CN114231606A - Method for rapidly analyzing CYP2C9 genotype - Google Patents
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- CN114231606A CN114231606A CN202111428462.XA CN202111428462A CN114231606A CN 114231606 A CN114231606 A CN 114231606A CN 202111428462 A CN202111428462 A CN 202111428462A CN 114231606 A CN114231606 A CN 114231606A
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Abstract
A method for rapidly analyzing the CYP2C9 genotype, comprising: (1) extracting a DNA sample; (2) preprocessing a sample: (2-1) breaking, repairing the tail end and adding a tail end A to the sample to obtain a DNA fragment; (2-2) carrying out pre-amplification and purification on the obtained DNA fragment to obtain a pre-amplification product; (3) and (3) performing hybridization capture on the pre-amplification product: (3-1) mixing all the obtained pre-amplification products, adding a pre-hybridization reagent, and performing hybridization amplification; (3-2) capturing and cleaning the obtained product, and amplifying and purifying; (4) library pooling and sequencing: (4-1) calculating the pooling volume of each library and then sorting, and mixing all the libraries in order; (4-2) performing on-machine sequencing on the mixed library; (5) and (4) preprocessing the sequencing data, analyzing the data and judging the CYP2C9 genotype. The invention can realize the simultaneous detection of a plurality of loci and analyze the sequence through analysis software to judge the genotype, thereby guiding the aim of clinical medication.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a method for rapidly analyzing CYP2C9 genotype.
Background
Cytochrome oxidase P450(CYP) is an important drug metabolizing enzyme system in human liver, can metabolize various endogenous substrates, drugs and exogenous compounds, and mainly comprises three super families: CYP1, CYP2, and CYP 3. Of these, CYP2C is the most extensively studied and well-defined subfamily. CYP2C9 is an isozyme in the CYP2C subfamily, and is distributed mainly in liver tissue, accounting for about 20% of the total amount of liver microsomal CYP enzymes. Approximately 10% of the clinically common drugs are oxidatively metabolized via CYP2C9, including tolbutamide, S-warfarin, phenytoin, glipizide, glyburide, tolazamide, losartan, irbesartan, and many non-steroidal anti-inflammatory drugs.
The CYP2C9 gene has genetic polymorphism, which causes the difference of CYP2C9 enzyme activity, and is one of the reasons for the difference between drug metabolism ethnicity and individuals. The functional gene mutation causes the reduction of the CYP2C9 enzyme activity, and can reduce the curative effect of CYP2C9 enzyme substrate drugs or generate more adverse reactions. In recent years, more and more single base mutations have been discovered, and 30 alleles have been discovered so far, and the distribution frequency and the functional significance of the new alleles in the human population are more and more concerned.
The human CYP2C9 gene promoter region and the gene coding region are highly polymorphic, and the frequency of different alleles is different in different ethnicities. When the drug treatment is carried out by CYP2C9 oxidative metabolism, individual differences exist in drug metabolism and drug efficacy, and this phenomenon is related to the existence of high-level gene polymorphism in CYP2C 9. In vivo and in vitro evidence suggests that CYP2C9 x 2, 3, 5, 11, 13, 14, 16, 24, 26, 28 and 30 can significantly impair the catalytic function of CYP2C9, but their effect on enzymatic activity presents substrate specificity. When CYP2C9 substrate drugs such as warfarin, glipizide and the like with narrow therapeutic indexes are used for treatment, severe drug toxicity reaction can occur for those CYP2C9 weak metabolites, especially CYP2C9 x 3 homozygote carriers. In contrast, when treated with prodrugs metabolized by CYP2C9 (e.g., losartan, cyclophosphamide, etc.), poorly metabolized CYP2C9 patients may experience inadequate drug efficacy or treatment failure.
Therefore, the CYP2C9 gene is detected before administration, so that doctors can be guided to adjust the dosage of CYP2C9 substrate medicines according to the genotype of patients, individualized medicine treatment is realized, the curative effect of the medicines is improved, and the incidence rate of adverse reactions is reduced.
