CN111808943A - Gene detection method for individual medication of mental - Google Patents
Gene detection method for individual medication of mental Download PDFInfo
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- CN111808943A CN111808943A CN202010559173.2A CN202010559173A CN111808943A CN 111808943 A CN111808943 A CN 111808943A CN 202010559173 A CN202010559173 A CN 202010559173A CN 111808943 A CN111808943 A CN 111808943A
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- ADIMAYPTOBDMTL-UHFFFAOYSA-N oxazepam Chemical compound C12=CC(Cl)=CC=C2NC(=O)C(O)N=C1C1=CC=CC=C1 ADIMAYPTOBDMTL-UHFFFAOYSA-N 0.000 description 1
- 229960004535 oxazepam Drugs 0.000 description 1
- 229960002296 paroxetine Drugs 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 229960000762 perphenazine Drugs 0.000 description 1
- 229960002036 phenytoin Drugs 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 229960004431 quetiapine Drugs 0.000 description 1
- URKOMYMAXPYINW-UHFFFAOYSA-N quetiapine Chemical compound C1CN(CCOCCO)CCN1C1=NC2=CC=CC=C2SC2=CC=CC=C12 URKOMYMAXPYINW-UHFFFAOYSA-N 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 201000000980 schizophrenia Diseases 0.000 description 1
- 229960002073 sertraline Drugs 0.000 description 1
- VGKDLMBJGBXTGI-SJCJKPOMSA-N sertraline Chemical compound C1([C@@H]2CC[C@@H](C3=CC=CC=C32)NC)=CC=C(Cl)C(Cl)=C1 VGKDLMBJGBXTGI-SJCJKPOMSA-N 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- KQKPFRSPSRPDEB-UHFFFAOYSA-N sumatriptan Chemical compound CNS(=O)(=O)CC1=CC=C2NC=C(CCN(C)C)C2=C1 KQKPFRSPSRPDEB-UHFFFAOYSA-N 0.000 description 1
- 229960003708 sumatriptan Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 229960002784 thioridazine Drugs 0.000 description 1
- 229960004380 tramadol Drugs 0.000 description 1
- TVYLLZQTGLZFBW-GOEBONIOSA-N tramadol Natural products COC1=CC=CC([C@@]2(O)[C@@H](CCCC2)CN(C)C)=C1 TVYLLZQTGLZFBW-GOEBONIOSA-N 0.000 description 1
- 229960000604 valproic acid Drugs 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 229960000607 ziprasidone Drugs 0.000 description 1
- MVWVFYHBGMAFLY-UHFFFAOYSA-N ziprasidone Chemical compound C1=CC=C2C(N3CCN(CC3)CCC3=CC=4CC(=O)NC=4C=C3Cl)=NSC2=C1 MVWVFYHBGMAFLY-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention provides a gene detection method for individual psychotropic medication, which screens 32 polymorphic sites of genes closely related to 41 antipsychotic drugs by analyzing the individual differences of the use, metabolism and drug effect of NCBI databases and clinical common drugs for mental patients, and prepares a primer composition for detecting the 32 polymorphic sites and a detection reagent using the composition. The problems of low sensitivity, complex operation, high cost and the like when detecting the drug gene locus of the psychosis in the prior art are solved through second-generation sequencing.
Description
Technical Field
The invention is applied to the technical field of gene detection, and particularly relates to a gene detection method for individual psychotropic medication.
Background
In recent years, mental diseases have gradually become one of the major diseases affecting human health and normal learning and living, and mental patients account for almost 1% of the total number of people worldwide, have an incidence rate of about 17% in China, and have a tendency of rising year by year. Psychosis not only presents in an abnormal way in the thinking and cognition level, but also is accompanied by a series of abnormal behaviors such as impulsion, aggressive behavior, excessive activity, difficult management and the like, and has become a huge medical burden in society due to the complexity of psychosis. At present, the main means for controlling mental disorder is drug therapy, but the drug therapy has obvious individual difference, which is mainly manifested by no obvious curative effect or unstable curative effect, and even causes a series of adverse reactions, such as impaired liver function, granulocytopenia, extrapyramidal system reaction (EPS), metabolic endocrine dyscrasia and the like. Clinical data indicate that of schizophrenic patients, about 1/3-1/2 patients respond poorly to typical and atypical antipsychotics; in addition, in the treatment of depression, less than 45% of patients experience complete remission of clinical symptoms under sufficient treatment with a sufficient course of antidepressant medication. The main causes of these differences in therapeutic effects and adverse reactions may be related to mutations in metabolic enzymes, receptors or transporters in the body. For example, the drug clozapine, which is a lead of atypical antipsychotics, accounts for a higher proportion of the use of numerous antipsychotics, particularly in patients with refractory psychosis. The curative effect range of clozapine is wide, and the clinical response of different individuals to clozapine shows larger difference, mainly because the activity of cytochrome P4501A 2(CYP1A2) enzyme has larger individual difference, the enzyme mainly catalyzes demethylation metabolism of chlorine nitrogen level to generate norclozapine and plays a main role in vivo metabolism, and researches show that the gene site mutation of the enzyme can cause the clinical curative effect of schizophrenic patients on the clozapine to be reduced; the research also shows that the gene polymorphism of CYP2D6 of Chinese Han nationality schizophrenia patient has certain correlation with the blood concentration of novel atypical antipsychotic drug risperidone and metabolite 9-hydroxyrisperidone, thereby influencing the drug effect of risperidone.
