CN112831557A - Primer group and method for detecting drug metabolism related SNP (Single nucleotide polymorphism) sites - Google Patents
Primer group and method for detecting drug metabolism related SNP (Single nucleotide polymorphism) sites Download PDFInfo
- Publication number
- CN112831557A CN112831557A CN202110260045.2A CN202110260045A CN112831557A CN 112831557 A CN112831557 A CN 112831557A CN 202110260045 A CN202110260045 A CN 202110260045A CN 112831557 A CN112831557 A CN 112831557A
- Authority
- CN
- China
- Prior art keywords
- detecting
- drug metabolism
- snp
- primer
- snp sites
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 28
- 230000036267 drug metabolism Effects 0.000 title claims abstract description 27
- 239000002773 nucleotide Substances 0.000 title description 3
- 125000003729 nucleotide group Chemical group 0.000 title description 3
- 239000003814 drug Substances 0.000 claims abstract description 38
- 238000006243 chemical reaction Methods 0.000 claims abstract description 35
- 108090000623 proteins and genes Proteins 0.000 claims description 22
- 238000007403 mPCR Methods 0.000 claims description 18
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 16
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 16
- 230000003321 amplification Effects 0.000 claims description 16
- 230000029087 digestion Effects 0.000 claims description 16
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 16
- 241000238557 Decapoda Species 0.000 claims description 13
- 238000012408 PCR amplification Methods 0.000 claims description 13
- 230000035772 mutation Effects 0.000 claims description 12
- 239000012634 fragment Substances 0.000 claims description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 claims description 6
- 230000004060 metabolic process Effects 0.000 claims description 6
- 230000002194 synthesizing effect Effects 0.000 claims description 6
- 241000143060 Americamysis bahia Species 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
- 108700036248 MT-RNR1 Proteins 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 101000666920 Streptomyces hygroscopicus subsp. limoneus Validoxylamine A 7'-phosphate phosphatase Proteins 0.000 claims description 2
- 108010000178 IGF-I-IGFBP-3 complex Proteins 0.000 claims 3
- 229940079593 drug Drugs 0.000 abstract description 19
- 230000000694 effects Effects 0.000 abstract description 9
- 206010067484 Adverse reaction Diseases 0.000 abstract description 4
- 230000006838 adverse reaction Effects 0.000 abstract description 4
- 230000000857 drug effect Effects 0.000 abstract description 3
- 230000004907 flux Effects 0.000 abstract description 3
- 238000012502 risk assessment Methods 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 18
- 238000004949 mass spectrometry Methods 0.000 description 12
- 239000000203 mixture Substances 0.000 description 11
- 238000010586 diagram Methods 0.000 description 10
- 238000001514 detection method Methods 0.000 description 7
- 150000002500 ions Chemical class 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000005684 electric field Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 210000005239 tubule Anatomy 0.000 description 2
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000020169 heat generation Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
Images
Classifications
-
- 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
Abstract
The invention discloses a primer group for detecting drug metabolism related SNP sites and a method thereof, belonging to the technical field of biology, wherein the method is used for detecting whether 14 SNP sites related to specific drug metabolism of any crowd are mutated, and the primer sequence is shown as SEQIDNo.1-42. The primer group and the method for detecting the SNP sites related to drug metabolism provided by the invention can not only provide accurate medication for sick people, but also provide personalized medication reminding from various aspects such as metering, drug effect and adverse reaction, improve the curative effect of patients and reduce or avoid the side effect of drugs; risk assessment can be provided for healthy people, and potential improper risks of medication can be effectively avoided. The method can achieve the effects of measuring 14 sites through one reaction, high accuracy, high flux, low cost, quickness and suitability for all people.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer group for detecting SNP sites related to drug metabolism and a method thereof.
Background
The world health organization makes statistics that 1/3 death cause of death cases is irrational medication. If all people can safely take the medicine, tens of millions of people can be prevented from dying every year.
The personalized medicine gene detection is to detect the genotype of the medicine sensitive site of different individuals by using a gene sequencing technology, and provide reference information related to the curative effect and metabolism of the medicine for diagnosis and medicine application, so that personalized 'medicine application to the symptom' is realized. Through genetic testing, the physician can: (1) according to the gene polymorphism condition of the metabolic enzyme or the drug action receptor or target point, guiding proper dosage; (2) identifying patients with certain genotypes as likely to develop adverse drug reactions; (3) patients who have confirmed certain genetic characteristics may benefit more readily with certain treatment regimens and may be guided in drug selection compliance and dose adjustments to achieve desired therapeutic effects; (4) virus resistance is detected and appropriate drugs are selected. By revealing the genetic difference of individuals and guiding the formulation of individual medical treatment schemes, the safety and effectiveness of medication are improved, serious adverse reactions are avoided, and the risk and cost of medication are reduced.