The application publication No. CN111118144A discloses a kit for simultaneously detecting CYP2C9 and VKORC1 genes and application thereof, which can simultaneously detect the polymorphism of CYP2C9 gene A1075C site and VKORC1 gene-1639G > A site, and adjust the maintenance dose by combining with an INR detection value, thereby being used as a reference for an initial dose and the maintenance dose of warfarin taken by a blood anticoagulation therapist, and reducing the side reaction and the risk of warfarin administration.
High-throughput sequencing is a revolutionary change to conventional sequencing, and sequences hundreds of thousands to millions of DNA molecules at a time, so some documents refer to next generation sequencing as a next generation sequencing technology (next generation sequencing) which is full of its epoch-making changes, and High-throughput sequencing makes it possible to perform detailed global analysis on transcriptome and genome of a species, and is also referred to as deep sequencing. The high-throughput sequencing technology greatly accelerates the progress of genomics research, the whole genome sequencing or the whole exon sequencing can provide more comprehensive genomics information, and the convenience and the welfare brought by the high-throughput sequencing technology are continuously shown along with the development of the high-throughput sequencing technology, and become hot spots for clinical diagnosis and scientific research.
The file with the authorization notice number of CN112029842B discloses a kit and a method for ABO blood group genotyping based on high-throughput sequencing, which can obtain sequencing data covering 90.92% of the full-length area of the ABO gene, including all 7 exon areas of the ABO gene, and can more accurately complete the ABO blood group genotyping. However, high-throughput sequencing detection of polymorphic sites of the CYP2C9 gene is not available at present.
Disclosure of Invention
In view of the above problems, the present invention provides a method for rapidly analyzing the CYP2C9 genotype, which can achieve the purpose of simultaneously detecting multiple loci and analyzing the sequence by analysis software to determine the genotype, thereby guiding clinical medication.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for rapidly analyzing CYP2C9 genotype at least comprises the following steps:
(1) extracting a DNA sample; furthermore, according to the amount of the sample, a proper extraction method and a proper reagent are selected for extraction, a small amount of samples can be subjected to DNA extraction by adopting a column extraction method, and an automatic extraction mode can be selected for DNA extraction under the condition of large amount of samples. Furthermore, a small amount of sample can be extracted by using an extraction kit (Tiangen organism), and a larger amount of sample can be extracted by using a nucleic acid purifier (Aikan organism).
(2) Pretreating a DNA sample: (2-1) breaking, repairing the tail end and adding a tail end A to the DNA sample to obtain a DNA fragment; (2-2) carrying out pre-amplification and purification on the DNA fragment obtained in the step (2-1) to obtain a pre-amplification product;
(3) and (3) performing hybridization capture on the pre-amplification product: (3-1) mixing all the obtained pre-amplification products, adding a pre-hybridization reagent, and performing hybridization amplification; (3-2) capturing and cleaning the product obtained in the step (3-1), and then amplifying and purifying;
(4) library pooling and sequencing: (4-1) calculating the pooling volume of each library and then sorting, and mixing all the libraries in order; (4-2) carrying out on-machine sequencing on the mixed library to obtain sequencing data;
(5) the sequencing data were pre-processed and then subjected to data analysis to determine the CYP2C9 genotype.
As a further preferred aspect of the present invention, the PCR reaction conditions for DNA cleavage in step (2-1) are: the temperature of a hot cover of the PCR instrument is 68-72 ℃, the reaction is carried out for 22min at 32 ℃, and the reaction is carried out for 30min at 65 ℃.
As a further preferred aspect of the present invention, the PCR reaction conditions for the pre-amplification in step (2-2) are: the temperature of a hot cover of the PCR instrument is 104-106 ℃, 98 ℃, 45s and 1 cycle; 8 cycles of 98 ℃, 15s, 60 ℃, 30s, 72 ℃ and 30 s; 72 ℃, 1min, 1 cycle; then the temperature is reduced to 4 ℃.