Therefore, gene detection means is utilized to clarify gene polymorphism of related drug metabolizing enzymes of an individual, medication is guided according to individual gene specificity and a corresponding treatment scheme is given, and finally the conversion from 'medication according to symptoms' to 'medication for human' is realized, namely individualized medication, which has extremely important effects on improving the drug effect and reducing adverse drug reaction events and has certain social and economic meanings.
At present, a plurality of technologies for detecting gene polymorphism are provided, commonly used technologies comprise a fluorescent quantitative PCR method based on a Tm value and a dissolution curve, first-generation sequencing and the like, some of the technologies are complex to operate, and some of the technologies cannot realize simultaneous detection of multiple sites, so that the application of the existing clinical polymorphism detection technology is still not ideal, and the clinical detection requirement cannot be met all the time. With the development of high-throughput sequencing technology, more and more applied gene detection projects are generated in the Next Generation Sequencing (NGS), and the second generation sequencing technology can efficiently complete the detection of a large number of sequences, and meanwhile, the precision can reach 99.99%. The second-generation sequencing technology can process thousands of samples at a time while ensuring the detection precision.
Disclosure of Invention
The invention provides a gene detection method for individual psychotropic medication, aiming at the problems of low sensitivity, complex operation, high cost and the like in the detection of psychotropic medication gene loci in the prior art. The invention is based on the second-generation sequencing technology, has the characteristics of high sensitivity, low cost, simple operation, high flux and the like, and can simultaneously detect 32 related gene loci at one time.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
a gene detection method for mental individualized medication comprises the following steps:
1) taking an anticoagulant blood sample containing EDTA to perform nucleic acid extraction;
2) pre-mixing CYP2D6(rs1080985, rs35742686, rs3892097, rs5030655), ABCB1(rs1128503), UGT2B7(rs12233719), CYP2C19(rs12248560, rs4244285, rs4986893), EPM2A (rs1415744), OPRM1(rs1799971), DRD2(rs1799978), ANKK1(rs1800497), NAT2(rs 1801281280), UGT2B15(rs1902023), UGT1A4(rs2011425), EPHX1(rs 2233022), CYP2B6(rs2279343), ADORA2A (rs2298383), SCN2A (rs 594014016), SACM1L (rs 2742424242435), PON30878787879 (rs 2249879), RST 84418446, FK 8446 RG 8427 (rs), FK 849, and FK 849, wherein the PCR amplification site pairs of CYP 9611, FKMC 48, FKR 9611 (rs 8646) are preferably NO, and the PCR 4746B NO, the PCR No. 7;
3) adding the DNA obtained in the step 1) and the premixed primer mixed solution obtained in the step 2) into a multiple PCR reaction solution together to perform multiple PCR amplification reaction;
4) purifying the amplification product by using a product purification kit to complete the preparation of a sequencing library;
5) quantifying the sequencing library prepared in the step 4), and then performing Next Generation Sequencing (NGS);
6) after sequencing is finished, a biological information statistical method is utilized, splitting is carried out according to the primer of each locus, the total number of the amplified sequences of the fragments corresponding to each locus is obtained, then the percentage of the number of the wild type homozygous/heterozygous/mutant homozygous sequences in the total number of the amplified sequences of the corresponding loci is counted, and genotyping interpretation is carried out according to the calculated percentage.
Further, the amplification primers comprise chip adaptor sequences for second-generation sequencing, and primer pairs of the 32 gene loci have the following sequences:
the primer pair PG1 for detecting the rs1080985 locus of the gene CYP2D6 is shown as SEQ ID NO.1-2,
the primer pair PG2 for detecting the gene CYP2D6 locus rs35742686 is shown as SEQ ID NO.3-4,
the primer pair PG3 for detecting the rs3892097 locus of the gene CYP2D6 is shown as SEQ ID NO.5-6,
the primer pair PG4 for detecting rs5030655 site of gene CYP2D6 is shown as SEQ ID NO.7-8,
the primer pair PG5 for detecting rs1128503 site of gene ABCB1 is shown as SEQ ID NO.9-10,
the primer pair PG6 for detecting rs12233719 at the locus of UGT2B7 is shown as SEQ ID NO.11-12,
the primer pair PG7 for detecting rs12248560 at site of CYP2C19 gene is shown as SEQ ID NO.13-14,
the primer pair PG8 for detecting rs4244285 site of gene CYP2C19 is shown as SEQ ID NO.15-16,
the primer pair PG9 for detecting the locus rs4986893 of the gene CYP2C19 is shown as SEQ ID NO.17-18,
the primer pair PG10 for detecting the locus rs1415744 of the gene EPM2A is shown as SEQ ID NO.19-20,
the primer pair PG11 for detecting the locus rs1799971 of the gene OPRM1 is shown as SEQ ID NO.21-22,
the primer pair PG12 for detecting the locus rs1799978 of the gene DRD2 is shown as SEQ ID NO.23-24,
the primer pair PG13 for detecting the locus rs1800497 of the gene ANKK1 is shown as SEQ ID NO.25-26,
the primer pair PG14 for detecting the locus rs1801280 of the gene NAT2 is shown as SEQ ID NO.27-28,
the primer pair PG15 for detecting rs1902023 site of UGT2B15 gene is shown as SEQ ID NO.29-30,
the primer pair PG16 for detecting rs2011425 site of UGT1A4 is shown as SEQ ID NO.31-32,
the primer pair PG17 for detecting the locus rs2234922 of the gene EPHX1 is shown as SEQ ID NO.33-34,
the primer pair PG18 for detecting rs2279343 at the site of CYP2B6 of the gene is shown as SEQ ID NO.35-36,
the primer pair PG19 for detecting gene ADORA2A site rs2298383 is shown as SEQ ID NO.37-38,
the primer pair PG20 for detecting locus rs2304016 of gene SCN2A is shown as SEQ ID NO.39-40,
the primer pair PG21 for detecting rs2742435 site of gene SACM1L is shown as SEQ ID NO.41-42,
the primer pair PG22 for detecting the gene POLG locus rs3087374 is shown as SEQ ID NO.43-44,
the primer pair PG23 for detecting the locus rs3812718 of the gene SCN1A is shown as SEQ ID NO.45-46,
the primer pair PG24 for detecting rs41271330 of gene BMP5 site is shown as SEQ ID NO.47-48,
the primer pair PG25 for detecting the locus rs4646425 of the gene CYP1A2 is shown as SEQ ID NO.49-50,
the primer pair PG26 for detecting the locus rs762551 of the gene CYP1A2 is shown as SEQ ID NO.51-52,
the primer pair PG27 for detecting rs4713916 site of gene FKBP5 is shown as SEQ ID NO.53-54,
the primer pair PG28 for detecting rs489693 site of gene MC4R is shown as SEQ ID NO.55-56,
the primer pair PG29 for detecting the rs5443 site of the gene GNB3 is shown as SEQ ID NO.57-58,
the primer pair PG30 for detecting rs6295 site of HTR1A gene is shown as SEQ ID NO.59-60,
the primer pair PG31 for detecting the locus rs678849 of the gene OPRD1 is shown as SEQ ID NO.61-62,
the primer pair PG32 for detecting the locus rs951439 of the gene RGS4 is shown as SEQ ID NO. 63-64.