The basic principle of the Sequenom SNP detection system is a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology. The method is mainly characterized in that SNP sequence specific extension primers are added into a product obtained after PCR amplification, and 1 base is extended on an SNP locus. The prepared sample analyte was then co-crystallized with the chip matrix, the crystal was placed into the vacuum tube of a mass spectrometer, and transient nanoseconds (10) were used-9s) strong laser excitation, because the energy absorbed by the matrix molecule by radiation leads to energy accumulation and rapid heat generation, thus leading the matrix crystal to sublimate, the nucleic acid molecule will be desorbed and converted into metastable state ion, the generated ion is mostly single charge ion, the single charge ion obtains the same kinetic energy in the accelerating electric field, and then is separated according to the mass-to-charge ratio in a non-electric field drift region, and flies in a vacuum tubule to reach the detector. MALDI-generated ions are often detected using a Time-of-Flight (TOF) detector, with the smaller the mass of the ion, the smaller the mass of the ionThe faster the arrival. Theoretically, there is no upper limit on the number of mass molecules that can be detected by the TOF detector, as long as the flight tube length is sufficient. MASSARRAY SNP mass spectra were measured in the range of 5000 to 8500 Dalton. Because the Mass spectrum technology is combined with the multi-primer extension technology and the Mass ARRAY technology for use, a plurality of mutation sites can be simultaneously detected in one reaction system, the workload is greatly reduced, the detection flux is improved, and the detection cost is reduced.
Disclosure of Invention
In view of the defects of unreasonable medication of human beings at present, the invention provides a primer group for detecting SNP sites related to drug metabolism and a method thereof
The SNP genotype analysis is carried out by extending the primer by a single base, and the effects of detecting 14 sites by one reaction, high accuracy, high flux, low cost, quickness and suitability for all people can be achieved.
In order to achieve the purpose, the invention adopts the following technical scheme:
a primer set for detecting a drug metabolism-associated SNP site, comprising: 12s rRNA, rs267606617, rs1876828, rs4244285, rs4986893, rs1799853, rs1057910, rs1065852, rs776746, rs37973, rs3909184, rs2844682, rs730012 and rs 3812718; the sequence of the primer group is shown as SEQ ID NO. 1-28.
According to one aspect of the invention, the primer set further comprises 14 single-base extension primers shown in SEQ ID NO. 29-42.
A method for detecting SNP sites related to drug metabolism comprises the following steps: synthesizing a primer sequence: synthesizing the primer set for detecting SNP sites related to drug metabolism according to any one of claims 1 to 2; amplifying SNP locus gene fragments by multiplex PCR: amplifying the synthesized primer group once through a multiplex PCR amplification system to cover the personalized medicine gene fragment for detecting the SNP locus related to the medicine metabolism; single base extension SNP site: extending SNP sites of personalized medicine genes at one time through a multiple single base extension system; detecting the genotype on a computer: and detecting data and analyzing the genotype of each mutation site by a matrix-assisted laser desorption ionization time-of-flight mass spectrometry method.
According to one aspect of the present invention, the method further comprises a shrimp alkaline phosphatase digestion reaction step of performing a shrimp alkaline phosphatase digestion reaction after amplifying the SNP site gene fragment by multiplex PCR, and performing a single base extension of the SNP site after performing the shrimp alkaline phosphatase digestion reaction.
According to one aspect of the present invention, the multiplex PCR amplification system is an amplification reaction system in which a plurality of components are mixed in a single well.
According to one aspect of the invention, the reagents required for the multiplex PCR amplification system include nucleic-Free Water, 10 XPCR Buffer, MgCl2dNTP Mix, PCR amplification primer Mix, genomic DNA and PCR Enzyme.
According to one aspect of the invention, the reagents required for the multiplex single base extension system include nucleic-Free Water, iPLX Buffer Plus, iPLX Termination Mix, single base extension primer Mix and iPLX Enzyme.
In accordance with one aspect of the invention, the reagents required for the Shrimp alkaline Phosphatase digestion reaction include Nuclear-Free Water, SAP Buffer, and Shrimp Alkal ine Phosphomutase Enzyme.
Compared with the prior art, the invention has the following beneficial effects:
1) the primer group provided by the invention is an SNP locus of a specific drug metabolism related gene of all people.
2) The invention can detect 14 SNP sites by one reaction.
