CN114703261A - A kind of multiplex PCR specific gene detection primer set, kit, method and application - Google Patents

Abstract

A multiplex PCR specific gene detection primer set, kit, method and application, which can be used to detect cardiovascular and cerebrovascular disease drug gene polymorphism sites; including 55 pairs of specific primers, divided into two Pools, and Pool-1 contains 23 pairs of primers, the nucleic acid sequences are shown in SEQ ID NO: 1-46; Pool-2 includes 32 pairs of primers, and the nucleic acid sequences are shown in SEQ ID NO: 47-110; the technical solution of the application has the ability to be used for cardiovascular and cerebrovascular diseases Gene detection of drug use can reduce the side effects of drugs, predict the efficacy of drugs, and use the advantages of precise drug dosage.

Description

A multiplex PCR specific gene detection primer set, kit, method and application

technical field

The invention belongs to the field of biomolecule detection, and in particular relates to a multiplex PCR specific gene detection primer set, kit, method and application, which can be used to detect the polymorphism sites of cardiovascular and cerebrovascular diseases drug genes.

Background technique

Cardiovascular and cerebrovascular diseases are a general term for cardiovascular and cerebrovascular diseases, which generally refer to ischemic or hemorrhagic diseases of the heart, brain and whole body caused by hyperlipidemia, blood viscosity, atherosclerosis, hypertension, etc. disease. Cardiovascular and cerebrovascular disease is a common disease that seriously threatens the health of human beings, especially the middle-aged and elderly people over 50 years old. The prevalence of cardiovascular disease in China continues to rise. It is estimated that the number of people suffering from cardiovascular diseases is 330 million, including 13 million strokes, 11 million coronary heart diseases, 5 million pulmonary heart diseases, 8.9 million heart failures, 2.5 million rheumatic heart diseases, 2 million congenital heart diseases, and lower extremity arteries. Disease 45.3 million, hypertension 245 million.

With the completion of the Human Genome Project and the advent of the post-genome era, truly personalized medicine has become a reality. People can use advanced molecular biology techniques to detect and interpret drug-related genes (drug metabolizing enzymes, transporters and receptor genes) of different individuals, and clinicians can implement dosing regimens according to patients' genotype data to improve the efficacy of drugs. Efficacy and reduction of toxic and side effects of drugs, while reducing the pain and economic burden of patients, this kind of gene-oriented individualized medicine, which represents the perfect combination of pharmacogenomics and clinical individualized drug treatment, is of epoch-making significance.

Genetic testing is a technology that detects DNA through blood, other body fluids or cells. Molecular information is used for detection, to predict the risk of disease in the body, and to analyze the various genes contained in it, so that people can understand their own genetic information, so as to avoid or delay the occurrence of diseases by improving their living environment and living habits.

On March 22, 2005, the U.S. Food and Drug Administration (FDA) issued guidelines for "Pharmacogenomic Data Submissions" for pharmaceutical companies. The guideline aims to urge pharmaceutical companies to provide the pharmacogenomic data of the drug according to specific circumstances when submitting new drug applications. Genetic status”, so that patients can obtain the maximum drug effect while only facing the minimum risk of adverse drug reactions. In 2007, the genomics detection method of warfarin was approved for the first time to determine its dosage and sensitivity. In February 2010, the drug insert of warfarin was revised. Gene testing for CYP2C9 and VKORC1 before prescribing Farin, and drug dosage adjustment for different genotypes. Since 2014, my country's CFDA and the National Health and Family Planning Commission have intensively launched policies related to gene sequencing. In March 2015, the Ministry of Science and Technology held the country's first precision medicine strategy expert meeting, and the precision medicine plan will be launched soon. On July 31, 2015, the Chinese National Health and Family Planning Commission individual The Expert Committee of Chemical Medicine Detection Technology, based on extensive consultation, formulated the "Technical Guidelines for Gene Detection of Drug Metabolizing Enzymes and Drug Action Targets (Trial)" and "Technical Guidelines for the Detection of Tumor Individualized Therapy (Trial)", and proposed Before 2030, China's precision medicine will invest 60 billion yuan, and domestic precision medicine will bring new changes to my country's medical methods driven by huge funds.

Next-generation sequencing "PCR+sequencing" method is the gold standard for molecular detection. Primers can be designed for the target region of drug-related genes and subjected to PCR amplification, and then the target region can be sequenced to obtain the results; however, this technology is time-consuming and labor-intensive, with low throughput and high price, which limits its clinical application. As a representative of high-throughput detection technology, biochip has become one of the important technology choices for multi-target detection due to its high-throughput, simple operation and cost advantages. However, the method contains a limited number of genes and loci and can only detect known variants. Time-of-flight mass spectrometry has a high throughput. Each instrument can detect 3000 samples per day. It can detect many sites and is efficient and accurate. However, it requires high personnel skills and the purity of the detected samples, and the instrument is expensive. General medical institutions to carry out, limiting its wide application. Recent applications of next-generation sequencing (NGS) have proven powerful in detecting genetic loci polymorphisms associated with personalized medicine. Multiplex PCR simultaneously amplifies multiple regions in the same reaction tube, resulting in significant time and reagent savings and more accurate diagnostic information.

And how to use next-generation sequencing as a technology platform to enable it to be used for genetic testing of drugs for cardiovascular and cerebrovascular diseases, to provide patients with drug side effects and drug efficacy predictions, so as to reduce the side effects of drug use, and to change the drug dose by changing the drug dose. To improve the therapeutic efficacy of drugs has become an urgent technical problem to be solved.