Further, the chip adaptor sequences for second-generation sequencing are respectively: AATGATACGGCGACCACCG AGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT, and CAAGCAGAAGACGGCATAC GAGATTACCGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC.
Furthermore, the amplification primer comprises an index sequence used for sequence analysis, and the primary splitting sequence and the secondary splitting sequence are TACCGAAT and TCGACGTC respectively.
Further, the molar ratio of each primer is PG 1: PG 2: PG 3: PG 4: PG 5: PG 6: PG 7: PG 8: PG 9: PG 10: PG 11: PG 12: PG 13: PG 14: PG 15: PG 16: PG 17: PG 18: PG 19: PG 20: PG 21: PG 22: PG 23: PG 24: PG 25: PG 26: PG 27: PG 28: PG 29: PG 30: PG 31: PG32 is 0.1-0.2: 0.15-0.2: 0.3-0.5: 0.15-0.2: 0.1-0.2: 0.15-0.2: 0.2-0.3: 0.4-0.5: 0.25-0.3: 0.1-0.2: 0.3-0.5: 0.1-0.2: 0.25-0.3: 0.3-0.4: 0.1-0.2: 0.3-0.2: 0.2-0.25: 0.1-0.2: 0.3-0.2: 0.3: 0.1-0.2: 0.1-0.2: 0.0.3: 0.0.2: 0.0.3: 0.0.1-0.2: 0.
Further, the molar ratio of each primer is PG 1: PG 2: PG 3: PG 4: PG 5: PG 6: PG 7: PG 8: PG 9: PG 10: PG 11: PG 12: PG 13: PG 14: PG 15: PG 16: PG 17: PG 18: PG 19: PG 20: PG 21: PG 22: PG 23: PG 24: PG 25: PG 26: PG 27: PG 28: PG 29: PG 30: PG 31: PG 32: 0.15:0.15:0.175:0.4:0.175:0.15:0.15:0.175:0.25:0.45:0.275:0.2:0.15:0.15:0.15:0.4:0.15:0.15:0.275:0.35:0.2:0.35:0.15:0.35:0.215:0.15:0.15:0.15: 0.175:0.25: 0.2.
Further, the configuration of the PCR reaction solution in step 3) is: multiplex PCR Buffer, 25. mu.L; multiplex PCR Enzyme Mix, 0.25. mu.L; primer Mix, 13.63 μ L; genomic DNA (template), 150 ng; sterilizing the deionized water.
Further, the multiplex PCR amplification reaction conditions in the step 3) are as follows:
pre-denaturation: 1 minute at 94 ℃;
35 amplification cycles: denaturation: 30 seconds at 94 ℃; annealing: 30 seconds at 60 ℃; extension: 72 ℃ for 30 seconds;
extension: 72 ℃ for 5 minutes.
Further, the step of purifying the product before sequencing in the step 4) comprises the following steps:
1) placing the magnetic beads stored at low temperature at room temperature for 5-10 min;
2) transiently dissociating the PCR product;
3) adding the PCR product subjected to the transient dissociation into 56uL of magnetic beads, uniformly blowing and sucking for 15 times, and standing at room temperature for 5 min;
4) placing the PCR tube on a magnetic frame, and discarding the supernatant after 2 min;
5) adding 250uL 80% ethanol solution into each tube for washing, removing supernatant after 30s, and repeating twice;
6) sucking off residual ethanol in the tube, standing at room temperature for 10min, and air drying the magnetic beads;
7) adding 50uL of eluent into each tube, uniformly mixing for 15 times by blowing and sucking, and standing for 5min at room temperature;
8) placing the sample on a magnetic frame for 5min, collecting 30uL of eluted liquid in a new centrifugal tube, detecting the concentration, diluting the liquid to 1-5ng/uL by using eluent, and then loading the liquid on a machine for sequencing.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention realizes the simultaneous detection of 32 polymorphic sites of the genes closely related to the 41 antipsychotics, pre-mixes and packages the primer pairs of the 32 psychotropic gene sites, is convenient for detection, realizes the multi-gene variation analysis of a reaction system, reduces the cost and greatly improves the detection flux and efficiency;
(2) the method has the characteristics of high sensitivity, low cost, simple and convenient operation, high detection flux and the like, and can quickly and accurately detect the gene polymorphism of the individual medication of the mental patients.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the following specific examples. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
In addition, unless otherwise specifically indicated, various starting materials, reagents, equipment and equipment used in the present invention may be commercially available or prepared by existing methods.