As a further preferred aspect of the present invention, the PCR reaction conditions in step (3-1) are: the temperature of a hot cover of the PCR instrument is 84-86 ℃, the reaction is carried out for 5min at 95 ℃; reacting for 1-4 h at 60 ℃.
As a further preferred aspect of the present invention, the PCR reaction conditions in step (3-2) are: the temperature of a hot cover of the PCR instrument is 104-106 ℃, 98 ℃, 45s and 1 cycle; at 98 deg.C, 15s, 60 deg.C, 30s, 72 deg.C, 30s, and 12 cycles; 72 ℃, 1min, 1 cycle; then the temperature is reduced to 4 ℃.
As a further preferable aspect of the present invention, the data analysis in step (5) comprises the steps of: (5-1) aligning the sequencing sequence to a human reference genome by using a sequence alignment module; (5-2) analyzing the point mutation and the indel mutation in the sample using a variation identification module; (5-3) SNP analysis and interpretation of the mutation sites using the database.
As a further preferred mode of the present invention, the sequence alignment module in step (5-1) is BWA software or GATK software.
As a further preferred embodiment of the present invention, the variance detecting module in the step (5-2) is VarScan software.
As a further preferred aspect of the present invention, the database in step (5-3) includes PharmGKB database, CLINVAR database, dbSNP database.
In conclusion, the invention has the following beneficial effects:
the invention realizes the purpose of rapidly detecting the polymorphic site of the CYP2C9 gene by adopting a high-throughput sequencing method, can detect only one site compared with a fluorescent quantitative PCR method, can simultaneously detect a plurality of sites by adopting the sequencing method, and analyzes the sequence by analysis software to judge the genotype of the site, thereby achieving the aim of guiding clinical medication.
Drawings
FIG. 1 is an electrophoretic gel of the present invention.
FIG. 2 is a detection peak diagram of the present invention.
Detailed Description
Examples
Primary reagents and instruments
Reagent: blood genome DNA extraction kit (Tiangen organism) and polygenic gene mutation detection kit (hybrid capture method Twist Bioscience)
The instrument comprises the following steps: a gene sequencer, a DNA quantitative analyzer, a PCR amplification instrument, Agilent 2100, a magnetic frame, a high-speed refrigerated centrifuge, a constant-temperature metal bath, an electric heating constant-temperature water tank, a vacuum drying instrument and the like.
The embodiment provides a method for rapidly analyzing CYP2C9 genotype, which mainly comprises the following steps:
1. whole blood (1-2 ml) of 16 volunteers was collected with EDTA anticoagulation vacuum blood collection tube, and severe hemolysis, coagulation samples, heparin anticoagulation samples were rejected.
2. DNA extraction for related gene detection
2.1 Take 200. mu.l of whole blood into a 1.5ml EP tube, add 20. mu.l proteinase K and 200. mu.l buffer GB, mix well by inversion, mix well at 56 ℃ for 10 min.
Standing at room temperature for 5min at 2.2, adding 350 μ l BD, and mixing; then all the solution was transferred to an adsorption column at 12000rpm for 30 seconds, and the waste solution was discarded.
2.3 adding 500 mul GDB into the adsorption column, 12000rpm for 30s, and discarding the waste liquid; adding PWB600 μ l to adsorption column at 12000rpm for 30s, discarding waste liquid, and repeating once; 12000rpm, 2min, adsorption column to new EP tube open cover and air drying for 2min, adding TB60 u l, room temperature placed for 2min, 12000rpm, 2min, DNA collection.
3. Construction of an enzymatic cleavage library
3.1 DNA disruption, end repair and A-tailing addition
Mix is prepared as follows, and is subpackaged into eight-connected pipes:
50ng DNA samples were added to tubes containing Master Mix, less than 35. mu.l ddH2O supplementing, pipe sealing and vortex homogenizing; the flash-off was placed in a PCR instrument with a hot lid at 70 ℃ under the following reaction conditions:
3.2 connect Universal adapter
Mix was prepared as follows:
to the 3.1 products were added 50. mu.l each of Mix, gently shaken and mixed, sealed, and placed in a PCR instrument after flash separation at 20 ℃ for 15min (at which time the lid was closed).