SUMMARY OF THE INVENTION

Aiming at the above-mentioned deficiencies of the prior art, the present application provides a multiple PCR-specific gene detection primer set that can be used for gene detection of drugs for cardiovascular and cerebrovascular diseases, reduces drug side effects, predicts drug efficacy, and uses precise drug doses.

In order to solve the above-mentioned technical problems, the technical solution adopted in the present application is: a multiplex PCR specific gene detection primer set, including 55 pairs of specific primers, divided into two Pools, Pool-1 includes 23 pairs of primers, and the nucleic acid sequence such as SEQ ID NO: 1-46; Pool-2 contains 32 pairs of primers, and the nucleic acid sequence is shown in SEQ ID NO: 47-110.

Further, the 55 pairs of specific primers described in this application, wherein: the primer pairs whose nucleic acid sequence is SEQ ID NO:1-2 corresponds to the VKORC1 gene; the primer pair whose nucleic acid sequence is SEQ ID NO:3-4 corresponds to the CYP3A5 gene; The primer pairs with nucleic acid sequences of SEQ ID NO:5-6 correspond to the ABCC8 gene; the primer pairs with the nucleic acid sequences of SEQ ID NOs:7-8 correspond to the APOE gene; the primer pairs with the nucleic acid sequences of SEQ ID NO:9-10 correspond to the DOT1L gene Nucleic acid sequence is the primer pair corresponding LTC4S gene of SEQ ID NO:11-12; Nucleic acid sequence is the primer pair corresponding ALDH2 gene of SEQ ID NO:13-14; Nucleic acid sequence is the primer pair corresponding APOA5 of SEQ ID NO:15-16 Gene; the primer pairs whose nucleic acid sequences are SEQ ID NOs: 17-18 correspond to the GP1BA gene; the primer pairs whose nucleic acid sequences are SEQ ID NOs: 19-20 correspond to the GNB3 gene; the primer pairs whose nucleic acid sequences are SEQ ID NOs: 21-22 correspond to KCNJ11 gene; primer pairs with nucleic acid sequences of SEQ ID NOs: 23-24 correspond to AGTR1 gene; primer pairs with nucleic acid sequences of SEQ ID NOs: 25-26 correspond to NPPA gene; primer pairs with nucleic acid sequences of SEQ ID NOs: 27-28 Corresponds to CYP2C19 gene; the primer pair whose nucleic acid sequence is SEQ ID NO: 29-30 corresponds to the ADD1 gene; the primer pair whose nucleic acid sequence is SEQ ID NO: 31-32 corresponds to the CETP gene; the primer pair whose nucleic acid sequence is SEQ ID NO: 33-34 Corresponds to COQ2 gene; the primer pair of nucleotide sequence is SEQ ID NO:35-36 is corresponding to APOE gene; The primer pair of nucleic acid sequence is SEQ ID NO:37-38 is corresponding to CYP2C19 gene; The primer pair of nucleic acid sequence is SEQ ID NO:39-40 Pair corresponding to NEDD4L gene; nucleic acid sequence is SEQ ID NO:41-42 primer pair corresponding SLCO1B1 gene; nucleic acid sequence is SEQ ID NO:43-44 primer pair corresponding SLC22A2 gene; nucleic acid sequence is SEQ ID NO:45-46 The primer pair corresponds to the SLCO1B1 gene; the primer pair with the nucleic acid sequence of SEQ ID NO:47-48 corresponds to the KCNIP1 gene; the primer pair with the nucleic acid sequence of SEQ ID NO:49-50 corresponds to the SLC22A1 gene; the nucleic acid sequence is SEQ ID NO:51-52 The primer pair corresponding to ABCG2 gene; The primer pair that nucleotide sequence is SEQ ID NO:53-54 is corresponding to CYP4F2 gene; The primer pair that nucleotide sequence is SEQ ID NO:55-56 is corresponding KIF6 gene; Nucleic acid sequence is SEQ ID NO:57-58 primer pairs corresponding to ABCB1 gene; primer pairs with nucleic acid sequences of SEQ ID NOs: 59-60 correspond to ABCB1 genes; primer pairs with nucleic acid sequences of SEQ ID NOs: 61-62 correspond to MTRR genes; primer pairs with nucleic acid sequences of SEQ ID NOs: 63-64 correspond to PPAGR gene; primer pairs with nucleic acid sequences of SEQ ID NO: 65-66 correspond to ADRB1 gene; primer pairs with nucleic acid sequences of SEQ ID NO: 67-68 correspond to MTHFR gene; primer pairs with nucleic acid sequences of SEQ ID NO: 69-70 Corresponding MTHFR gene; nucleic acid sequence is the primer pair of SEQ ID NO:71-72 corresponding to NAT2 gene; nucleic acid sequence is the primer pair of SEQ ID NO:73-74 corresponding NAT2 gene; nucleic acid sequence is the primer of SEQ ID NO:75-76 The pair corresponds to the NAT2 gene; the primer pair with the nucleic acid sequence of SEQ ID NO:77-78 corresponds to the CYP2C9 gene; the primer pair with the nucleic acid sequence of SEQ ID NO:79-80 corresponds to the ACE gene; the nucleic acid sequence is the pair of primers of SEQ ID NO:81-82 The primer pair corresponds to the PRKCA gene; the primer pair with the nucleic acid sequence of SEQ ID NO:83-84 corresponds to the KCNK3 gene; the primer pair with the nucleic acid sequence of SEQ ID NO:85-86 corresponds to the CYP2C19 gene; the nucleic acid sequence is SEQ ID NO:87-88 The primer pair corresponding to NAT2 gene; the primer pair corresponding to the nucleotide sequence of SEQ ID NO:89-90 is the corresponding GPIa gene; the primer pair corresponding to the nucleotide sequence of SEQ ID NO:91-92 is the corresponding C11ORF65 gene; the nucleic acid sequence is SEQ ID NO:93-94 The primer pair corresponding to NOS1AP gene; the primer pair corresponding to the nucleotide sequence of SEQ ID NO:95-96 is the corresponding CYP2D6 gene; the primer pair corresponding to the nucleotide sequence of SEQ ID NO:97-98 is the corresponding GPIa gene; the nucleotide sequence is SEQ ID NO:99- The primer pair of 100 corresponds to the CYP2C9 gene; the primer pair of nucleotide sequence SEQ ID NO:101-102 corresponds to the NOS1AP gene; the primer pair of nucleotide sequence SEQ ID NO:103-104 corresponds to the ABCB1 gene; the nucleic acid sequence is SEQID NO:105- The primer pair of 106 corresponds to the ADRB2 gene; the primer pair with the nucleic acid sequence of SEQ ID NO: 107-108 corresponds to the NAT2 gene; the primer pair with the nucleic acid sequence of SEQ ID NO: 109-110 corresponds to the COX-1 gene.