Example 1: preparation of detection reagent
1. Selection of drug and test Gene targets
The inventor screens 32 polymorphic sites of genes closely related to 41 antipsychotic drugs by analyzing the use, metabolism and individual difference of drug effect of NCBI databases and clinical common drugs for mental patients, prepares a primer composition for detecting the 32 polymorphic sites and a detection reagent using the composition, and establishes an accurate detection method for realizing effective medication guidance. The 41 antipsychotic agents are carbamazepine, phenytoin, valproic acid, lamotrigine, mephenytoin, venlafaxine, paroxetine, citalopram, amitriptyline, clomipramine, bupropion, modafinil, imipramine, escitalopram, nortriptyline, desipramine, fluoxetine, sertraline, doxepin, fluvoxamine, venlafaxine, mirtazapine, risperidone, olanzapine, clozapine, perphenazine, haloperidol, quetiapine, aripiprazole, ziprasidone, amisulpride, chlorpromazine, thioridazine, fluphenazine, oxazepam, diazepam, lorazepam, sumatriptan, tramadol, buprenorphine and caffeine, respectively. Drug and gene association analyses are shown in table 1:
TABLE 1 drug and Gene Association analysis
The kit is used for specifically detecting related 32-locus gene polymorphisms of CYP2D6, ABCB1, UGT2B7, CYP2C19, EPM2A, OPRM1, DRD2, ANKK1, NAT2, UGT2B15, UGT1A4, EPHX1, CYP2B6, ADORA2A, SCN2A, SACM1L, POLG, SCN1A, BMP5, CYP1A2, FKBP5, MC4R, GNB3, HTR1A, OPRD1, RGS4 and other genes in a sample, and is shown in Table 2:
TABLE 2 detection sites in PCR reaction System
2. Detection target amplification primer design
The invention utilizes CYP2D6(rs1080985, rs35742686, rs3892097, rs5030655), ABCB1(rs1128503), UGT2B7(rs12233719), CYP2C19(rs12248560, rs4244285, rs4986893), EPM2A (rs1415744), OPRM1(rs1799971), DRD2(rs1799978), ANKK1(rs1800497), 2(rs 1801281281281280), UGT2B15(rs 1902022022023), UGT1A4(rs2011425), EPHX1(rs 19020222), CYP2B6(rs2279343), ADORA2A (rs2298383), SCN2A (rs 5736), SACM1L (rs 2730435), PON87878787879, N5441849 (rs 41849), PCR accession No. the invention designs multiple primers RG 849, PCR primers for multiple amplification of PCR accession numbers of CYP2D 369641849, PCR accession No. the primers No.7, RS 41849, PCR accession No. 7(rs 8441849), PCR accession No.7, RS 368646 NO. 7(rs 364746), PCR accession No. 7(rs 849), PCR accession No. 4:
TABLE 3 PCR amplification primer sequences
The amplification primers comprise chip joint sequences for second-generation sequencing, and the chip joint sequences are respectively as follows: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT, and CAAGCAGAAGACGGCA TACGAGATTACCGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC.
The amplification primer also comprises an index sequence used for sequence analysis, and the preferred primary resolution sequence and the preferred secondary resolution sequence are TACCGAAT and TCGACGTC respectively.
3. Sequencing library construction
(1) The invention adopts a multiplex PCR one-step method to prepare a sequencing library. The amounts of each primer pair used in the primer mixture system are shown in Table 4:
TABLE 4 primer mix System
| Primer name | Primer concentration (μ M) | Amount of primer used (ul) | Primer name | Primer concentration (μ M) | Amount of primer used (ul) |
| rs1080985-F | 10 | 0.15 | rs1080985-R | 10 | 0.15 |
| rs1128503-F | 10 | 0.15 | rs1128503-R | 10 | 0.15 |
| rs12233719-F | 10 | 0.175 | rs12233719-R | 10 | 0.175 |
| rs12248560-F | 10 | 0.4 | rs12248560-R | 10 | 0.4 |
| rs1415744-F | 10 | 0.175 | rs1415744-R | 10 | 0.175 |
| rs1799971-F | 10 | 0.15 | rs1799971-R | 10 | 0.15 |
| rs1799978-F | 10 | 0.15 | rs1799978-R | 10 | 0.15 |
| rs1800497-F | 10 | 0.175 | rs1800497-R | 10 | 0.175 |
| rs1801280-F | 10 | 0.25 | rs1801280-R | 10 | 0.25 |
| rs1902023-F | 10 | 0.45 | rs1902023-R | 10 | 0.45 |
| rs2011425-F | 10 | 0.275 | rs2011425-R | 10 | 0.275 |
| rs2234922-F | 10 | 0.2 | rs2234922-R | 10 | 0.2 |
| rs2279343-F | 10 | 0.15 | rs2279343-R | 10 | 0.15 |
| rs2298383-F | 10 | 0.15 | rs2298383-R | 10 | 0.15 |
| rs2304016-F | 10 | 0.15 | rs2304016-R | 10 | 0.15 |
| rs2742435-F | 10 | 0.4 | rs2742435-R | 10 | 0.4 |
| rs3087374-F | 10 | 0.15 | rs3087374-R | 10 | 0.15 |
| rs35742686-F | 10 | 0.15 | rs35742686-R | 10 | 0.15 |
| rs3812718-F | 10 | 0.275 | rs3812718-R | 10 | 0.275 |
| rs3892097-F | 10 | 0.35 | rs3892097-R | 10 | 0.35 |
| rs41271330-F | 10 | 0.2 | rs41271330-R | 10 | 0.2 |
| rs4244285-F | 10 | 0.35 | rs4244285-R | 10 | 0.35 |
| rs4646425-F | 10 | 0.15 | rs4646425-R | 10 | 0.15 |
| rs4713916-F | 10 | 0.35 | rs4713916-R | 10 | 0.35 |
| rs489693-F | 10 | 0.215 | rs489693-R | 10 | 0.215 |
| rs4986893-F | 10 | 0.15 | rs4986893-R | 10 | 0.15 |