3.3 first step magnetic bead purification
A1.5 ml EP tube was prepared and labeled and dispensed with magnetic beads, 80. mu.l/tube.
And (3) transferring all the 3.2 products into corresponding marked EP tubes, uniformly mixing the products by light vortexes, incubating the products for 5min at room temperature, standing the products for 5min on a magnetic frame, and removing the supernatant.
Fresh 80% ethanol 200 μ l, standing for 1min, discarding the supernatant, and repeating once.
Instantaneously separating, sucking up residual liquid, and drying until the magnetic beads do not reflect light, and avoiding dry cracking.
Adding H2O, 18 mul/tube, mixing, incubating at room temperature for 5min, and standing by a magnetic frame for 5 min.
3.4 PCR amplification of UDI primers
KaPA HiFi HotStartStreadyMix was dispensed into eight tubes at 25. mu.l/tube, and 10. mu.l of different UDI primers were added to each well of the eight tubes.
Taking 3.3 purified supernatant, adding 15 mul/sample into a corresponding tube, mixing uniformly by vortex, placing in a PCR instrument after instantaneous separation, covering a hot cover at 105 ℃, and reacting under the following conditions:
3.5 second purification
A1.5 ml EP tube was prepared and labeled and dispensed with magnetic beads, 50. mu.l/tube.
Transferring all PCR products to a corresponding marked EP tube, mixing uniformly by a gentle vortex, and incubating for 5min at room temperature; standing on a magnetic frame for 5min, and discarding the supernatant.
Fresh 80% ethanol 200 μ l, standing for 1min, and removing supernatant; and repeating the steps once.
Instantaneously separating, sucking up residual liquid, and drying until the magnetic beads do not reflect light, and avoiding dry cracking.
Adding H2O, 24 mul/tube, mixing evenly, and incubating for 5min at room temperature; the magnetic frame is kept still for 5 min.
Mu.l of the supernatant was diluted 10-fold and subjected to the determination of the concentration of Qubit, and the remaining 20. mu.l of the supernatant was transferred to a 1.5ml EP tube and temporarily stored at-20 ℃.
4. Hybrid Capture
4.1 library hybridization
Calculating the entering amount of a library pool according to the concentration of the library DNA, and mixing to obtain the library pool; the total amount of the DNA in the culture medium pool is 1.5-4 mug, and the culture medium pool is established by the same mass sample; the DNA library was used as follows:
each library was mixed in a 1.5ml centrifuge tube, gently shaken, and immediately centrifuged for use.
Respectively adding the following prehybridization reagents into the mixed samples, and uniformly mixing to avoid generating bubbles as much as possible; the proportion of the prehybridization reagent is as follows:
opening the cover, sealing the opening with a sealing film, puncturing the hole, balancing in a vacuum drying instrument, and drying at normal temperature until the product is complete.
Fast Hybridization Mix was incubated at 65 ℃ until all pellets were dissolved.
After oven drying, Fast Hybridization Mix was vortexed rapidly and 20. mu.l was added to each tube (without returning the Hybridization solution to room temperature), gently mixed, and allowed to stand at room temperature for 5 min. The solutions in each tube were transferred to a new PCR tube.
30. mu.l Hybridization Enhancer was added to the surface of each PCR tube, and air bubbles were removed by flash separation.
The program was run with the PCR instrument pre-heated, hot lid 85 ℃: 95 ℃ for 5 min; incubate at 60 ℃ for 2h (at this point, remove the streptomycin, buffer and DNA purification beads, vortex them evenly, equilibrate to room temperature for at least 30 min).