3) The SNP sites selected by the method are genetic materials existing in human cells, polymorphism exists in people, the extended product fragments are between 15 and 30bp, and the genotype of 14 SNP sites is analyzed according to the flight time of the extended product in a vacuum tubule.
4) The invention not only can provide accurate medication for the sick people, give personalized medication reminding from various aspects such as metering, drug effect and adverse reaction, improve the curative effect of patients and reduce or avoid the side effect of the drug, but also can provide risk assessment for healthy people and effectively avoid the improper risk of potential medication.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a diagram of mass spectrometry of a normal sample of the SNP site of rs 1057910;
FIG. 2 is a diagram of mass spectrometry of a sample of a heterozygous mutation at the SNP site of rs1057910 in example 2;
FIG. 3 is a diagram of mass spectrometry of a normal sample of the SNP site of rs 1065852;
FIG. 4 is a diagram of mass spectrometry of a sample of a heterozygous mutation at the SNP site of rs1065852 in example 2;
FIG. 5 is a diagram of mass spectrometry of a normal sample of the SNP site of rs 2844682;
FIG. 6 is a diagram of mass spectrometry of a sample of a heterozygous mutation at the SNP site of rs2844682 in example 2;
FIG. 7 is a diagram of mass spectrometry of a normal sample of the SNP site of rs 37973;
FIG. 8 is a diagram of mass spectrometry of a sample of a heterozygous mutation at the SNP site of rs37973 in example 2;
FIG. 9 is a mass spectrometry plot of a normal sample of the SNP site of rs 3812718;
FIG. 10 is a diagram of mass spectrometry of a sample of a heterozygous mutation at the SNP site of rs3812718 in example 2;
fig. 11 is a mass spectrometry plot of a normal sample of the SNP site of rs 730012;
fig. 12 is a diagram of mass spectrometry of a heterozygous mutation sample at the SNP point of rs730012 in example 2.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A primer set for detecting a drug metabolism-associated SNP site, comprising: 12s rRNA, rs267606617, rs1876828, rs4244285, rs4986893, rs1799853, rs1057910, rs1065852, rs776746, rs37973, rs3909184, rs2844682, rs730012 and rs3812718, wherein the sequence of the primer group is shown as SEQ ID NO. 1-28; the primer group also comprises 14 single-base extension primers shown in SEQ ID NO. 29-42.
A method for detecting SNP sites related to drug metabolism comprises the following steps: synthesizing a primer sequence: synthesizing the primer set for detecting SNP sites related to drug metabolism according to any one of claims 1 to 2; amplifying SNP locus gene fragments by multiplex PCR: amplifying the synthesized primer group once through a multiplex PCR amplification system to cover the personalized medicine gene fragment for detecting the SNP locus related to the medicine metabolism; single base extension SNP site: extending SNP sites of personalized medicine genes at one time through a multiple single base extension system; detecting the genotype on a computer: and detecting data and analyzing the genotype of each mutation site by a matrix-assisted laser desorption ionization time-of-flight mass spectrometry method.
According to one aspect of the present invention, the method further comprises a shrimp alkaline phosphatase digestion reaction step of performing a shrimp alkaline phosphatase digestion reaction after amplifying the SNP site gene fragment by multiplex PCR, and performing a single base extension of the SNP site after performing the shrimp alkaline phosphatase digestion reaction.
The PCR amplification primer and the single base extension primer can amplify a plurality of SNPs sites and extend a plurality of SNPs sites with equal efficiency in one hole, the detection genes related to the personalized medicine are shown in table 1, 14 SNP sites are related, and the PCR amplification primer and the single base extension primer used in 14 sites are shown in table 2. Wherein, the PCR amplification primers are shown in Table 3; single base extension primers are shown in Table 4.
TABLE 1 personalized medicine correspondence detection genes
PCR amplification primers and Single base extension primers for the 214 SNP sites in Table
TABLE 3 PCR amplification primer sequences
The multiplex PCR amplification system of the present application is an amplification reaction system in which a plurality of components are mixed in a single well.
TABLE 4 Single-base extension primers
Example 1
A kit for detecting whether 14 SNP sites related to personalized medicine metabolism are mutated or not by one person comprises the following components:
(1) PCR amplification primers for 14 SNP sites, 1OD for each primer, and the primer sequences are shown in Table 3.
(2) Primers were extended for a single base at 14 SNP sites, 1OD per primer, and the primer sequences are shown in Table 4.
(3) Reagent: PCR Reagent Set, Large (agena bioscience),
Gold Reagent Set, large (agena bioscience), absolute ethanol.
The kit is stored at-20 ℃ and repeated freeze thawing is reduced as much as possible.
