8 data
Interpretation is carried out to genotyping result in genemapper4.0 after the completion of 3730xl sequenator parting, analysis method is adopted It is arranged with software default, panel is preset according to the base distribution in each site and extension primer size, according to peak position out The site allele is judged with appearance color.
Sample site genotyping result see the table below in Fig. 2:
Sample site genotyping result see the table below in Fig. 3:

Claims (3)

1. a kind of SNaPShot serotype specific primer of cardiovascular drugs medication guide, it is characterised in that: detect cardiovascular related drugs The SNP site of medication include: rs776746, rs6720173, rs12041331, rs4244285, rs2072183, rs1065852、rs9923231、rs2306283、rs12248560、rs5186、rs4149056、rs5065、rs662799、 rs4986893、rs5443、rs1057910、rs1799853、rs3808607、rs671、rs5918。
2. the detection method of SNaPShot serotype specific primer as described in claim 1, which is characterized in that the angiocarpy related drugs Including diuretics NPPA；Angiotensin converting enzyme inhibitors ACE；Beta-blocker ADRB1, CYP2D6；Angiotensin II Receptor antagonist AGTR1, CYP2C9；Ca2+ overloading CYP3A5, NPPA；1 receptor blocker GNB3 of α；Statins SLCO1B1；Bei Te Class APOA5；Ezetimibe NPC1L1；Resinae CYP7A1；Antioxidant ABCG5；Clopidogrel CYP2C19；Aspirin GPIIIa,PEAR1；Warfarin CYP2C9, VKORC1；Nitroglycerin ALDH2.
3. the detection method of SNaPShot serotype specific primer as claimed in claim 1 or 2, which is characterized in that use following steps:
(1) according to SNAPSHOT typing method, 20 SNP point primers are designed comprising amplimer, extension primer；
(2) DNA is extracted from EDTA anticoagulated blood sample and buccal swab sample；
(3) multiple SNAPSHOT parting: including multiplexed PCR amplification；PCR product purifying；Multiple extension；Extension products are pure Change；
(4) Capillary Electrophoresis is detected for the experimental result to multiple SNAPSHOT parting；
(5) data are analyzed.
CN201810486949.5A 2018-05-21 2018-05-21 SNaPShot serotype specific primer and its detection method Pending CN109306377A (en)

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Publication number Priority date Publication date Assignee Title
CN110423803A (en) * 2019-08-07 2019-11-08 郑州大学第一附属医院 Clopidogrel, statin and aspirin relevant drug metabolism Genotyping detection composite amplification system and kit
CN111593106A (en) * 2019-02-21 2020-08-28 葛猛 Artificial mimic nucleic acid molecular beacon and kit for detecting polymorphism of rs12041331 locus of PEAR1 gene
CN111621553A (en) * 2020-05-28 2020-09-04 长沙都正生物科技有限责任公司 Reagent for detecting NPC1L1 mutant genotyping and application thereof
CN116515994A (en) * 2023-06-26 2023-08-01 广州凯普医药科技有限公司 Primer group and kit for detecting cardiovascular disease drug genes

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CN106893783A (en) * 2017-04-05 2017-06-27 李爱娟 It is a kind of for the accurate early warning of cardiovascular and cerebrovascular disease risk and the detection method and primer special of accurate medication
CN107022611A (en) * 2017-04-05 2017-08-08 李爱娟 It is a kind of to be used for the method and primer special of 4 kinds of accurate medications of common clinical cardiovascular and cerebrovascular disease medicine of detection

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111593106A (en) * 2019-02-21 2020-08-28 葛猛 Artificial mimic nucleic acid molecular beacon and kit for detecting polymorphism of rs12041331 locus of PEAR1 gene
CN110423803A (en) * 2019-08-07 2019-11-08 郑州大学第一附属医院 Clopidogrel, statin and aspirin relevant drug metabolism Genotyping detection composite amplification system and kit
CN111621553A (en) * 2020-05-28 2020-09-04 长沙都正生物科技有限责任公司 Reagent for detecting NPC1L1 mutant genotyping and application thereof
WO2021239081A1 (en) * 2020-05-28 2021-12-02 长沙都正生物科技股份有限公司 Reagent capable of being used for detecting npc1l1 mutant genotyping, kit, usage method therfor and application thereof
CN116515994A (en) * 2023-06-26 2023-08-01 广州凯普医药科技有限公司 Primer group and kit for detecting cardiovascular disease drug genes

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