| rs5030655-F | 10 | 0.15 | rs5030655-R | 10 | 0.15 |
| rs5443-F | 10 | 0.15 | rs5443-R | 10 | 0.15 |
| rs6295-F | 10 | 0.15 | rs6295-R | 10 | 0.15 |
| rs678849-F | 10 | 0.175 | rs678849-R | 10 | 0.175 |
| rs762551-F | 10 | 0.25 | rs762551-R | 10 | 0.25 |
| rs951439-F | 10 | 0.2 | rs951439-R | 10 | 0.2 |
(2) The DNA of the sample to be tested and the primer mixture premixed according to the table 4 are added into the multiplex PCR reaction solution together to carry out the multiplex PCR amplification reaction. The Multiplex PCR amplification reaction of the invention adopts a Multiplex PCRAssay Kit Ver.2 Kit produced by TaKaRa to carry out amplification. The multiplex PCR reaction was prepared as shown in Table 5:
TABLE 5 PCR reaction solution
(3) The PCR amplification conditions in the invention are as follows:
(4) the amplification product was purified using a product purification kit (AgencourtAmpure XP 60mL kit from MECKMAN COULTER used in the present invention), and the sequencing library preparation was completed.
4. Second generation sequencing and results analysis
The detection platform used in the invention is a CN500 high-throughput sequencer produced by Iillumina company, and the second-generation sequencing (NGS) is carried out after the prepared sequencing library is quantified; after sequencing is finished, splitting according to the primer of each locus by using a biological information statistical method to obtain the total number of the amplified sequences of the segments corresponding to each locus, then counting the percentage of the number of the wild type homozygous/heterozygous/mutant homozygous sequences in the total number of the amplified sequences of the corresponding loci, and carrying out genotyping interpretation according to the calculated percentage.
Example 2: gene detection for individual drug use in mental
In this example, 2 cases of peripheral blood of patients were collected, and respectively numbered as sample 1 and sample 2, and the accuracy of the results was verified by a one-generation sequencing. 2 exception weekly samples were obtained from the ninth national hospital of Chongqing, and 5ml of blood samples were collected using a blood sampling tube containing EDTA anticoagulant. Standing the sample at room temperature for 30 minutes, centrifuging at 1500-2000 rpm for 10 minutes, and respectively collecting cells in blood plasma and blood in a sterile screw plastic tube.
1. Sample processing
The invention uses HiPureblood DNA Mini Kit produced by Meiji organism to extract nucleic acid from the sample by using a commercial Kit. Mu.l of Proteinase K was added to a 1.5ml centrifuge tube and 10-250. mu.l of anticoagulated blood was transferred to the centrifuge tube containing protease. Mix by gentle shaking, add 250 μ l Buffer AL to the sample, reverse 3-5 times mix, vortex mix at maximum speed for 30 seconds, water bath at 70 ℃ for 10 minutes, vortex mix once in between. Add 250. mu.l of absolute ethanol to the sample, vortex for 30 seconds and mix, centrifuge briefly to collect droplets on the tube wall. The DNA column was loaded into a fresh collection tube and the mixture was transferred to the column. Centrifugation at 10000 Xg for 1 min, discarding the collection tube and the effluent. The DNA column was loaded into a fresh collection tube, 500. mu.l Buffer DW1 was added to the column, the mixture was inverted and mixed several times, centrifuged at 10000 Xg for 30-60 seconds, the effluent was decanted, and the column was reloaded into the collection tube. 650. mu.l Buffer DW2 (diluted with ethanol) was added to the column, centrifuged at 10000 Xg for 30-60 seconds, the effluent was decanted, and the column was replaced in the collection tube. Centrifugation at 10000 Xg for 2min can completely remove ethanol remained in the column. The column was transferred to a new 1.5ml centrifuge tube. 30-100. mu.l of Elution Buffer or Buffer TE preheated to 70 ℃ was added to the center of the membrane of the column, left for 3 minutes, and then centrifuged at 10000 Xg for 1 minute. Adding 30-100 μ l of solution Buffer or Buffer TE preheated to 70 deg.C to the center of the membrane of the column, standing for 3 min, centrifuging at 10000 Xg for 1 min, discarding the DNA binding column, detecting the concentration of DNA liquid, storing the DNA at 2-8 deg.C, and storing at-20 deg.C for a long time.
2. PCR reaction
Reaction system preparation the reaction system was prepared according to the PCR reaction solution in example 1, DNA extracted from 2 samples was added to the prepared reaction system with an amount of template of about 150ng, and multiplex PCR amplification reaction was performed.