4.2 library Capture and Wash
Preparing and subpackaging an enrichment reagent (single reaction dosage) into a 1.5ml EP tube, wherein the reagent ratio is as follows:
taking 100 mu l of Binding Beads which are vibrated and mixed evenly into a 1.5mL tube; adding 200 mul of Fast Binding Buffer, blowing and mixing evenly.
Placing on a magnetic frame until the solution is clear, discarding the supernatant, and taking down the centrifuge tube.
Repeating the above washing steps 2 times for 3 times; then 200. mu.l of Fast Binding Buffer was added, and the mixture was resuspended by shaking to mix well.
After the hybridization is finished, opening a hybridization PCR tube on a PCR instrument, quickly transferring all the hybridization solution into the treated magnetic bead solution, and immediately blowing, beating and uniformly mixing.
Centrifuge the tubes on a metal bath at 25 ℃ 1200rpm for 30 min. Centrifuging the tube rapidly, mounting on magnetic frame for 1min, and removing supernatant.
200 μ l of preheated Fast Wash Buffer 1 (not removed from the metal bath) was added, flushed well and incubated at 66 ℃ for 5 min.
After removing the supernatant, 200 mul of preheated Fast Wash Buffer 1 is added again, and after being blown and mixed evenly, the mixture is quickly placed at 66 ℃ for incubation for 5 min.
Transfer all liquid in the tube to a new 1.5ml tube, place on magnetic rack for 1min, remove supernatant.
Add 200. mu.l of preheated Wash Buffer 2 (not removed from the bath), blow down and mix well and incubate quickly at 48 ℃ for 5 min.
After removing the supernatant, the magnetic frame was set on a magnetic stand for 1min, and the washing was repeated 2 times with Wash Buffer 2 for three times.
The tube was instantaneously detached and placed on a magnetic stand, and the residue was completely aspirated by a 10. mu.l pipette.
Add 45. mu. l H2O, mix well by pipetting, incubate on ice (for example, in the case of transient PCR, the mixture of magnetic beads can be frozen at-20 ℃).
4.3 PCR amplification
To each PCR tube was added (25. mu.l KAPA HiFi HotStartStreadyMix + 2.5. mu.l Amplification Primers = 27.5. mu.l).
And (3) putting the enriched 22.5 mu L of magnetic bead suspension into a PCR tube, mixing the magnetic bead suspension in a flick mode, instantaneously separating the mixture in a PCR instrument, covering a hot cover at 105 ℃, and operating the program:
note: after the PCR is finished, the PCR product can be temporarily frozen at-20 ℃.
4.4 magnetic bead purification
A1.5 ml EP tube was prepared and labeled and aliquoted with equilibrated magnetic beads, 90. mu.l/tube.
After the transient dissociation of the PCR product, the PCR product is transferred to a corresponding marked EP tube as completely as possible, mixed evenly by a gentle vortex and incubated for 5min at room temperature.
Standing on magnetic frame for 5min, and discarding the supernatant.
Fresh 80% ethanol 200 u l, standing for 30s, abandoning the supernatant, repeat once.
Instantaneously separating, sucking up residual liquid, and drying until the magnetic beads do not reflect light, and avoiding dry cracking.
Adding H2O, 32. mu.l/tube, mixed well and incubated for 5min at room temperature.
Standing for 5min on a magnetic rack after instantaneous separation; the results of taking 1. mu.l for the Qubit concentration determination and taking 1u for the biochip analysis (Agilent 2100) are shown in figures 1 and 2, which can prove that the library prepared in this example has no obvious degradation and good integrity.
The remaining library was transferred to 1.5ml EP tubes and stored at-20 ℃.