Example 2
The use step of the kit for detecting whether 14 SNP sites related to personalized medicine metabolism are mutated or not by one person comprises the following steps:
(1) extracting genomic DNA of a sample
Genomic DNA samples (blood, buccal swabs, dried blood slides) from 1 adult male (45 years old) were extracted and assayed for concentration using Nanodrop. Diluted with Nuclean-Free Water to a final concentration of 10-20 ng/ul. The diluted samples were stored at-20 ℃. Before use, the mixture can be thawed by centrifugation at 2000rpm for 2-5 min.
(2) Preparation of PCR amplification primer Mix and single base extension primer Mix
The primer dry powder in Table 2 was centrifuged at 12000rpm for 2min, and nucleic-Free Water was added as a primer synthesis solution. Diluting all PCR amplification primers to 100uM, diluting single base extension primers to 400uM, fully shaking and mixing uniformly, standing at room temperature for 15-30min, shaking, mixing uniformly again, centrifuging, and storing at-20 ℃.
Preparing a PCR amplification primer Mix: the final concentration of PCR primers Mix was 0.5uM, i.e., each 100uM primer was diluted 200-fold. Shaking, mixing, centrifuging, and storing at-20 deg.C.
Preparation of single-base extension primer Mix: each single base extension (UEP) primer was stored at a concentration of 400 uM. Then, the calculation was carried out according to the "single-base extension primer dilution Table". Shaking, mixing, centrifuging, and storing at-20 deg.C.
(3) Multiplex PCR amplification of SNP locus gene fragment
The multiplex PCR amplification system is shown in table 5:
TABLE 5 multiplex PCR amplification System
| Reagent | Reaction volume (uL) |
| Nuclease-FreeWater | 1.8 |
| 10×PCRBuffer | 0.5 |
| MgCl2 | 0.4 |
| dNTPMix | 0.1 |
| PCR amplification primer Mix | 1 |
| Genomic DNA (10-20ng/ul) | 1 |
| PCREnzyme | 0.2 |
| Total amount of | 5 |
The multiplex PCR amplification reaction conditions are shown in table 6:
TABLE 6 multiplex PCR amplification reaction conditions
(4) Shrimp alkaline phosphatase digestion (SAP) reaction
The shrimp alkaline phosphatase digestion reaction system is shown in table 7:
TABLE 7 shrimp alkaline phosphatase digestion reaction System
| Reagent | Reaction volume (uL) |
| Nuclease-FreeWater | 1.53 |
| SAPBuffer | 0.17 |
| ShrimpAlkalinePhosphataseEnzyme | 0.3 |
| Total amount of | 2 |
The shrimp alkaline phosphatase digestion reaction conditions are shown in table 8:
TABLE 8 shrimp alkaline phosphatase digestion reaction conditions
| Reaction temperature | Reaction time | Number of cycles |
| 37℃ | 40min | 1 |
| 85℃ | 5min | 1 |
| 4℃ | hold | 1 |
(5) Single base extension reaction
The single-base extension reaction system is shown in Table 9:
TABLE 9 Single-base extension reaction System
The conditions for the single-base extension reaction are shown in Table 10:
TABLE 10 Single-base extension reaction conditions
(6) Detecting the genotype by using a mass spectrometer: and detecting data and analyzing the genotype of each mutation site by a matrix-assisted laser desorption ionization time-of-flight mass spectrometry method.
The single base extension product was subjected to resin purification desalting reaction and centrifugation to deposit the resin on the bottom of 384 well plates. The 384 well plate is then placed on the automatic spotting machine and the chip is placed in the corresponding position. 60uL of sample is taken from each well and added to the corresponding position of the chip for detecting the quality of the chip and the sample application used in the experiment.
And adding the sample in the 384-well to the corresponding position of the chip, then placing the chip after adding the sample in a mass spectrometer, editing the hole to be detected, and clicking START until the whole process is finished.
(7) Analysis of results
And opening the software Mass ARRAY Typers 4.0, checking experimental detection data and performing data analysis. Analyzing a mass spectrum analysis chart of the SNP locus genotype according to a signal value detected by a mass spectrometer, wherein a map result shows that 6 SNP locus mutations exist in the experimenter, and the 6 mutated SNP loci are respectively as follows: rs1057910, rs1065852, rs2844682, rs37973, rs3812718 and rs 730012. Among them, 6 mutated SNP sites are shown in FIG. 2, FIG. 4, FIG. 6, FIG. 8, FIG. 10 and FIG. 12, respectively.