3. Purification of product before sequencing
The purification of PCR products before sequencing is carried out in the experiment by adopting a commercial Beckmann kit, and the steps are as follows:
1) placing the magnetic beads stored in a refrigerator with the temperature of 4 ℃ at room temperature for 5-10 min;
2) second round PCR products were transiently detached (this step is not required if no residue on the wall);
3) adding the PCR product subjected to the transient dissociation into 56uL of magnetic beads, uniformly blowing and sucking for 15 times, and standing at room temperature for 5 min;
4) placing the PCR tube on a magnetic frame, and discarding the supernatant after 2 min;
5) adding 250uL 80% ethanol into each tube, and discarding the supernatant after 30 s; repeating the steps twice;
6) absorbing residual ethanol in the tube; standing at room temperature for 10min until the magnetic beads are dried;
7) adding 50uL of eluent into each tube, uniformly mixing for 15 times by blowing and sucking, and standing for 5min at room temperature;
8) placing the magnetic frame for 5 min; and collecting 30uL of eluted liquid in a new centrifugal tube, detecting the concentration, diluting the liquid to 1-5ng/uL by using eluent, and then loading the liquid on a machine for sequencing.
4. Sequencing results
And counting the result by using a biological information method and judging the genotyping result by using the off-line data after the reaction is finished. The detection method of the invention adopted by the samples 1 and 2 is completely consistent with the first generation sequencing structure, and the detection results are shown in Table 6.
TABLE 6 test results
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-described embodiments. It will be understood by those skilled in the art that various changes, substitutions of equivalents, and improvements may be made without departing from the spirit and scope of the invention.
Sequence listing
<110> Chongqing Puluotong Gene medical research institute Co., Ltd
<120> gene detection method for mental individualized medication
<160>64
<170>SIPOSequenceListing 1.0
<210>1
<211>79
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcgagg cacccaatc 79
<210>2
<211>86
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatccgtat caggtagcag tggctc 86
<210>3
<211>82
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcagtg agctgctaac tg 82
<210>4
<211>82
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcttcct cctgacgctc aa 82
<210>5
<211>87
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtctcac ggctttgtcc aagagac 87
<210>6
<211>81
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatctgatg ggcagaggca c 81
<210>7
<211>80
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcgtgg ttggcgaagg 80
<210>8
<211>81
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcgcgag cagacgcttc t 81
<210>9
<211>82
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtctcca ctctgcacct tc 82
<210>10
<211>86
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcttaaa ccgaacagtc agttcc 86
<210>11
<211>83
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcgcat cttcagcttc cat 83
<210>12
<211>85
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatctctga ccattaatct gttgc 85
<210>13
<211>84
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtctctg aggtcttctg atgc 84
<210>14
<211>86
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcttaaa caggtgaatg tggtat 86
<210>15
<211>87
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtctgca ataattttcc cactatc 87
<210>16
<211>85
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcgttgt tgatgttcga ttctt 85
<210>17
<211>87
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcgaca tcaggattgt aagcacc 87
<210>18
<211>85
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcaatgt acttcgcttg gtcaa 85
<210>19
<211>84
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtccgtt cacatgctga cttt 84
<210>20
<211>83
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcaagga acaacttagc tgg 83
<210>21
<211>86
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcctgt caacttgtcc cactta 86
<210>22
<211>82
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcatgat cgtgaccgtg at 82
<210>23
<211>81
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtccgca accctcgacc g 81
<210>24
<211>83
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcaaccc ttggctgagt cct 83
<210>25
<211>84
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcgcac agccatcctc aaag 84
<210>26
<211>83
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcttgcc ctctaaggac atg 83
<210>27
<211>83
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcttgg ttcaccttct cct 83
<210>28
<211>81
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcgcacc tgagatcctt c 81
<210>29
<211>87
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtctcta tcgagaattt tcagaag 87
<210>30
<211>84
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcggtga ctgtgacatc ttcg 84
<210>31
<211>83
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtctgtt ggtgcccact gat 83
<210>32
<211>85
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatctctct tcttgtgcat attca 85
<210>33
<211>84
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtccaca tccacttcat ccac 84
<210>34
<211>86
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatccagtc aggaggatga tcttat 86
<210>35
<211>83
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcgtaa ccctggaccc cag 83
<210>36
<211>82
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcccatc ttccccttcc ta 82
<210>37
<211>86
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcgatg aaattgccag atcctg 86
<210>38
<211>83
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcgcaca gagaacagca gca 83
<210>39
<211>86
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcctac agggtggctg aagtgt 86
<210>40
<211>82
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcgcatc tgagttgcca ca 82
<210>41
<211>86
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcgact tcatttgaac cacagc 86
<210>42
<211>88
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcatatg taaaacctgc acaatgtg 88
<210>43
<211>82
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>43
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcaccc aggcagtgct at 82
<210>44
<211>84
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>44
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatctcttg aggtatccta acca 84
<210>45
<211>88
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>45
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcgtag gtacaaagag cctatcct 88
<210>46
<211>86
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>46
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatccaggt ttcagaattt gtaaac 86
<210>47
<211>84
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>47
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtctttc ccacaagacc agca 84
<210>48
<211>83
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcctttt caaccctcag aat 83
<210>49
<211>84
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>49
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtctacg ttgcttccct gtgt 84
<210>50
<211>80
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>50
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcgcagg agctagcagc 80
<210>51