5. Library pooling and sequencing
5.1 library pooling
a. Taking out the library from the refrigerator layer at-20 deg.C, thawing completely at room temperature, shaking, mixing, centrifuging instantly, and freezing. A clean ribozyme-free 1.5ml low adsorption centrifuge tube was then placed in an ice box for future use.
b. Calculating the Pooling volume of each library in the order from large to small, reordering the libraries, performing library posing in the order from large to small on a posing table after confirming no errors, and sequentially and accurately sucking each library into a 1.5ml centrifugal tube in an ice box (the posing process needs double rechecking, the libraries after finishing the sample sucking need to be placed in another row, and the libraries can not be placed back to the original position, and each sampled library needs to be marked with a square mark in a confirmation column corresponding to the library in the posing table).
c. After sampling all the libraries, fully mixing the libraries by using a vortex oscillator, performing instantaneous centrifugation, and putting the libraries back into an ice box for later use.
5.2 library pretreatment
a. Preparing a denaturing agent: 5 mul of 1N NaOH solution is taken, 45 mul of non-enzyme water is added to prepare 0.2N library denaturation liquid, the mixture is evenly mixed by vortex, and the mixture is centrifuged for a short time and is placed at room temperature for standby.
b. Preparing a neutralization reagent: mu.l Tris-HCl (1M) was added to 45. mu.l enzyme-free water to prepare 200mM Tris-HCl, vortexed, centrifuged briefly, and allowed to stand at room temperature.
c. Library denaturation: mu.l of the pooled library (4 nM) was taken to the bottom of the low adsorption tube, 5. mu.l of freshly prepared 0.2N library denaturant was added, mixed gently with shaking, centrifuged instantaneously, incubated at room temperature for 5min, and then 5. mu.l of freshly prepared Tris-HCl (200mM) was immediately added to the tube.
d. Dilution of the library: adding 985 mu l of hybridization buffer HT1 precooled on ice into the neutralized 15 mu l library, mixing uniformly, quickly centrifuging, taking 117 mu l of hybridization buffer into another new 1.5ml centrifuge tube, supplementing 1183 mu l of HT1 solution, fully shaking and mixing uniformly to obtain 1300 mu l of on-machine library with the concentration of 1.8pM, and keeping on ice for later use.
e. 1300. mu.l of the prepared upper library was completely injected into the Load Samples well by piercing the foil seal of the Load Samples well with a clean 1mL tip. Avoiding contact with the foil seal.
f. After loading the sample, the wells were checked for air bubbles, and if any, the kit was gently tapped on a bench to release the air bubbles.
5.3 sequencing on machine
Transferring the sequencing kit loaded with the Library to a sequencing area through a transfer window, putting the sequencing kit into NextSeq CN500, and performing on-machine sequencing according to 'NextSeq CN500 high-throughput sequencer standard operating procedures', wherein the on-machine sequencing adopts a PE150 mode, and run name and Library ID are uniformly set as: and (4) loading the machine with date and chip number.
6. Bioinformatic analysis and result interpretation
6.1 data preprocessing
Analyzing Sequencing quality Q30 base ratio of each batch of data by using Illumina Sequencing Analysis Viewer v1.8 software, and if the base ratio of the batch of data Q30 is more than or equal to 75%, passing quality control; if the base percentage of the batch data Q30 is less than 75%, the quality control is not passed. The BCL files generated by MiSeqDx sequencing were then converted into FastQ files corresponding to the samples using BCL2FastQ v2.19 software from Illumina. The adaptor sequences introduced during the library construction and low quality base fragments are removed using correlation analysis software such as Trimmomatic-0.36 software.
6.2 data alignment
The sequence alignment module (such as BWA v0.7 and GATK v3.4 software) is used for aligning the base sequence in the FastQ file to hg19(GRCh37) human reference genome to generate BAM file, and the BAM file is ordered according to genome coordinates, and then sequence alignment optimization is carried out on the genome complex region.
6.3 data quality control
And (3) calculating parameters such as the Q30 base ratio, the ratio of sequence alignment to a reference genome, the average sequencing depth of a target region and the like of each sample by using related data quality control software. If the base ratio of Q30 is more than or equal to 75%, the sequence alignment is carried out until the ratio of the reference genome is more than or equal to 90%, and the average sequencing depth is more than or equal to 100X, the quality control of the sample data is passed. And if the data quality control fails, judging that the experiment fails and needing to be performed again.