The invention can determine 14 SNP loci of the specific drug metabolism related genes through a reaction, provide accurate medication for a detector by detecting whether the 14 SNP loci of the specific drug metabolism related genes are mutated or not, give personalized medication reminding from multiple aspects such as metering, drug effect and adverse reaction, improve the curative effect of patients and reduce or avoid the side effect of drugs; risk assessment can be provided for healthy people, and potential improper risks of medication can be effectively avoided.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention disclosed herein are intended to be covered by the scope of the present invention.
Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Sequence listing
<110> Shanghai Pudong decoding Life sciences research institute
<120> primer set for detecting drug metabolism-related SNP (single nucleotide polymorphism) sites and method thereof
<160> 42
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 1
acgttggatg agttgaacag ggccctgaag 30
<210> 2
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 2
acgttggatg cactttccag tacacttacc 30
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 3
acgttggatg agttgaacag ggccctgaag 30
<210> 4
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 4
acgttggatg cactttccag tacacttacc 30
<210> 5
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 5
acgttggatg agcagcatac ccctagggac 30
<210> 6
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 6
acgttggatg gactggcgat tgtctagagc 30
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 7
acgttggatg gcaataattt tcccactatc 30
<210> 8
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 8
acgttggatg tccatcgatt cttggtgttc 30
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 9
acgttggatg aacatcagga ttgtaagcac 30
<210> 10
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 10
acgttggatg ctgtaagtgg tttctcagg 29
<210> 11
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 11
acgttggatg catgacgctg cggaattttg 30
<210> 12
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 12
acgttggatg cagtgatatg gagtagggtc 30
<210> 13
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 13
acgttggatg atgcaagaca ggagccacat 30
<210> 14
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 14
acgttggatg tgtcacaggt cactgcatgg 30
<210> 15
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 15
acgttggatg tgttctggaa gtccacatgc 30
<210> 16
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 16
acgttggatg tggacctgat gcaccggcg 29
<210> 17
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 17
acgttggatg gtaatgtggt ccaaacaggg 30
<210> 18
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 18
acgttggatg atgtaccacc cagcttaacg 30
<210> 19
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 19
acgttggatg caaatgttag gctgtaagag 30
<210> 20
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 20
acgttggatg aagaacttct ggtgatcagg 30
<210> 21
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 21
acgttggatg tgatgagccc ccgtttaatc 30
<210> 22
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 22
acgttggatg cagaaagcca acatgtgctc 30
<210> 23
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 23
acgttggatg taaaatgggt caggatgggc 30
<210> 24
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 24
acgttggatg ctctgtgctc ttttctctgg 30
<210> 25
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 25
acgttggatg ttccgcagag gagggtttg 29
<210> 26
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 26
acgttggatg actcctccac ccaccttatc 30
<210> 27
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 27
acgttggatg gtaggtacaa agagcctatc 30
<210> 28
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 28
acgttggatg ctcttcgcac tttcagagtc 30
<210> 29
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 29
ggacacaccg cccgtcac 18
<210> 30
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 30
acacttacca tgttacgact tg 22
<210> 31
<211> 15
<212> DNA
<213> Artificial Sequence
<400> 31
<210> 32
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 32
ttaagtaatt tgttatgggt tcc 23
<210> 33
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 33
ggagtgtaag caccccctg 19
<210> 34
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 34
cgaggagcat tgaggac 17
<210> 35
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 35
acgaggtcca gagatac 17
<210> 36
<211> 15
<212> DNA
<213> Artificial Sequence
<400> 36
<210> 37
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 37
ctacagagct cttttgtctt tca 23
<210> 38
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 38
ctgtaagagt attttacttt gttca 25
<210> 39
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 39
cgtttaatct atttttccca c 21
<210> 40
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 40
gtgaccatct ctatggccaa t 21
<210> 41
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 41
ccaccttatc tgttccc 17
<210> 42
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 42
cctatccttt actctaatca ctt 23
Claims (8)
1. A primer set for detecting a drug metabolism-associated SNP site, comprising: 12s rRNA, rs267606617, rs1876828, rs4244285, rs4986893, rs1799853, rs1057910, rs1065852, rs776746, rs37973, rs3909184, rs2844682, rs730012 and rs 3812718; the sequence of the primer group is shown as SEQ ID NO. 1-28.
2. The primer set for detecting SNP sites related to drug metabolism according to claim 1, wherein the primer set further comprises 14 single-base extension primers shown in SEQ ID No.29 to 42.