<211>84
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>51
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtctgat gctcaaaggg tgag 84
<210>52
<211>86
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>52
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatctcatt catttcagtc atttga 86
<210>53
<211>88
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>53
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtccacc tctctggact cctacatt 88
<210>54
<211>86
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>54
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatccacaa gggtttgtag agatta 86
<210>55
<211>89
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>55
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtccatc atgtcttaat tctgttgtc 89
<210>56
<211>86
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>56
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcagctg agtcaggatg tatgag 86
<210>57
<211>83
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>57
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtccaag agcatcatct gcg 83
<210>58
<211>84
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>58
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcacacg ctcattcatg gagt 84
<210>59
<211>86
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>59
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcattt ggaagaagac cgagtg 86
<210>60
<211>83
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>60
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcttgtt gttgtcgtcg ttg 83
<210>61
<211>88
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>61
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcaagg tgtccttctt accatagt 88
<210>62
<211>83
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>62
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcatgag aactgcctcc aat 83
<210>63
<211>82
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>63
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcttcc tgctgtgtgg ct 82
<210>64
<211>85
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>64
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcgtttg aagttttaag ttcct 85
Claims (9)
1. A gene detection method for mental individualized medication is characterized by comprising the following steps:
1) taking an anticoagulant blood sample containing EDTA to perform nucleic acid extraction;
2) pre-mixing CYP2D6(rs1080985, rs35742686, rs3892097, rs5030655), ABCB1(rs1128503), UGT2B7(rs12233719), CYP2C19(rs12248560, rs4244285, rs4986893), EPM2A (rs1415744), OPRM1(rs1799971), DRD2(rs1799978), ANKK1(rs1800497), NAT2(rs 1801281280), UGT2B15(rs1902023), UGT1A4(rs2011425), EPHX1(rs 2233022), CYP2B6(rs2279343), ADORA2A (rs2298383), SCN2A (rs 594014016), SACM1L (rs 2742424242435), PON30878787879 (rs 2249879), RST 84418446, FK 8446 RG 8427 (rs), FK 849, and FK 849, wherein the PCR amplification site pairs of CYP 9611, FKMC 48, FKR 9611 (rs 8646) are preferably NO, and the PCR 4746B NO, the PCR No. 7;
3) adding the DNA obtained in the step 1) and the premixed primer mixed solution obtained in the step 2) into a multiple PCR reaction solution together to perform multiple PCR amplification reaction;
4) purifying the amplification product by using a product purification kit to complete the preparation of a sequencing library;
5) quantifying the sequencing library prepared in the step 4), and then performing Next Generation Sequencing (NGS);
6) after sequencing is finished, splitting according to the primer of each locus by using a biological information statistical method to obtain the total number of the amplified sequences of the segments corresponding to each locus, then counting the percentage of the number of the wild type homozygous/heterozygous/mutant homozygous sequences in the total number of the amplified sequences of the corresponding loci, and carrying out genotyping interpretation according to the calculated percentage.
2. The gene detection method for the psychotropic personalized medicine according to claim 1, wherein: the amplification primers comprise chip joint sequences for second-generation sequencing, and the sequences of primer pairs of the 32 gene loci are as follows:
the primer pair PG1 for detecting the rs1080985 locus of the gene CYP2D6 is shown as SEQ ID NO.1-2,
the primer pair PG2 for detecting the gene CYP2D6 locus rs35742686 is shown as SEQ ID NO.3-4,
the primer pair PG3 for detecting the rs3892097 locus of the gene CYP2D6 is shown as SEQ ID NO.5-6,
the primer pair PG4 for detecting rs5030655 site of gene CYP2D6 is shown as SEQ ID NO.7-8,
the primer pair PG5 for detecting rs1128503 site of gene ABCB1 is shown as SEQ ID NO.9-10,
the primer pair PG6 for detecting rs12233719 at the locus of UGT2B7 is shown as SEQ ID NO.11-12,
the primer pair PG7 for detecting rs12248560 at site of CYP2C19 gene is shown as SEQ ID NO.13-14,
the primer pair PG8 for detecting rs4244285 site of gene CYP2C19 is shown as SEQ ID NO.15-16,
the primer pair PG9 for detecting the locus rs4986893 of the gene CYP2C19 is shown as SEQ ID NO.17-18,
the primer pair PG10 for detecting the locus rs1415744 of the gene EPM2A is shown as SEQ ID NO.19-20,
the primer pair PG11 for detecting the locus rs1799971 of the gene OPRM1 is shown as SEQ ID NO.21-22,
the primer pair PG12 for detecting the locus rs1799978 of the gene DRD2 is shown as SEQ ID NO.23-24,
the primer pair PG13 for detecting the locus rs1800497 of the gene ANKK1 is shown as SEQ ID NO.25-26,
the primer pair PG14 for detecting the locus rs1801280 of the gene NAT2 is shown as SEQ ID NO.27-28,
the primer pair PG15 for detecting rs1902023 site of UGT2B15 gene is shown as SEQ ID NO.29-30,
the primer pair PG16 for detecting rs2011425 site of UGT1A4 is shown as SEQ ID NO.31-32,
the primer pair PG17 for detecting the locus rs2234922 of the gene EPHX1 is shown as SEQ ID NO.33-34,
the primer pair PG18 for detecting rs2279343 at the site of CYP2B6 of the gene is shown as SEQ ID NO.35-36,
the primer pair PG19 for detecting gene ADORA2A site rs2298383 is shown as SEQ ID NO.37-38,
the primer pair PG20 for detecting locus rs2304016 of gene SCN2A is shown as SEQ ID NO.39-40,
the primer pair PG21 for detecting rs2742435 site of gene SACM1L is shown as SEQ ID NO.41-42,
the primer pair PG22 for detecting the gene POLG locus rs3087374 is shown as SEQ ID NO.43-44,
the primer pair PG23 for detecting the locus rs3812718 of the gene SCN1A is shown as SEQ ID NO.45-46,
the primer pair PG24 for detecting rs41271330 of gene BMP5 site is shown as SEQ ID NO.47-48,
the primer pair PG25 for detecting the locus rs4646425 of the gene CYP1A2 is shown as SEQ ID NO.49-50,
the primer pair PG26 for detecting the locus rs762551 of the gene CYP1A2 is shown as SEQ ID NO.51-52,
the primer pair PG27 for detecting rs4713916 site of gene FKBP5 is shown as SEQ ID NO.53-54,
the primer pair PG28 for detecting rs489693 site of gene MC4R is shown as SEQ ID NO.55-56,
the primer pair PG29 for detecting the rs5443 site of the gene GNB3 is shown as SEQ ID NO.57-58,
the primer pair PG30 for detecting rs6295 site of HTR1A gene is shown as SEQ ID NO.59-60,
the primer pair PG31 for detecting the locus rs678849 of the gene OPRD1 is shown as SEQ ID NO.61-62,
the primer pair PG32 for detecting the locus rs951439 of the gene RGS4 is shown as SEQ ID NO. 63-64.
3. The gene detection method for the psychotropic personalized medicine according to claim 2, wherein: the chip joint sequences for the second-generation sequencing are respectively as follows: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT, and CAAGCAGAAGACGGCATACGAGATTACCGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC.
4. The gene detection method for the psychotropic personalized medicine according to claim 2, wherein: the amplification primer comprises an index sequence used for sequence analysis, and the primary splitting sequence and the secondary splitting sequence are TACCGAAT and TCGACGTC respectively.
5. The gene detection method for the psychotropic personalized medicine according to claim 2, wherein: the molar ratio of each primer is PG 1: PG 2: PG 3: PG 4: PG 5: PG 6: PG 7: PG 8: PG 9: PG 10: PG 11: PG 12: PG 13: PG 14: PG 15: PG 16: PG 17: PG 18: PG 19: PG 20: PG 21: PG 22: PG 23: PG 24: PG 25: PG 26: PG 27: PG 28: PG 29: PG 30: PG 31: PG32 is 0.1-0.2: 0.15-0.2: 0.3-0.5: 0.15-0.2: 0.1-0.2: 0.15-0.2: 0.2-0.3: 0.4-0.5: 0.25-0.3: 0.1-0.2: 0.3-0.5: 0.1-0.2: 0.25-0.3: 0.3-0.4: 0.1-0.2: 0.3-0.2: 0.2-0.25: 0.1-0.2: 0.3-0.2: 0.3: 0.1-0.2: 0.1-0.2: 0.0.3: 0.0.2: 0.0.3: 0.0.1-0.2: 0.
6. The method for gene detection of psychotropic personalized medicine according to claim 5, wherein: the molar ratio of each primer is PG 1: PG 2: PG 3: PG 4: PG 5: PG 6: PG 7: PG 8: PG 9: PG 10: PG 11: PG 12: PG 13: PG 14: PG 15: PG 16: PG 17: PG 18: PG 19: PG 20: PG 21: PG 22: PG 23: PG 24: PG 25: PG 26: PG 27: PG 28: PG 29: PG 30: PG 31: PG 32: 0.15:0.15:0.175:0.4:0.175:0.15:0.15:0.175:0.25:0.45:0.275:0.2:0.15:0.15:0.15:0.4:0.15:0.15:0.275:0.35:0.2:0.35:0.15:0.35:0.215:0.15:0.15:0.15: 0.175:0.25: 0.2.
7. The gene detection method for the psychotropic personalized medicine according to claim 1, wherein: the configuration of the PCR reaction solution in the step 3) is as follows: multiplex PCR Buffer, 25. mu.L; multiplex PCR Enzyme Mix, 0.25. mu.L; primer Mix, 13.63 μ L; genomic DNA (template), 150 ng; and (5) sterilizing the deionized water.
8. The gene detection method for the psychotropic personalized medicine according to claim 1, wherein: the multiple PCR amplification reaction conditions in the step 3) are as follows:
pre-denaturation: 1 minute at 94 ℃;
35 amplification cycles: denaturation: 30 seconds at 94 ℃; annealing: 30 seconds at 60 ℃; extension: 72 ℃ for 30 seconds;
extension: 72 ℃ for 5 minutes.
9. The gene detection method for the psychotropic personalized medicine according to claim 1, wherein: the purification steps of the products before sequencing in the step 4) are as follows:
1) placing the magnetic beads stored at low temperature at room temperature for 5-10 min;
2) transiently dissociating the PCR product;
3) adding the PCR product subjected to the transient dissociation into 56uL of magnetic beads, uniformly blowing and sucking for 15 times, and standing at room temperature for 5 min;
4) placing the PCR tube on a magnetic frame, and discarding the supernatant after 2 min;
5) adding 250uL 80% ethanol solution into each tube for washing, removing supernatant after 30s, and repeating twice;
6) sucking off residual ethanol in the tube, standing at room temperature for 10min, and air drying the magnetic beads;
7) adding 50uL of eluent into each tube, uniformly mixing for 15 times by blowing and sucking, and standing for 5min at room temperature;
8) placing the sample on a magnetic frame for 5min, collecting 30uL of eluted liquid in a new centrifugal tube, detecting the concentration, diluting the liquid to 1-5ng/uL by using eluent, and then loading the liquid on a machine for sequencing.
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