6.4 mutation analysis
Point mutations and indel mutations in the samples were analyzed using a variation identification module (e.g., VarScan v2.3 software).
6.5SNP analysis
And carrying out SNP analysis and interpretation on the mutation sites by using PharmGKB database, CLINVAR database, dbSNP database and other related databases.
6.6 genotype determination
After the analysis process of the 6.1-6.5 steps, the mutation result meeting the quality index requirement is subjected to related genotype judgment.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (9)
1. A method for rapidly analyzing the CYP2C9 genotype, comprising at least the steps of:
(1) extracting a DNA sample;
(2) pretreating a DNA sample:
(2-1) breaking, repairing the tail end and adding a tail end A to the DNA sample to obtain a DNA fragment;
(2-2) carrying out pre-amplification and purification on the DNA fragment obtained in the step (2-1) to obtain a pre-amplification product;
(3) and (3) performing hybridization capture on the pre-amplification product:
(3-1) mixing all the obtained pre-amplification products, adding a pre-hybridization reagent, and performing hybridization amplification;
(3-2) capturing and cleaning the product obtained in the step (3-1), and then amplifying and purifying;
(4) library pooling and sequencing:
(4-1) calculating the pooling volume of each library and then sorting, and mixing all the libraries in order;
(4-2) carrying out on-machine sequencing on the mixed library to obtain sequencing data;
(5) the sequencing data were pre-processed and then subjected to data analysis to determine the CYP2C9 genotype.
2. The method for rapid analysis of CYP2C9 genotype according to claim 1, wherein the PCR reaction conditions for DNA disruption in step (2-1) are: the temperature of a hot cover of the PCR instrument is 68-72 ℃, the reaction is carried out for 22min at 32 ℃, and the reaction is carried out for 30min at 65 ℃.
3. The method for rapid analysis of CYP2C9 genotype according to claim 1, wherein the PCR reaction conditions for pre-amplification in step (2-2) are: the temperature of a hot cover of the PCR instrument is 104-106 ℃, 98 ℃, 45s and 1 cycle; 8 cycles of 98 ℃, 15s, 60 ℃, 30s, 72 ℃, 30 s; 72 ℃, 1min, 1 cycle; then the temperature is reduced to 4 ℃.
4. The method for rapid analysis of CYP2C9 genotype according to claim 1, wherein said PCR reaction conditions of step (3-1) are: the temperature of a hot cover of the PCR instrument is 84-86 ℃, the reaction is carried out for 5min at 95 ℃; reacting for 1-4 h at 60 ℃.
5. The method for rapid analysis of CYP2C9 genotype according to claim 1, wherein said PCR reaction conditions of step (3-2) are: the temperature of a hot cover of the PCR instrument is 104-106 ℃, 98 ℃, 45s and 1 cycle; 12 cycles of 98 ℃, 15s, 60 ℃, 30s, 72 ℃, 30 s; 72 ℃, 1min, 1 cycle; then the temperature is reduced to 4 ℃.
6. The method for rapid analysis of CYP2C9 genotype according to claim 1, wherein said data analysis in step (5) comprises the steps of:
(5-1) aligning the sequencing sequence to a human reference genome by using a sequence alignment module;
(5-2) analyzing the point mutation and the indel mutation in the sample using a variation identification module;
(5-3) SNP analysis and interpretation of the mutation sites using the database.
7. The method for rapid analysis of CYP2C9 genotype of claim 7, wherein said sequence alignment module in step (5-1) is BWA software or GATK software.
8. The method for rapid analysis of CYP2C9 genotype according to claim 7, wherein said mutation identification module in step (5-2) is VarScan software.
9. The method for rapid analysis of CYP2C9 genotype according to claim 7, wherein said database in step (5-3) comprises PharmGKB database, CLINVAR database, dbSNP database.
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