Further, the specific gene detection primer sets described in this application are all modified primers, no dimer is formed between the primers, the primers themselves do not form a hairpin structure, and no mismatch occurs between the primers and the template, In addition, there is no mutation site in the primer design region. At the same time, during the primer design, the annealing temperature difference between each pair of primers is less than 2 degrees Celsius, and the length of the amplified product is between 200-300 bp.

The present application provides a kit for detecting individualized drug-related gene loci polymorphisms based on multiple PCR and high-throughput sequencing technology, the kit includes the nucleic acid sequences of SEQ ID NOs: 1-110. , at least one of the 55 pairs of specific primers.

Further, the kit for detecting individualized drug-related gene loci polymorphisms based on multiplex PCR and high-throughput sequencing technology includes specific primers, Index, enzyme mixture, PCR Enhancer, and nuclease-free water; In order to amplify the sequence containing the target polymorphism site, Index is used to mark the amplification products of the target region of different samples to be tested, and it is an 8-base nucleotide sequence (Index R5 and Index R7 are the raw material). The enzyme mixture includes: high-fidelity DNA polymerase, PCR Buffer, dNTP mixture; the kit can also include quality control products and blank quality control products, of which the quality control products are Normal human genomic DNA, blank quality control is nuclease-free water.

Further, the described kit based on multiplex PCR and high-throughput sequencing technology for detecting the polymorphism of individualized medicine related gene loci, the specific components are as follows:

The application provides a method for detecting gene polymorphism sites based on multiplex PCR and high-throughput sequencing technology, comprising the following steps:

(1) Nucleic acid extraction and quality control of the samples to be tested;

(2) at least one of the 55 pairs of specific primers whose nucleotide sequences are SEQ ID NOs: 1-110, carry out PCR amplification to the target region of the extracted DNA, for subsequent use;

(3) Use the magnetic bead method to purify the PCR amplification products, use the Fluorometer Qubit (Qubit Fluorometer fluorescence quantitative instrument) to measure the concentration of the purified amplicon library, record the library concentration, and compare the pool-1 and pool-2 The purified products were mixed with equal quality to obtain a library; then the library was subjected to fragment length determination (using Agilent 4200TapeStation or Agilent 2100Bioanalyzer system). If the fragment length is mainly distributed in 300-500bp, the main peak is 380±10bp, the quality inspection is qualified, and the mixed sample is quantitatively mixed. , on-machine sequencing; if the length distribution exceeds 300-500bp, or the main peak is not between 380±10bp, repeat the library construction and quality inspection. If it fails again, the test will be terminated, and the blank quality control group should be unqualified, otherwise it means that there is nucleic acid contamination , no sequencing is required.

Further, the PCR reaction system used in the described PCR amplification:

Further, the procedure of described PCR amplification is:

Further, the samples to be detected include but are not limited to blood, paraffin-embedded tissue, frozen sectioned tissue, and the like.

Further, the described on-machine sequencing is high-throughput sequencing, and the high-throughput sequencing platforms used include but are not limited to sequencing such as Illumina HiSeq/MiSeq, Ion Torrent/Proton platform of Life, BGISEQ-500/50 of BGI platform.

The present application also provides an application of a multiplex PCR-specific gene detection primer set in the gene detection of drugs for cardiovascular and cerebrovascular diseases.

Advantages and beneficial effects of the present application:

1. The specific primer set of this application can detect the genes of drug use in cardiovascular and cerebrovascular diseases and predict the metabolic phenotype, reduce adverse drug reactions (ADRs) and drug toxicity, predict the efficacy, and guide clinical individualized drug use , which can effectively improve the detection efficiency and accuracy, while reducing the cost, one-time detection, lifetime use.

2. The specificity of the specific primer set of the present application is high, no dimer is formed between the primer and the primer, the primer itself does not form a hairpin structure, no mismatch occurs between the primer and the template, and there is no mutation in the primer design region At the same time, in the primer design, the annealing temperature difference between each pair of primers is less than 2 degrees Celsius, and the length of the amplified product is between 200-300bp.

3. The present invention is aimed at the detection of common drug-related gene polymorphism sites, which can effectively improve detection efficiency and accuracy, while reducing costs and simplifying operation steps, and is a site-specific gene detection primer for detecting drug-related gene polymorphisms. Innovation on set.

4. The primer set is divided into primer pool-1 and primer pool-2 in this application; because the number of specific primers for drug-related gene loci is large, the above scheme can avoid the occurrence of primer dimers to a certain extent, and is divided into two primer pools, PCR was performed separately.