3. A method for detecting SNP sites associated with drug metabolism, characterized in that the method for detecting SNP sites associated with drug metabolism comprises the following steps:
synthesizing a primer sequence: synthesizing the primer set for detecting SNP sites related to drug metabolism according to any one of claims 1 to 2;
amplifying SNP locus gene fragments by multiplex PCR: amplifying the synthesized primer group once through a multiplex PCR amplification system to cover the personalized medicine gene fragment for detecting the SNP locus related to the medicine metabolism;
single base extension SNP site: extending SNP sites of personalized medicine genes at one time through a multiple single base extension system;
detecting the genotype on a computer: and detecting data and analyzing the genotype of each mutation site by a matrix-assisted laser desorption ionization time-of-flight mass spectrometry method.
4. The method for detecting SNP sites associated with drug metabolism according to claim 3, wherein: the method also comprises a shrimp alkaline phosphatase digestion reaction step, wherein the shrimp alkaline phosphatase digestion reaction is carried out after the SNP locus gene fragment is amplified by multiple PCR, and the SNP locus is extended by single base after the shrimp alkaline phosphatase digestion reaction is carried out.
5. The method for detecting SNP sites associated with drug metabolism according to claim 3, wherein: the multiplex PCR amplification system is an amplification reaction system for mixing a plurality of components in a single hole.
6. The method for detecting SNP sites associated with drug metabolism according to claim 3, wherein: the reagents required by the multiplex PCR amplification system comprise nucleic-Free Water, 10 XPCR Buffer and MgCl2dNTP Mix, PCR amplification primer Mix, genomic DNA and PCR Enzyme.
7. The method for detecting SNP sites associated with drug metabolism according to claim 3, wherein: reagents required for the multiplex single base extension system include nucleic-Free Water, iPLEX Buffer Plus, iPLEX Termination Mix, single base extension primer Mix and iPLEX Enzyme.
8. The method for detecting SNP sites associated with drug metabolism according to claim 4, wherein: reagents required for the Shrimp alkaline Phosphatase digestion reaction include nucleic-Free Water, SAP Buffer, and Shrimp Alkal ine Phosphomutase Enzyme.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110260045.2A CN112831557B (en) | 2021-03-10 | 2021-03-10 | Primer composition for detecting SNP locus related to drug metabolism and method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110260045.2A CN112831557B (en) | 2021-03-10 | 2021-03-10 | Primer composition for detecting SNP locus related to drug metabolism and method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112831557A true CN112831557A (en) | 2021-05-25 |
| CN112831557B CN112831557B (en) | 2024-03-15 |
Family
ID=75929964
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110260045.2A Active CN112831557B (en) | 2021-03-10 | 2021-03-10 | Primer composition for detecting SNP locus related to drug metabolism and method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112831557B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115961024A (en) * | 2022-11-16 | 2023-04-14 | 中国人民解放军海军第九七一医院 | SNP locus and kit for predicting sorafenib adverse reaction |
Citations (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108192966A (en) * | 2018-02-12 | 2018-06-22 | 北京天平永达科技发展有限公司 | For detecting the primer set of drug metabolic enzyme gene SNP site and its application |
| CN110511993A (en) * | 2019-09-06 | 2019-11-29 | 江苏先声医疗器械有限公司 | For detecting primer sets, application, product and the method for children's drug metabolism associated SNP positions |
| CN111172268A (en) * | 2018-11-13 | 2020-05-19 | 北京毅新博创生物科技有限公司 | Method for distinguishing individual drug administration of verapamil by detecting product and performing mass spectrometry |
| CN111172266A (en) * | 2018-11-13 | 2020-05-19 | 北京毅新博创生物科技有限公司 | Kit for detecting drug-using guide gene of antihypertensive drug verapamil |
| CN111187822A (en) * | 2018-11-15 | 2020-05-22 | 北京毅新博创生物科技有限公司 | Detection product for distinguishing individual drug type of Nicholol |
| CN111187820A (en) * | 2018-11-15 | 2020-05-22 | 北京毅新博创生物科技有限公司 | Method for distinguishing Nistrol personalized medicine by detecting product and performing mass spectrometry |
| CN111187824A (en) * | 2018-11-15 | 2020-05-22 | 北京毅新博创生物科技有限公司 | Nicholol medication guide gene detection kit for antihypertensive drug |