Description of drawings

FIG. 1 is a peak diagram of the distribution of library fragments constructed in Example 1. FIG.

Detailed ways

The present invention will be described below through specific embodiments, but the present invention is not limited thereto. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified, and the following examples are not intended to limit the scope of the invention. , all equivalent implementations or changes that do not depart from the present invention shall be included in the protection scope of this patent.

Example 1

The composition of the kit of the present embodiment is specifically shown in Table 1 below:

Table 1 Composition of the kit of this example

Specific Primer Pool-1 and Specific Primer Pool-2 are used in the above reagents (Pool-1 contains 23 pairs of primers, and the nucleic acid sequences are shown in the following table SEQ ID: 1-46; Pool-2 contains 32 pairs of primers, and the nucleic acid sequences are shown in the following table SEQ ID: : 47-110), the corresponding specific sequence is shown in Table 2 below:

Table 2 Primer sequences of each gene locus

Note: This application involves the sequences of multiple PCR-specific gene detection primer sets. The sequences in Table 1 above shall prevail;

The size of the kit is for 12 people.

Example 2 Detection of 10 whole blood samples

Unconventional instruments required: ABI 7500, PCR instrument, magnetic stand, Agilent 2100 bioanalyzer, Qubit3.0, NextSeq CN500;

Sample source: The sample comes from the laboratory, and is a whole blood sample stored in an EDTA anticoagulation tube. The extraction volume is 200ul, and the elution volume is 30ul.

1. Library construction

Due to the large number of specific primers for drug-related gene loci, in order to avoid the occurrence of primer-dimers to a certain extent, it is necessary to divide into two primer pools and perform PCR separately.

a) Prepare a sterile, nuclease-free 200ul PCR tube, place it on an ice box, and prepare a library amplification system according to Table 3 below:

Table 3 Library amplification system

reaction system volume 2x PCR Mix 15ul Specific Primer Pool-1/-2 1ul Index R5 2ul Index R7 2ul Enhancer 1ul DNA (50ng) 2ul DNase-free water 7ul Total 30ul

Note: Different R7 primers should be used for different samples; sample libraries sequenced on the same batch should not use the same combination of tags.

The concentration of 10 whole blood samples was diluted to 25ng/ul;

b) Gently mix by pipetting, centrifuge briefly and place on ice;

c) Put the tube in the PCR machine, run the program according to Table 4, set the hot cover to 105°C, and the reaction volume is 30ul;

Table 4 PCR program running parameters

d) After the operation is finished, take out the 200ul PCR tube and proceed to the next step;

e) Take out the magnetic beads and equilibrate at room temperature for 30 minutes;

f) The amplified product was supplemented to 50 μL with nuclease-free water, transferred to a 1.5 mL centrifuge tube without nuclease contamination, added with 47.5 μL of purified magnetic beads, mixed by pipetting for 10 times, centrifuged briefly, and allowed to stand for 5 minute;

g) Place the centrifuge tube on the magnetic rack for 5 minutes until the liquid is completely clear;

h) Prepare 80% ethanol solution, 400 μL per reaction;

i) Keep the centrifuge tube on the magnetic stand, use a pipette to remove the clear liquid, add 200 μL of 80% ethanol solution, let stand for 30 seconds at room temperature, and repeat this step once;

j) Use a pipette to suck off the liquid to ensure that there is no liquid residue in the tube, and leave it at room temperature for 3 to 5 minutes;

k) Remove the centrifuge tube from the magnetic stand, resuspend the magnetic beads with 20 μL of 10 mM Tris-HCl (pH 8.0), and place at room temperature for 3 minutes;

l) Place the centrifuge tube on the magnetic stand again and let it stand for 2 minutes at room temperature or the liquid becomes clear;

m) Transfer the clear liquid to a new centrifuge tube. If the next step is not performed immediately, store it in a -20±5°C refrigerator for no more than 1 month, and freeze-thaw times no more than 3 times;

n) Use Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Cat. No.: Q32851 or Q32854) to detect the concentration of the library. If the library concentration measured by Qubit is ≤0.5ng/μL, the library construction fails and needs to be rebuilt;

o) Mix the two reaction tubes (Tubel and Tube2) of a sample with equal quality to obtain the final library of the sample;

p) Use the Agilent High Sensitivity DNA Kit (produced by Agilent, Cat. No. 5067-4626) to detect the concentration and fragment size distribution of the library. The target fragments should be concentrated at 200-350bp. If they are inconsistent, the library construction fails and needs to be rebuilt;

2. On-board sequencing

After the library was quantified by an Agilent 2100 bioanalyzer, the on-board detection was carried out according to the standard operating procedures of the instrument. The sequencing mode is 150PE, and the paired-end index recognition mode is used. The amount of library data is not less than 0.3Gb. The recommended concentration of NextSeq CN500 is 1.0～1.5pM. Note: The following process is the process of using sequencing primers (CIS Primer) to identify double-ended Index. If you only need to identify single-ended Index, you do not need to use sequencing primer.

a) Take out the Reagent Cartridge, HT1 and Flow Cell in advance, place the Flow Cell at room temperature for equilibration for 30 minutes, place the Reagent Cartridge in a water bath at room temperature for 1 hour, and shake and mix the HT1 after standing at room temperature to thaw;

b) Mix all the sample libraries to be sequenced according to the amount of sequencing data, use the nucleic acid quantification kit HighSensitivity DNA c) Kit (Agilent) and supporting instruments to measure the concentration of the mixed library, dilute the mixed library with HT1 to 4nM, and the mixing volume is greater than 5L ;

c) Prepare fresh 0.2N NaOH, which is obtained by adding 10 μL 2N NaOH to 90 μL purified water and mixing;

d) Take 5μL of 4nM mixed library into a new 1.5mL centrifuge tube, add 5μL of freshly prepared 0.2N NaOH, mix by pipetting and let stand for 5 minutes at room temperature for denaturation, add 5μL of 200mM Tris to neutralize NaOH after denaturation, and add after mixing 985μL HT1 to dilute the mixed library to 20pM, volume 1000μL;

e) Select the appropriate final concentration on the machine, use HT1 for dilution, and the final volume is 1300 μL;

f) Take out the Reagent Cartridge thawed in a water bath at room temperature, dry it with dust-free paper, and invert 5 times to mix the reagents.

g) Use a clean 1mL pipette tip to pierce the foil seal of the No. 10 well with the Load Samples label on the Reagent Cartridge, and inject 1300 μL of the mixed library into the No. 10 well;

h) Poke hole 22 with a clean 1mL pipette tip, add 9μL of sequencing primer when using the Illumina NextSeq500 HighOutput v2 Reagent kit, add 5μL of sequencing primer when using the Illumina NextSeq 500Mid Output v2Reagent kit, and then use a 1mL pipette to lightly Mix by gently pipetting 10 times.

i) Carry out the reagent loading operation according to the sequencer operation setting prompt. After the sequencer self-check is completed, click the "Start" button to perform sequencing.

3. Bioinformatics analysis

After the sequencing is completed, the sequencing off-machine data is analyzed.

a) Quality control of the original off-line data, use fastp (v0.21.0) software to automatically find and trim the adapter sequence, and use the sliding window to calculate the average quality value of the base, and then directly trim the sliding window that does not meet; quality control; After completion, statistics of relevant indicators are performed on the valid reads data set to evaluate the quality of this sequencing data; see Table 5 for details:

table 5

b) Reference genome comparison, the clean data after quality control is compared with the human reference genome hg19 version using BWA (v 0.7.17) software, and the reads are pasted back to the genome to provide the former data bam file for subsequent mutation detection.

c) Use Picard (v 2.25.4) software to count the sequencing depth of each probe coverage area to evaluate the capture capability of the target area; see Table 6 for details:

Table 6

d) Variation detection, the bam file obtained after comparison, use the UnifiedGenotyper in GATK (v 3.3) to perform mutation detection to obtain the vcf file, and each mutation site and genotype of each sample will give an accurate posterior probability ;

e) Perform genotyping analysis on each site on the genome, record the genotype mutation frequency and effective sequencing depth of each variant site, and summarize the genotype information of all samples in an excel sheet.

The quality control data are as follows:

a) Target area coverage: 100% of the designed amplified fragments of the panel are amplified;

b) Uniformity of sequencing data: the coverage of amplified fragments on 96% is greater than 20% of the average sequencing depth;

c) The mapping ratio of the qualified data in the target region after removing the adapters from the sequencing data is 93%.

Fig. 1 is a peak map of the distribution of library fragments constructed in the above-mentioned example, indicating that the library is obtained by the scheme of the present application.

4. Analyze the test results

After the detection result is obtained, the gene locus interpretation information is obtained according to the pharmacogenome database, and the corresponding drug use guidance information of the gene locus is given according to the interpretation information.

sequence listing

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<213> artificial sequence()

<400> 40

ggggcctgct gaccactgaa 20

<210> 41

<211> 20

<212> DNA

<213> artificial sequence()

<400> 41

ccatctggct gagcaatgtt 20

<210> 42

<211> 24

<212> DNA

<213> artificial sequence()

<400> 42

accctggaaa gagctggagt aatt 24

<210> 43

<211> 21

<212> DNA

<213> artificial sequence()

<400> 43

accttgatgc atgttccatg c 21

<210> 44

<211> 20

<212> DNA

<213> artificial sequence()

<400> 44

aaagggtcca gtcacgaaga 20

<210> 45

<211> 24

<212> DNA

<213> artificial sequence()

<400> 45

acacacacac acacacacac tttt 24

<210> 46

<211> 24

<212> DNA

<213> artificial sequence()

<400> 46

tcagtgcagt tgagaggatt aaag 24

<210> 47

<211> 24

<212> DNA

<213> artificial sequence()

<400> 47

acaggaaaca ggcagtgagg atac 24

<210> 48

<211> 22

<212> DNA

<213> artificial sequence()

<400> 48

gccactcaag cggtcctctc at 22

<210> 49

<211> 24

<212> DNA

<213> artificial sequence()

<400> 49

atagagcaag aagaagaagt tggg 24

<210> 50

<211> 24

<212> DNA

<213> artificial sequence()

<400> 50

cagaatttgt tgggcggaga tatc 24

<210> 51

<211> 20

<212> DNA

<213> artificial sequence()

<400> 51

ttctctgtgc ctaaccctgt 20

<210> 52

<211> 21

<212> DNA

<213> artificial sequence()

<400> 52

aaatgagtca gagggtttcc c 21

<210> 53

<211> 22

<212> DNA

<213> artificial sequence()

<400> 53

cctcagcaca atcggggagt cc 22

<210> 54

<211> 23

<212> DNA

<213> artificial sequence()

<400> 54

gcaatcccca tctccaggct ctc 23

<210> 55

<211> 24

<212> DNA

<213> artificial sequence()

<400> 55

atcttggccc cgctactgtt agaa 24

<210> 56

<211> 23

<212> DNA

<213> artificial sequence()

<400> 56

actttgtcaa atggcgtccg agc 23

<210> 57

<211> 24

<212> DNA

<213> artificial sequence()

<400> 57

ggaaactgga ggtatacttt catc 24

<210> 58

<211> 22

<212> DNA

<213> artificial sequence()

<400> 58

ccattctgtt tactcctgct ta 22

<210> 59

<211> 23

<212> DNA

<213> artificial sequence()

<400> 59

gtacgacaca cagcaacctt agg 23

<210> 60

<211> 24

<212> DNA

<213> artificial sequence()

<400> 60

ccacgtatgt accacccagc ttaa 24

<210> 61

<211> 24

<212> DNA

<213> artificial sequence()

<400> 61

tcctctgttg aaggtggggt taat 24

<210> 62

<211> 24

<212> DNA

<213> artificial sequence()

<400> 62

ggcctattag caccaaaact tacc 24

<210> 63

<211> 24

<212> DNA

<213> artificial sequence()

<400> 63

tgctgacacg gaaagatcac taag 24

<210> 64

<211> 24

<212> DNA

<213> artificial sequence()

<400> 64

cagagcctag aacagtgcca ggta 24

<210> 65

<211> 22

<212> DNA

<213> artificial sequence()

<400> 65

agaggcagat tgggagggtc ag 22

<210> 66

<211> 23

<212> DNA

<213> artificial sequence()

<400> 66

tcctaccctg ctccccacat tct 23

<210> 67

<211> 24

<212> DNA

<213> artificial sequence()

<400> 67

gcggaagaat gtgtcagcct caaa 24

<210> 68

<211> 24

<212> DNA

<213> artificial sequence()

<400> 68

ggtctcttca tccctcgcct tgaa 24

<210> 69

<211> 24

<212> DNA

<213> artificial sequence()

<400> 69

ccccactcca gcatcactca cttt 24

<210> 70

<211> 24

<212> DNA

<213> artificial sequence()

<400> 70

gcaattcctc ttcccctgcc tttg 24

<210> 71

<211> 24

<212> DNA

<213> artificial sequence()

<400> 71

cgtgccattt acaaagctgc tgat 24

<210> 72

<211> 23

<212> DNA

<213> artificial sequence()

<400> 72

cttgcttttg agccgagttt act 23

<210> 73

<211> 24

<212> DNA

<213> artificial sequence()

<400> 73

cctccgagat gctacctgga taat 24

<210> 74

<211> 22

<212> DNA

<213> artificial sequence()

<400> 74

gcaaaatccc agcacattta gt 22

<210> 75

<211> 24

<212> DNA

<213> artificial sequence()

<400> 75

gaggttgaag aagtgctgag aaat 24

<210> 76

<211> 23

<212> DNA

<213> artificial sequence()

<400> 76

aacgtgaggg tagagaggat atc 23

<210> 77

<211> 23

<212> DNA

<213> artificial sequence()

<400> 77

acgtgaaaag tgaaccgagt act 23

<210> 78

<211> 24

<212> DNA

<213> artificial sequence()

<400> 78

tgtctgatat caaggctcac actt 24

<210> 79

<211> 24

<212> DNA

<213> artificial sequence()

<400> 79

tggagaggga gaggagggac attt 24

<210> 80

<211> 24

<212> DNA

<213> artificial sequence()

<400> 80

tgtaggatag gatgtgggcg aaat 24

<210> 81

<211> 24

<212> DNA

<213> artificial sequence()

<400> 81

cagttgagag acacttgtgt tgtt 24

<210> 82

<211> 24

<212> DNA

<213> artificial sequence()

<400> 82

aacatgtact gctggtggga attc 24

<210> 83

<211> 24

<212> DNA

<213> artificial sequence()

<400> 83

gcttgcatct tggattgtct tttc 24

<210> 84

<211> 24

<212> DNA

<213> artificial sequence()

<400> 84

accagcatcg tgtcagagga atca 24

<210> 85

<211> 23

<212> DNA

<213> artificial sequence()

<400> 85

gggatggggga agaggagcat tga 23

<210> 86

<211> 24

<212> DNA

<213> artificial sequence()

<400> 86

agatatggcc acccctgaaa tgtt 24

<210> 87

<211> 24

<212> DNA

<213> artificial sequence()

<400> 87

aagcaatctc aaggtccctg atct 24

<210> 88

<211> 24

<212> DNA

<213> artificial sequence()

<400> 88

aggagagatt aatgaaccca agag 24

<210> 89

<211> 20

<212> DNA

<213> artificial sequence()

<400> 89

agcttctggt gggcgacgaa 20

<210> 90

<211> 24

<212> DNA

<213> artificial sequence()

<400> 90

acccaatagc ggcaagacaa gtga 24

<210> 91

<211> 24

<212> DNA

<213> artificial sequence()

<400> 91

tcagccagca ttcatagggt taaa 24

<210> 92

<211> 24

<212> DNA

<213> artificial sequence()

<400> 92

tcgggagctt gtgaacgtgt gtat 24

<210> 93

<211> 24

<212> DNA

<213> artificial sequence()

<400> 93

aaagagctgt atctgcccca ttaa 24

<210> 94

<211> 24

<212> DNA

<213> artificial sequence()

<400> 94

ccagtggggc ttcaagagag aaaa 24

<210> 95

<211> 24

<212> DNA

<213> artificial sequence()

<400> 95

ccactcccat cctttctccc attt 24

<210> 96

<211> 23

<212> DNA

<213> artificial sequence()

<400> 96

tggccatcac attcgtcaga tct 23

<210> 97

<211> 24

<212> DNA

<213> artificial sequence()

<400> 97

ggcacatatc ttgataggct atga 24

<210> 98

<211> 24

<212> DNA

<213> artificial sequence()

<400> 98

gccatttaca agcctaagtc tcag 24

<210> 99

<211> 24

<212> DNA

<213> artificial sequence()

<400> 99

caaaatgcca accctagccc caag 24

<210> 100

<211> 23

<212> DNA

<213> artificial sequence()

<400> 100

ctggagtgca gtggtgtgat cat 23

<210> 101

<211> 24

<212> DNA

<213> artificial sequence()

<400> 101

agaggcaggg aggaggaaga gaat 24

<210> 102

<211> 24

<212> DNA

<213> artificial sequence()

<400> 102

gcacccagag agatgtgtcc taga 24

<210> 103

<211> 24

<212> DNA

<213> artificial sequence()

<400> 103

agcatagtaa gcagtaggga gtaa 24

<210> 104

<211> 24

<212> DNA

<213> artificial sequence()

<400> 104

ggcaactaac actgttactc ttag 24

<210> 105

<211> 19

<212> DNA

<213> artificial sequence()

<400> 105

tgcgctcacc tgccagact 19

<210> 106

<211> 23

<212> DNA

<213> artificial sequence()

<400> 106

cagacgctcg aacttggcaa tgg 23

<210> 107

<211> 24

<212> DNA

<213> artificial sequence()

<400> 107

tttgttcctt gcagacccca gaag 24

<210> 108

<211> 22

<212> DNA

<213> artificial sequence()

<400> 108

caggtttggg cacgagattt ct 22

<210> 109

<211> 23

<212> DNA

<213> artificial sequence()

<400> 109

cctcggccac ctcactgact tac 23

<210> 110

<211> 23

<212> DNA

<213> artificial sequence()

<400> 110

acgcctctct ccatgtgcag tag 23

Claims (12)

1. a multiple PCR specific gene detection primer set is characterized in that: comprise 55 pairs of specific primers, be divided into two Pools, and Pool-1 comprises 23 pairs of primers, and the nucleic acid sequence is as shown in SEQ ID NO:1-46; Pool-2 contains 32 pairs of primers, and the nucleic acid sequences are shown in SEQ ID NOs: 47-110. 2. multiple PCR specific gene detection primer set according to claim 1, is characterized in that: described 55 pairs of specific primers, wherein: nucleic acid sequence is the primer pair of SEQ ID NO:1-2 corresponding VKORC1 gene; The primer pairs with nucleic acid sequence of SEQ ID NO:3-4 correspond to CYP3A5 gene; the primer pairs with nucleic acid sequence of SEQ ID NO:5-6 are corresponding to ABCC8 gene; the primer pairs with nucleic acid sequence of SEQ ID NO:7-8 are corresponding to APOE gene; The primer pairs with nucleic acid sequence of SEQ ID NO:9-10 correspond to DOT1L gene; the primer pairs with nucleic acid sequence of SEQ ID NO:11-12 are corresponding to LTC4S gene; the primer pairs with nucleic acid sequence of SEQ ID NO:13-14 are corresponding to ALDH2 gene; The primer pairs with nucleic acid sequence of SEQ ID NO:15-16 correspond to APOA5 gene; the primer pairs with nucleic acid sequence of SEQ ID NO:17-18 correspond to GP1BA gene; the primer pairs with nucleic acid sequence of SEQ ID NO:19-20 correspond to GNB3 gene Nucleic acid sequence is the primer pair corresponding KCNJ11 gene of SEQ ID NO:21-22; Nucleic acid sequence is the primer pair corresponding AGTR1 gene of SEQ ID NO:23-24; Nucleic acid sequence is the primer pair corresponding NPPA of SEQ ID NO:25-26 Gene; primer pairs with nucleic acid sequence of SEQ ID NO:27-28 correspond to CYP2C19 gene; primer pairs with nucleic acid sequence of SEQ ID NO:29-30 correspond to ADD1 gene; primer pairs with nucleic acid sequence of SEQ ID NO:31-32 correspond to CETP Gene; the primer pairs whose nucleic acid sequence is SEQ ID NO:33-34 corresponds to COQ2 gene; the primer pair whose nucleic acid sequence is SEQ ID NO:35-36 corresponds to APOE gene; the primer pair whose nucleic acid sequence is SEQ ID NO:37-38 corresponds to CYP2C19 gene; primer pairs with nucleic acid sequences of SEQ ID NO: 39-40 correspond to NEDD4L gene; primer pairs with nucleic acid sequences of SEQ ID NO: 41-42 correspond to SLCO1B1 gene; primer pairs with nucleic acid sequences of SEQ ID NO: 43-44 correspond to SLC22A2 gene; primer pairs with nucleic acid sequences of SEQ ID NO: 45-46 correspond to SLCO1B1 gene; primer pairs with nucleic acid sequences of SEQ ID NO: 47-48 correspond to KCNIP1 gene; primer pairs with nucleic acid sequences of SEQ ID NO: 49-50 correspond to SLC22A1 gene; primer pairs with nucleic acid sequences of SEQ ID NOs: 51-52 correspond to ABCG2 gene; primer pairs with nucleic acid sequences of SEQ ID NOs: 53-54 correspond to CYP4F2 genes; primer pairs with nucleic acid sequences of SEQ ID NOs: 55-56 Corresponds to KIF6 gene; nucleic acid sequence The primer pairs listed as SEQ ID NOs: 57-58 correspond to the ABCB1 gene; the primer pairs with nucleotide sequences of SEQ ID NO: 59-60 correspond to the ABCB1 gene; the primer pairs with nucleotide sequences of SEQ ID NO: 61-62 correspond to the MTRR gene; The primer pairs with nucleic acid sequences of SEQ ID NOs: 63-64 correspond to the PPAGR gene; the primer pairs with nucleic acid sequences of SEQ ID NOs: 65-66 correspond to the ADRB1 gene; the primer pairs with nucleic acid sequences of SEQ ID NOs: 67-68 correspond to the MTHFR gene Nucleic acid sequence is the primer pair corresponding MTHFR gene of SEQ ID NO:69-70; Nucleic acid sequence is the primer pair corresponding NAT2 gene of SEQ ID NO:71-72; Nucleic acid sequence is the primer pair corresponding NAT2 gene of SEQ ID NO:73-74 Nucleic acid sequence is that the primer pair of SEQ ID NO:75-76 is corresponding to NAT2 gene; Nucleic acid sequence is that the primer pair of SEQ ID NO:77-78 is corresponding to CYP2C9 gene; Nucleic acid sequence is that the primer pair of SEQ ID NO:79-80 is corresponding to ACE Gene; primer pairs with nucleic acid sequence of SEQ ID NO:81-82 correspond to PRKCA gene; primer pairs with nucleic acid sequence of SEQ ID NO:83-84 correspond to KCNK3 gene; primer pairs with nucleic acid sequence of SEQ ID NO:85-86 correspond to CYP2C19 Gene; the primer pairs whose nucleic acid sequences are SEQ ID NOs: 87-88 correspond to the NAT2 gene; the primer pairs whose nucleic acid sequences are SEQ ID NOs: 89-90 correspond to the GPIa gene; the primer pairs whose nucleic acid sequences are SEQ ID NOs: 91-92 correspond to C11ORF65 gene; primer pairs with nucleic acid sequences of SEQ ID NO: 93-94 correspond to NOS1AP gene; primer pairs with nucleic acid sequences of SEQ ID NO: 95-96 correspond to CYP2D6 gene; primer pairs with nucleic acid sequences of SEQ ID NO: 97-98 correspond to GPIa gene; primer pairs with nucleic acid sequences of SEQ ID NOs: 99-100 correspond to CYP2C9 genes; primer pairs with nucleic acid sequences of SEQ ID NOs: 101-102 correspond to NOS1AP genes; primer pairs with nucleic acid sequences of SEQ ID NOs: 103-104 Corresponds to ABCB1 gene; the primer pair whose nucleic acid sequence is SEQ ID NO:105-106 corresponds to ADRB2 gene; the primer pair whose nucleic acid sequence is SEQ ID NO:107-108 corresponds to NAT2 gene; the primer pair whose nucleic acid sequence is SEQ ID NO:109-110 For the corresponding COX-1 gene. 3. multiple PCR specific gene detection primer set according to claim 1, is characterized in that: when described primer set is in primer design, the annealing temperature difference between each pair of primers is less than 2 degrees Celsius, and the length of amplified product is 2°C. Between 200-300bp. 4. a test kit based on multiple PCR and high-throughput sequencing technology to detect the polymorphism of individualized medicine related gene loci, it is characterized in that: described test kit comprises described nucleic acid sequence is SEQ ID NO:1- At least one of 110 of 55 specific primer pairs. 5. the test kit based on multiple PCR and high-throughput sequencing technology to detect the polymorphism of individualized medicine related gene loci according to claim 4, is characterized in that: described test kit comprises nucleic acid sequence and is SEQ ID NO: 1-110's, 55 pairs of specific primers, Index, enzyme mix, PCR Enhancer, and nuclease-free water. 6. the test kit based on multiple PCR and high-throughput sequencing technology to detect the polymorphism of individualized medicine related gene loci according to claim 5, is characterized in that: the specific components of described test kit are as follows:

7. a method for detecting gene polymorphism sites based on multiple PCR and high-throughput sequencing technology, is characterized in that: comprise the following steps: (1) Nucleic acid extraction and quality control of the samples to be tested; (2) at least one of the 55 pairs of specific primers whose nucleotide sequences are SEQ ID NOs: 1-110, carry out PCR amplification to the target region of the extracted DNA, for subsequent use; (3) Use the magnetic bead method to purify the PCR amplification products, use the FluorometerQubit to measure the concentration of the purified amplicon library, record the library concentration, and mix the purified products of pool-1 and pool-2 of equal quality to obtain Library; then measure the fragment length of the library, if the fragment length is mainly distributed in 300-500bp, the main peak is 380±10bp, the quality inspection is qualified, quantitative mixed sample, on-board sequencing; if the length distribution exceeds 300-500bp, or the main peak is not at 380 Between ±10bp, if the library construction is repeated and the quality inspection fails again, the test will be terminated. 8. the method for detecting gene polymorphism sites based on multiple PCR and high-throughput sequencing technology according to claim 7, is characterized in that: the PCR reaction system that described PCR amplification adopts is:

9. the method for detecting gene polymorphism sites based on multiple PCR and high-throughput sequencing technology according to claim 7, is characterized in that: the program of described PCR amplification is:

10. The method for detecting gene polymorphism sites based on multiplex PCR and high-throughput sequencing technology according to claim 7, wherein the sample to be detected includes but is not limited to blood, paraffin-embedded tissue and frozen A type of sliced tissue. 11. the method for detecting gene polymorphism sites based on multiplex PCR and high-throughput sequencing technology according to claim 7, is characterized in that: described on-machine sequencing is high-throughput sequencing, and the high-throughput sequencing adopted Platforms include but are not limited to Illumina HiSeq/MiSeq, Life's Ion Torrent/Proton platform, and BGI Gene's BGISEQ-500/50 sequencing platform. 12. The application of a multiplex PCR-specific gene detection primer set in the gene detection of drugs for cardiovascular and cerebrovascular diseases.

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