| CN111187823A (en) * | 2018-11-15 | 2020-05-22 | 北京毅新博创生物科技有限公司 | Primer composition for distinguishing Nichol individual medicine type |
| CN111187821A (en) * | 2018-11-15 | 2020-05-22 | 北京毅新博创生物科技有限公司 | Method for distinguishing Nicol lol personalized medicine by using primer composition to perform mass spectrometry |
| CN111206086A (en) * | 2018-11-22 | 2020-05-29 | 北京毅新博创生物科技有限公司 | Method for distinguishing trandolapril personalized medicine by mass spectrometry through product detection |
| CN111206087A (en) * | 2018-11-22 | 2020-05-29 | 北京毅新博创生物科技有限公司 | Method for distinguishing trandolapril personalized medicine by using primer composition to perform mass spectrometry |
| CN111206088A (en) * | 2018-11-22 | 2020-05-29 | 北京毅新博创生物科技有限公司 | Detection product for distinguishing trandolapril individual medication type |
| CN111206089A (en) * | 2018-11-22 | 2020-05-29 | 北京毅新博创生物科技有限公司 | Primer composition for distinguishing trandolapril individual medicine type |
| CN111206085A (en) * | 2018-11-22 | 2020-05-29 | 北京毅新博创生物科技有限公司 | Trandolapril medication guide gene detection kit for antihypertensive drug |
| CN111235256A (en) * | 2018-11-29 | 2020-06-05 | 北京毅新博创生物科技有限公司 | Detection kit for lacidipine medication guidance gene of antihypertensive drug |
| CN111235257A (en) * | 2018-11-29 | 2020-06-05 | 北京毅新博创生物科技有限公司 | Method for distinguishing lacidipine personalized medicine by mass spectrometry through product detection |
| CN111235252A (en) * | 2018-11-28 | 2020-06-05 | 北京毅新博创生物科技有限公司 | Method for distinguishing individual medication of nitrendipine by mass spectrometry through detecting product |
| CN111235251A (en) * | 2018-11-28 | 2020-06-05 | 北京毅新博创生物科技有限公司 | Kit for detecting gene of nitrendipine individualized medication guidance of antihypertensive drug |
| CN111235258A (en) * | 2018-11-29 | 2020-06-05 | 北京毅新博创生物科技有限公司 | Method for distinguishing lacidipine personalized medicine by using primer composition to perform mass spectrometry |
-
2021
- 2021-03-10 CN CN202110260045.2A patent/CN112831557B/en active Active
Patent Citations (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108192966A (en) * | 2018-02-12 | 2018-06-22 | 北京天平永达科技发展有限公司 | For detecting the primer set of drug metabolic enzyme gene SNP site and its application |
| CN111172266A (en) * | 2018-11-13 | 2020-05-19 | 北京毅新博创生物科技有限公司 | Kit for detecting drug-using guide gene of antihypertensive drug verapamil |
| CN111172268A (en) * | 2018-11-13 | 2020-05-19 | 北京毅新博创生物科技有限公司 | Method for distinguishing individual drug administration of verapamil by detecting product and performing mass spectrometry |
| CN111187821A (en) * | 2018-11-15 | 2020-05-22 | 北京毅新博创生物科技有限公司 | Method for distinguishing Nicol lol personalized medicine by using primer composition to perform mass spectrometry |
| CN111187822A (en) * | 2018-11-15 | 2020-05-22 | 北京毅新博创生物科技有限公司 | Detection product for distinguishing individual drug type of Nicholol |
| CN111187820A (en) * | 2018-11-15 | 2020-05-22 | 北京毅新博创生物科技有限公司 | Method for distinguishing Nistrol personalized medicine by detecting product and performing mass spectrometry |
| CN111187824A (en) * | 2018-11-15 | 2020-05-22 | 北京毅新博创生物科技有限公司 | Nicholol medication guide gene detection kit for antihypertensive drug |
| CN111187823A (en) * | 2018-11-15 | 2020-05-22 | 北京毅新博创生物科技有限公司 | Primer composition for distinguishing Nichol individual medicine type |
| CN111206087A (en) * | 2018-11-22 | 2020-05-29 | 北京毅新博创生物科技有限公司 | Method for distinguishing trandolapril personalized medicine by using primer composition to perform mass spectrometry |
| CN111206086A (en) * | 2018-11-22 | 2020-05-29 | 北京毅新博创生物科技有限公司 | Method for distinguishing trandolapril personalized medicine by mass spectrometry through product detection |
| CN111206088A (en) * | 2018-11-22 | 2020-05-29 | 北京毅新博创生物科技有限公司 | Detection product for distinguishing trandolapril individual medication type |
| CN111206089A (en) * | 2018-11-22 | 2020-05-29 | 北京毅新博创生物科技有限公司 | Primer composition for distinguishing trandolapril individual medicine type |
| CN111206085A (en) * | 2018-11-22 | 2020-05-29 | 北京毅新博创生物科技有限公司 | Trandolapril medication guide gene detection kit for antihypertensive drug |
| CN111235252A (en) * | 2018-11-28 | 2020-06-05 | 北京毅新博创生物科技有限公司 | Method for distinguishing individual medication of nitrendipine by mass spectrometry through detecting product |
| CN111235251A (en) * | 2018-11-28 | 2020-06-05 | 北京毅新博创生物科技有限公司 | Kit for detecting gene of nitrendipine individualized medication guidance of antihypertensive drug |
| CN111235256A (en) * | 2018-11-29 | 2020-06-05 | 北京毅新博创生物科技有限公司 | Detection kit for lacidipine medication guidance gene of antihypertensive drug |
| CN111235257A (en) * | 2018-11-29 | 2020-06-05 | 北京毅新博创生物科技有限公司 | Method for distinguishing lacidipine personalized medicine by mass spectrometry through product detection |
| CN111235258A (en) * | 2018-11-29 | 2020-06-05 | 北京毅新博创生物科技有限公司 | Method for distinguishing lacidipine personalized medicine by using primer composition to perform mass spectrometry |
| CN110511993A (en) * | 2019-09-06 | 2019-11-29 | 江苏先声医疗器械有限公司 | For detecting primer sets, application, product and the method for children's drug metabolism associated SNP positions |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115961024A (en) * | 2022-11-16 | 2023-04-14 | 中国人民解放军海军第九七一医院 | SNP locus and kit for predicting sorafenib adverse reaction |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112831557B (en) | 2024-03-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112831557B (en) | Primer composition for detecting SNP locus related to drug metabolism and method thereof | |
| CN111197078A (en) | Method for distinguishing nifedipine personalized medicine by mass spectrometry through product detection | |
| CN111197080A (en) | Detection product for distinguishing nifedipine individualized medication type | |
| CN111197077A (en) | Kit for detecting medication guidance gene of nifedipine serving as antihypertensive drug | |
| CN111206083A (en) | Detection kit for medication guidance gene of telmisartan medicament for reducing hypertension | |
| CN111235258A (en) | Method for distinguishing lacidipine personalized medicine by using primer composition to perform mass spectrometry | |
| CN111235257A (en) | Method for distinguishing lacidipine personalized medicine by mass spectrometry through product detection | |
| CN111206087A (en) | Method for distinguishing trandolapril personalized medicine by using primer composition to perform mass spectrometry | |
| CN111100914A (en) | Method for carrying out perindopril personalized medication guidance by detecting product | |
| CN111187815A (en) | Method for distinguishing individual administration of losartan by detecting product and performing mass spectrometry | |
| CN111172268A (en) | Method for distinguishing individual drug administration of verapamil by detecting product and performing mass spectrometry | |
| CN111235256A (en) | Detection kit for lacidipine medication guidance gene of antihypertensive drug | |
| CN111197079A (en) | Method for distinguishing nifedipine personalized medicine by using primer composition to perform mass spectrometry | |
| CN111197081A (en) | Primer composition for distinguishing nifedipine personalized medicine type | |
| CN111073971A (en) | Method for carrying out individualized medication guidance of antihypertensive drug benazepril by detecting product | |
| CN111073970A (en) | Method for conducting personalized medication guidance of antihypertensive drug benazepril through primer composition | |
| CN111073969A (en) | Method for distinguishing individual administration of benazepril by detecting product and performing mass spectrometry | |
| CN111073965A (en) | Antihypertensive drug benazepril medication guidance and gene detection kit | |
| CN116622886A (en) | Candida albicans drug resistance detection kit based on nucleic acid mass spectrum detection | |
| CN111073967A (en) | Detection product prepared by specific primer composition and used for distinguishing individualized administration of benazepril | |
| CN111187817A (en) | Method for carrying out losartan personalized medication guidance by detecting product | |
| CN111073966A (en) | Primer composition for distinguishing individual medicine type of antihypertensive drug benazepril | |
| CN111073968A (en) | Method for distinguishing individual administration of benazepril by using primer composition to perform mass spectrometry | |
| CN111088334A (en) | Method for carrying out captopril personalized medication guidance by detecting product | |
| CN111172264A (en) | Method for distinguishing individual drug administration of verapamil by using primer composition through mass spectrometry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20210607 Address after: 201500 room 1054, building 1, No.22, Lane 121, Songyin Yucai Road, Tinglin Town, Jinshan District, Shanghai Applicant after: Shanghai Dashan Biotechnology Co.,Ltd. Address before: Room 516, No.667 Zaozhuang Road, Pudong New Area, Shanghai, 200136 Applicant before: SHANGHAI GENOMEPILOT INSTITUTES OF GENOMICS AND HUMAN HEALTH |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |