CN112111573B

CN112111573B - Gene polymorphism detection kit and detection method for guiding medication of depression

Info

Publication number: CN112111573B
Application number: CN202011292960.1A
Authority: CN
Inventors: 李雅雅; 温景敏
Original assignee: Shanghai Youming Autometric Medical Technology Co ltd
Current assignee: Shanghai Yaoming Aozhe Medical Laboratory Co ltd; Shanghai Youming Autometric Medical Technology Co ltd; Suzhou Yaoming Zekang Biotechnology Co ltd
Priority date: 2020-11-18
Filing date: 2020-11-18
Publication date: 2021-02-19
Anticipated expiration: 2040-11-18
Also published as: CN112111573A

Abstract

The invention discloses a gene polymorphism detection kit and a detection method for guiding drug administration for depression, wherein the kit comprises an amplification primer and an extension primer for detecting 55 gene polymorphism sites for guiding drug administration for depression, a PCR reaction reagent, a PCR product purification reagent, a single base extension reaction reagent, a negative quality control product and a positive quality control product; the 5 'end of the amplification primer comprises a protective base sequence, and the 5' end of the extension primer comprises a base sequence; the detection method comprises the following steps: (1) multiplex PCR, amplifying the DNA region where the gene polymorphism site is; (2) purifying the PCR product; (3) performing multiplex single base extension on the purified PCR product to obtain an extension product; (4) purifying the extension product; (5) detecting by a mass spectrometer; (6) judging the result; the invention can comprehensively detect a plurality of gene polymorphism sites for guiding the medication of depression at one time.

Description

Gene polymorphism detection kit and detection method for guiding medication of depression

Technical Field

The invention belongs to the field of biological medicine, and relates to a detection kit and a detection method for gene polymorphism sites for guiding depression medication.

Background

By 2015, more than 3 million people worldwide have depression, accounting for about 4.4% of the global population, with over 800,000 depression patients suicided each year. Wherein the number of Chinese depression patients is more than 5000 ten thousands, which is the first to live in the whole world.

Patients with depression often face a long drug selection period (generally, about 1 year is needed to determine a proper drug, so that the optimal treatment time is possibly delayed), a poor treatment effect or a risk of drug toxic and side effects (the effective rate of the drug is about 30% -50%), treatment cost is greatly increased, and meanwhile, a great burden is caused to the country and the society.

Pharmacogenomic testing helps physicians find individuals at risk of adverse reactions and treatment failure when first diagnosing a patient, as it helps to select appropriate drugs and formulate scientific starting doses; in the treatment process, the reasons of adverse reaction, no response or low response rate are searched from the gene perspective, so that the treatment scheme is adjusted in time, and the treatment effect is improved. According to the existing socioeconomic evaluation data of gene detection of multiple anti-depression drugs, pharmacogenomics is greatly helpful in saving medical cost, reducing the number of times of medical treatment and improving the treatment effect.

At present, the mainstream of genetic polymorphism detection in the market is high throughput sequencing (NGS), but the sequencing cost is high, the time consumption is long, and therefore, the clinical application has certain limitation; the real-time fluorescent quantitative PCR can also be adopted for multi-hole reaction detection, although the time-effect problem is met, the flux is low, and when more than 10 sites need to be detected, the practical applicability is not strong.

The technical problems existing at present are as follows: common detection technologies, such as sequencing, fluorescent quantitative PCR and the like, all need to detect the sites one by one, and when the sites are more, the operation is complex and the cost is higher; the existing nucleic acid mass spectrometry based on multiple PCR detects that the like products are single, the selection and combination modes of gene loci are few compared with detection products of the traditional sequencing, real-time fluorescence quantitative PCR and other methods, and a method and a product capable of simultaneously and comprehensively detecting multiple gene polymorphisms for guiding depression medication at one time are lacked.

Disclosure of Invention

The invention aims to provide a method for detecting gene polymorphism for guiding medication of depression, which can detect a plurality of SNPs of the same individual in the same system;

the invention also aims to provide a gene polymorphism detection kit for guiding depression medication, which is used by matching with the detection method, contains all reagents required by detection, is convenient to operate, saves time and improves efficiency;

the invention also aims to provide a reference basis for reasonable medication of the depression by using the detection method and the kit.

In order to achieve the purpose, the invention provides a gene polymorphism detection kit for guiding depression medication, which guides reasonable selection of medicament dosage and medicament type by carrying out qualitative typing detection on 26 genes and 55 sites related to medicament metabolic enzymes, transporters and receptors related to the action of mental disease medicaments, thereby improving medicament curative effect and avoiding side reaction, and mainly relates to mental depression related diseases. The gene polymorphism detection kit for guiding the medication of the depression comprises: the amplification primer and the extension primer for detecting 55 gene polymorphism sites for guiding the medication of depression are respectively as follows:

rs1049353 site of CNR1 gene, wherein the amplification primer is SEQ ID No. 1-2, and the extension primer is SEQ ID No. 111; the rs1057868 locus of the POR gene, the amplification primer is SEQ ID No. 3-4, and the extension primer is SEQ ID No. 112; the site rs12248560 of CYP2C19 gene, the amplification primer is SEQ ID No. 5-6, the extension primer is SEQ ID No. 113; an rs1414334 site of the HTR2C gene, wherein an amplification primer is SEQ ID No. 7-8, and an extension primer is SEQ ID No. 114; locus rs1799732 of DRD2 gene, wherein the amplification primer is SEQ ID No. 9-10, and the extension primer is SEQ ID No. 115; the locus rs1799978 of DRD2 gene, the amplification primer is SEQ ID No. 11-12, the extension primer is SEQ ID No. 116; the site rs1800544 of the ADRA2A gene has an amplification primer of SEQ ID No. 13-14 and an extension primer of SEQ ID No. 117; the rs1902023 locus of UGT2B15 gene, the amplification primer is SEQ ID No. 15-16, the extension primer is SEQ ID No. 118; rs1954787 site of GRIK4 gene, wherein the amplification primer is SEQ ID No. 17-18, and the extension primer is SEQ ID No. 119; the rs28371706 site of CYP2D6 gene, the amplification primer is SEQ ID No. 19-20, the extension primer is SEQ ID No. 120; the site rs3211371 of CYP2B6 gene, the amplification primer is SEQ ID No 21-22, the extension primer is SEQ ID No 121; the site rs34223104 of CYP2B6 gene has the amplification primer of SEQ ID No. 23-24 and the extension primer of SEQ ID No. 122; the site rs8192709 of the CYP2B6 gene, the amplification primer is SEQ ID No. 25-26, and the extension primer is SEQ ID No. 123; the rs35599367 site of the CYP3A4 gene has an amplification primer of SEQ ID No. 27-28 and an extension primer of SEQ ID No. 124; the site rs35742686 of CYP2D6 gene, the amplification primer is SEQ ID No. 29-30, the extension primer is SEQ ID No. 125; the site rs3745274 of CYP2B6 gene has the amplification primer of SEQ ID No. 31-32 and the extension primer of SEQ ID No. 126; the site rs3892097 of the CYP2D6 gene has an amplification primer of SEQ ID No. 33-34 and an extension primer of SEQ ID No. 127; the rs5030865 locus of the CYP2D6 gene has an amplification primer of SEQ ID No. 35-36 and an extension primer of SEQ ID No. 128; the rs1135824 site of CYP2D6 gene has amplification primer of SEQ ID No. 37-38 and extension primer of SEQ ID No. 129; the rs5030655 site of the CYP2D6 gene has an amplification primer of SEQ ID No. 39-40 and an extension primer of SEQ ID No. 130; rs4713916 locus of FKBP5 gene, wherein the amplification primer is SEQ ID No. 41-42, and the extension primer is SEQ ID No. 131; rs489693 locus of MC4R gene, wherein the amplification primer is SEQ ID No 43-44, and the extension primer is SEQ ID No 132; an rs6313 locus of an HTR2A gene, wherein an amplification primer is SEQ ID No. 45-46, and an extension primer is SEQ ID No. 133; the site rs776746 of CYP3A5 gene, the amplification primer is SEQ ID No 47-48, the extension primer is SEQ ID No 134; an rs7997012 site of HTR2A gene, wherein an amplification primer is SEQ ID No. 49-50, and an extension primer is SEQ ID No. 135; rs1000940 site of RABEP1 gene, wherein the amplification primer is SEQ ID No. 51-52, and the extension primer is SEQ ID No. 136; the locus rs10042486 of the HTR1A gene has an amplification primer of SEQ ID No. 53-54 and an extension primer of SEQ ID No. 137; the rs1065852 site of the CYP2D6 gene has an amplification primer of SEQ ID No. 55-56 and an extension primer of SEQ ID No. 138; the rs10245483 site of ABCB1 gene, the amplification primer is SEQ ID No. 57-58, and the extension primer is SEQ ID No. 139; the site rs12769205 of CYP2C19 gene, the amplification primer is SEQ ID No 59-60, the extension primer is SEQ ID No 140; an rs130058 locus of the HTR1B gene, wherein an amplification primer is SEQ ID No. 61-62, and an extension primer is SEQ ID No. 141; rs1800497 site of ANKK1 gene, the amplification primer is SEQ ID No. 63-64, the extension primer is SEQ ID No. 142; the rs2279343 site of CYP2B6 gene, the amplification primer is SEQ ID No. 65-66, the extension primer is SEQ ID No. 143; the site rs28371725 of the CYP2D6 gene, the amplification primer is SEQ ID No. 67-68, the extension primer is SEQ ID No. 144; the rs16947 locus of the CYP2D6 gene has an amplification primer of SEQ ID No. 69-70 and an extension primer of SEQ ID No. 145; the rs28399499 locus of CYP2B6 gene, the amplification primer is SEQ ID No. 71-72, the extension primer is SEQ ID No. 146; the rs28399504 locus of CYP2C19 gene, the amplification primer is SEQ ID No. 73-74, the extension primer is SEQ ID No. 147; the rs4148739 locus of ABCB1 gene has the amplification primer of SEQ ID No. 75-76 and the extension primer of SEQ ID No. 148; ZNF385D gene rs4261893 locus, the amplification primer is SEQ ID No 77-78, the extension primer is SEQ ID No 149; the rs61888800 locus of the BDNF gene has an amplification primer of SEQ ID No. 79-80 and an extension primer of SEQ ID No. 150; an rs6295 site of an HTR1A gene, wherein an amplification primer is SEQ ID No. 81-82, and an extension primer is SEQ ID No. 151; an rs6311 locus of an HTR2A gene, wherein an amplification primer is SEQ ID No. 83-84, and an extension primer is SEQ ID No. 152; an rs6318 site of the HTR2C gene, wherein an amplification primer is SEQ ID No. 85-86, and an extension primer is SEQ ID No. 153; the rs762551 site of CYP1A2 gene, the amplification primer is SEQ ID No. 87-88, the extension primer is SEQ ID No. 154; rs1045642 site of ABCB1 gene, wherein the amplification primer is SEQ ID No. 89-90, and the extension primer is SEQ ID No. 155; the rs1135840 site of CYP2D6 gene has amplification primer of SEQ ID No. 91-92 and extension primer of SEQ ID No. 156; the rs1135835 site of CYP2D6 gene has amplification primer of SEQ ID No 93-94 and extension primer of SEQ ID No 157; rs1360780 site of FKBP5 gene, wherein the amplification primer is SEQ ID No. 95-96, and the extension primer is SEQ ID No. 158; the site rs1799853 of the CYP2C9 gene has an amplification primer of SEQ ID No. 97-98 and an extension primer of SEQ ID No. 159; the rs2032583 site of ABCB1 gene, the amplification primer is SEQ ID No. 99-100, the extension primer is SEQ ID No. 160; the rs2032582 site of the ABCB1 gene, the amplification primer is SEQ ID No. 101-102, and the extension primer is SEQ ID No. 161; the rs2235040 site of ABCB1 gene has the amplification primer of SEQ ID No. 103-104 and the extension primer of SEQ ID No. 162; the site rs324420 of the FAAH gene, the amplification primer is SEQ ID No. 105-106, and the extension primer is SEQ ID No. 163; the site rs3888190 of the SH2B1 gene, wherein the amplification primer is SEQ ID No. 107-108, and the extension primer is SEQ ID No. 164; the site rs4986893 of the CYP2C19 gene has an amplification primer of SEQ ID No. 109-110 and an extension primer of SEQ ID No. 165.

In a specific example, the 5' end of the amplification primer comprises a protective base sequence, so that the molecular weight of the amplification primer is increased, and the detection effect can be prevented from being interfered when the remaining primers enter the mass spectrometry detection process. Preferably, the protective base sequence comprises 5-10 bases; as a specific example, the protective base sequence is tag of 10 bp: AGCTTGGAAG are provided.

In a specific example, the 5' end of the extension primer comprises a base sequence, and the base sequence is increased to improve the detection effect by adding bases to one or both of the extension primers when the molecular weights of the extension primers and the products corresponding to the two genetic polymorphic sites are close, so as to change the molecular weights of the primers and the products and increase the difference between the molecular weights of the other extension primers and the products; however, the molecular weight of the primer and the product should not exceed the detection window after increasing the base sequence. As a specific example, the base sequence is the same as the aforementioned protected base sequence.

Specifically, the gene polymorphism detection kit for depression medication assistance further comprises:

(1) a PCR reaction reagent comprising: high temperature resistant DNA polymerase, dNTPs and PCR reaction buffer solution;

(2) a PCR product purification reagent comprising: alkaline phosphatase, enzyme digestion buffer solution;

(3) a single base extension reaction reagent comprising: specific extension primer of PCR product, high temperature resistant single base extension enzyme, ddNTPs and extension reaction buffer solution.

Specifically, the gene polymorphism detection kit for depression medication assistance further comprises: negative quality control product, positive quality control product.

Specifically, the gene polymorphism detection kit for depression medication assistance further comprises: resin for purifying extension products, target sheet for sample application and mass spectrum detection and human genome DNA extraction reagent.

The invention also provides a detection method of the gene polymorphism detection kit based on the medication assistance of the depression, which comprises the following steps:

(1) multiplex PCR: using specific amplification primers to simultaneously amplify DNA regions where 55 gene polymorphic sites for guiding the medication of the depression are located in three reaction systems to obtain a PCR product containing 55 gene polymorphic sites; wherein, the reaction system 1 comprises the following amplification primers and extension primers of gene polymorphic sites:

rs1049353 site of CNR1 gene, wherein the amplification primer is SEQ ID No. 1-2, and the extension primer is SEQ ID No. 111; the rs1057868 locus of the POR gene, the amplification primer is SEQ ID No. 3-4, and the extension primer is SEQ ID No. 112; the site rs12248560 of CYP2C19 gene, the amplification primer is SEQ ID No. 5-6, the extension primer is SEQ ID No. 113; an rs1414334 site of the HTR2C gene, wherein an amplification primer is SEQ ID No. 7-8, and an extension primer is SEQ ID No. 114; locus rs1799732 of DRD2 gene, wherein the amplification primer is SEQ ID No. 9-10, and the extension primer is SEQ ID No. 115; the locus rs1799978 of DRD2 gene, the amplification primer is SEQ ID No. 11-12, the extension primer is SEQ ID No. 116; the site rs1800544 of the ADRA2A gene has an amplification primer of SEQ ID No. 13-14 and an extension primer of SEQ ID No. 117; the rs1902023 locus of UGT2B15 gene, the amplification primer is SEQ ID No. 15-16, the extension primer is SEQ ID No. 118; rs1954787 site of GRIK4 gene, wherein the amplification primer is SEQ ID No. 17-18, and the extension primer is SEQ ID No. 119; the rs28371706 site of CYP2D6 gene, the amplification primer is SEQ ID No. 19-20, the extension primer is SEQ ID No. 120; the site rs3211371 of CYP2B6 gene, the amplification primer is SEQ ID No 21-22, the extension primer is SEQ ID No 121; the site rs34223104 of CYP2B6 gene has the amplification primer of SEQ ID No. 23-24 and the extension primer of SEQ ID No. 122; the site rs8192709 of the CYP2B6 gene, the amplification primer is SEQ ID No. 25-26, and the extension primer is SEQ ID No. 123; the rs35599367 site of the CYP3A4 gene has an amplification primer of SEQ ID No. 27-28 and an extension primer of SEQ ID No. 124; the site rs35742686 of CYP2D6 gene, the amplification primer is SEQ ID No. 29-30, the extension primer is SEQ ID No. 125; the site rs3745274 of CYP2B6 gene has the amplification primer of SEQ ID No. 31-32 and the extension primer of SEQ ID No. 126; the site rs3892097 of the CYP2D6 gene has an amplification primer of SEQ ID No. 33-34 and an extension primer of SEQ ID No. 127; the rs5030865 locus of the CYP2D6 gene has an amplification primer of SEQ ID No. 35-36 and an extension primer of SEQ ID No. 128; the rs1135824 site of CYP2D6 gene has amplification primer of SEQ ID No. 37-38 and extension primer of SEQ ID No. 129; the rs5030655 site of the CYP2D6 gene has an amplification primer of SEQ ID No. 39-40 and an extension primer of SEQ ID No. 130; rs4713916 locus of FKBP5 gene, wherein the amplification primer is SEQ ID No. 41-42, and the extension primer is SEQ ID No. 131; rs489693 locus of MC4R gene, wherein the amplification primer is SEQ ID No 43-44, and the extension primer is SEQ ID No 132; an rs6313 locus of an HTR2A gene, wherein an amplification primer is SEQ ID No. 45-46, and an extension primer is SEQ ID No. 133; the site rs776746 of CYP3A5 gene, the amplification primer is SEQ ID No 47-48, the extension primer is SEQ ID No 134; an rs7997012 site of HTR2A gene, wherein an amplification primer is SEQ ID No. 49-50, and an extension primer is SEQ ID No. 135;

the reaction system 2 comprises the following amplification primers and extension primers of gene polymorphic sites:

rs1000940 site of RABEP1 gene, wherein the amplification primer is SEQ ID No. 51-52, and the extension primer is SEQ ID No. 136; the locus rs10042486 of the HTR1A gene has an amplification primer of SEQ ID No. 53-54 and an extension primer of SEQ ID No. 137; the rs1065852 site of the CYP2D6 gene has an amplification primer of SEQ ID No. 55-56 and an extension primer of SEQ ID No. 138; the rs10245483 site of ABCB1 gene, the amplification primer is SEQ ID No. 57-58, and the extension primer is SEQ ID No. 139; the site rs12769205 of CYP2C19 gene, the amplification primer is SEQ ID No 59-60, the extension primer is SEQ ID No 140; an rs130058 locus of the HTR1B gene, wherein an amplification primer is SEQ ID No. 61-62, and an extension primer is SEQ ID No. 141; rs1800497 site of ANKK1 gene, the amplification primer is SEQ ID No. 63-64, the extension primer is SEQ ID No. 142; the site rs2279343 of CYP2B6 gene, the amplification primer is SEQ ID No. 65-66, the extension primer is SEQ ID No. 143, the site rs28371725 of CYP2D6 gene, the amplification primer is SEQ ID No. 67-68, and the extension primer is SEQ ID No. 144; the rs16947 locus of the CYP2D6 gene has an amplification primer of SEQ ID No. 69-70 and an extension primer of SEQ ID No. 145; the rs28399499 locus of CYP2B6 gene, the amplification primer is SEQ ID No. 71-72, the extension primer is SEQ ID No. 146; the rs28399504 locus of CYP2C19 gene, the amplification primer is SEQ ID No. 73-74, the extension primer is SEQ ID No. 147; the rs4148739 locus of ABCB1 gene has the amplification primer of SEQ ID No. 75-76 and the extension primer of SEQ ID No. 148; ZNF385D gene rs4261893 locus, the amplification primer is SEQ ID No 77-78, the extension primer is SEQ ID No 149; the rs61888800 locus of the BDNF gene has an amplification primer of SEQ ID No. 79-80 and an extension primer of SEQ ID No. 150; an rs6295 site of an HTR1A gene, wherein an amplification primer is SEQ ID No. 81-82, and an extension primer is SEQ ID No. 151; an rs6311 locus of an HTR2A gene, wherein an amplification primer is SEQ ID No. 83-84, and an extension primer is SEQ ID No. 152; an rs6318 site of the HTR2C gene, wherein an amplification primer is SEQ ID No. 85-86, and an extension primer is SEQ ID No. 153; the rs762551 site of CYP1A2 gene, the amplification primer is SEQ ID No. 87-88, the extension primer is SEQ ID No. 154;

the reaction system 3 comprises the following amplification primers and extension primers of gene polymorphic sites:

rs1045642 site of ABCB1 gene, wherein the amplification primer is SEQ ID No. 89-90, and the extension primer is SEQ ID No. 155; the rs1135840 site of CYP2D6 gene has amplification primer of SEQ ID No. 91-92 and extension primer of SEQ ID No. 156; the rs1135835 site of CYP2D6 gene has amplification primer of SEQ ID No 93-94 and extension primer of SEQ ID No 157; rs1360780 site of FKBP5 gene, wherein the amplification primer is SEQ ID No. 95-96, and the extension primer is SEQ ID No. 158; the site rs1799853 of the CYP2C9 gene has an amplification primer of SEQ ID No. 97-98 and an extension primer of SEQ ID No. 159; the rs2032583 site of ABCB1 gene, the amplification primer is SEQ ID No. 99-100, the extension primer is SEQ ID No. 160; the rs2032582 site of the ABCB1 gene, the amplification primer is SEQ ID No. 101-102, and the extension primer is SEQ ID No. 161; the rs2235040 site of ABCB1 gene has the amplification primer of SEQ ID No. 103-104 and the extension primer of SEQ ID No. 162; the site rs324420 of the FAAH gene, the amplification primer is SEQ ID No. 105-106, and the extension primer is SEQ ID No. 163; the site rs3888190 of the SH2B1 gene, wherein the amplification primer is SEQ ID No. 107-108, and the extension primer is SEQ ID No. 164; the site rs4986893 of the CYP2C19 gene, the amplification primer is SEQ ID No. 109-110, and the extension primer is SEQ ID No. 165;

(2) and (3) PCR product purification: respectively purifying PCR products obtained in the step (1) in an original system to reduce interference on subsequent reactions;

(3) single base extension: performing multiple single-base extension on the purified PCR product obtained in the step (2) in the reaction system to obtain an extension product by using 55 specific extension primers, wherein the extension primers extend one nucleotide at a corresponding SNP site, and the nucleotide is complementarily paired with the genotype at the SNP site;

(4) and (3) purification of an extension product: purifying the extension product obtained in the step (3) to obtain a high-purity extension product, and avoiding the influence of impurities such as salt ions on subsequent detection;

(5) mass spectrometer detection: spotting the high-purity extension product obtained in the step (4) on a target sheet containing a matrix, and putting the target sheet into a mass spectrometer for detection;

(6) and (5) judging the result.

As a specific example, the kit and the detection method are applied to guide the medication of depression.

The invention provides a scheme for detecting gene polymorphism for guiding medication of depression by combining a multiplex PCR (polymerase chain reaction) technology, a single base extension technology and a mass spectrum detection technology. Wherein: simultaneously amplifying products of up to 55 SNPs in multiplex PCR; in the single-base extension process, performing multiple single-base extension on the purified product of the multiple PCR, and extending one nucleotide at 55 SNP sites by the extension primer respectively to ensure that the type of the extended nucleotide is related to the genotype of the gene polymorphic site respectively; in the mass spectrometric detection process, one or more mass spectrometric results are determined by comparing the mass spectrometric results with a map of 55 SNP sites prepared in advance, so as to detect the corresponding SNP types. Compared with the prior art, the invention has the following beneficial effects:

1. the sensitivity is high: the invention integrates the technologies of multiplex PCR, single base extension, mass spectrum detection and the like, can amplify a detection template through the PCR technology and detect a trace sample through the mass spectrum technology, so that the invention has high sensitivity;

2. the specificity is good: the single base extension used in the invention is also called micro sequencing, and the DNA molecules are identified by using a specific probe, so that the method has the advantages of high accuracy, good specificity and low false positive of a sequencing technology, is different from the method for extending hundreds of bases by using the sequencing technology, only extends a single base by using the technology, and has lower error probability;

3. simple and safe: the operation is simple and safe, the automation degree is high, and the pollution is prevented;

4. and (3) fast: the speed is high, the flux is high, and detection of hundreds of samples can be completed within 8-9 hours;

5. the invention can detect a plurality of known patients to respectively obtain detection results with different SNPs, wherein the patients can have single SNP mutation sites or a plurality of SNP mutation sites;

6. the invention overcomes the defect of the prior art that the SNP sites are too few to be detected at one time and has low cost.

Drawings

FIG. 1 is a diagram of typing effect of human DNA sample detection using the kit of the present invention, wherein D-F are reaction system 1, reaction system 2 and reaction system 3 from top to bottom; circles (original image is green) from left to right of each row respectively represent No. 1-5 sample holes, 5 clinical samples respectively, and rhombuses (original image is red) represent NTC holes;

FIG. 2 is a graph showing NTC total typing results of the reaction system 1 using the kit of the present invention;

FIG. 3 is a graph showing NTC total typing results of reaction system 2 using the kit of the present invention;

FIG. 4 is a graph showing NTC typing results of the reaction system 3 using the kit of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

The embodiment of the invention provides a gene polymorphism detection kit for guiding medication of depression, which comprises: the amplification primers and extension primers for detecting 55 gene polymorphism sites for guiding the medication of depression have the following sequences: (relevant primers were synthesized in Jinzhi Biotechnology, Inc., Suzhou).

The first embodiment is as follows: primer sequences

。

Example two: use of the kit

The following is an example to illustrate the method of using the gene polymorphism detection kit for guiding the medication of depression of the present invention.

Sampling and extracting sample DNA

By EDTANA₂Anticoagulated tubes venous Blood of 5 clinical depression patients was collected, and human genomic DNA was extracted from 200. mu.L of whole Blood of each patient using a nucleic acid extraction kit (DNeasy Blood and Tissue kit, QIAGEN Co.), quantified with NanoDrop 2000 (Thermo Co.), and normalized to 30 ng/. mu.l.

Multiplex PCR

The DNA regions of 55 gene polymorphic sites related to medication guidance of depression are amplified by using an ABI 9700 type PCR instrument. To avoid interference between different primer sequences, the above 55 sites were performed in 3 reaction systems as shown below.

The reaction system 1 comprises the following amplification primers and extension primers of gene polymorphic sites:

rs1049353 site of CNR1 gene, wherein the amplification primer is SEQ ID No. 1-2, and the extension primer is SEQ ID No. 111; the rs1057868 locus of the POR gene, the amplification primer is SEQ ID No. 3-4, and the extension primer is SEQ ID No. 112; the site rs12248560 of CYP2C19 gene, the amplification primer is SEQ ID No. 5-6, the extension primer is SEQ ID No. 113; an rs1414334 site of the HTR2C gene, wherein an amplification primer is SEQ ID No. 7-8, and an extension primer is SEQ ID No. 114; locus rs1799732 of DRD2 gene, wherein the amplification primer is SEQ ID No. 9-10, and the extension primer is SEQ ID No. 115; the locus rs1799978 of DRD2 gene, the amplification primer is SEQ ID No. 11-12, the extension primer is SEQ ID No. 116; the site rs1800544 of the ADRA2A gene has an amplification primer of SEQ ID No. 13-14 and an extension primer of SEQ ID No. 117; the rs1902023 locus of UGT2B15 gene, the amplification primer is SEQ ID No. 15-16, the extension primer is SEQ ID No. 118; rs1954787 site of GRIK4 gene, wherein the amplification primer is SEQ ID No. 17-18, and the extension primer is SEQ ID No. 119; the rs28371706 site of CYP2D6 gene, the amplification primer is SEQ ID No. 19-20, the extension primer is SEQ ID No. 120; the site rs3211371 of CYP2B6 gene, the amplification primer is SEQ ID No 21-22, the extension primer is SEQ ID No 121; the site rs34223104 of CYP2B6 gene has the amplification primer of SEQ ID No. 23-24 and the extension primer of SEQ ID No. 122; the site rs8192709 of the CYP2B6 gene, the amplification primer is SEQ ID No. 25-26, and the extension primer is SEQ ID No. 123; the rs35599367 site of the CYP3A4 gene has an amplification primer of SEQ ID No. 27-28 and an extension primer of SEQ ID No. 124; the site rs35742686 of CYP2D6 gene, the amplification primer is SEQ ID No. 29-30, the extension primer is SEQ ID No. 125; the site rs3745274 of CYP2B6 gene has the amplification primer of SEQ ID No. 31-32 and the extension primer of SEQ ID No. 126; the site rs3892097 of the CYP2D6 gene has an amplification primer of SEQ ID No. 33-34 and an extension primer of SEQ ID No. 127; the rs5030865 locus of the CYP2D6 gene has an amplification primer of SEQ ID No. 35-36 and an extension primer of SEQ ID No. 128; the rs1135824 site of CYP2D6 gene has amplification primer of SEQ ID No. 37-38 and extension primer of SEQ ID No. 129; the rs5030655 site of the CYP2D6 gene has an amplification primer of SEQ ID No. 39-40 and an extension primer of SEQ ID No. 130; rs4713916 locus of FKBP5 gene, wherein the amplification primer is SEQ ID No. 41-42, and the extension primer is SEQ ID No. 131; rs489693 locus of MC4R gene, wherein the amplification primer is SEQ ID No 43-44, and the extension primer is SEQ ID No 132; an rs6313 locus of an HTR2A gene, wherein an amplification primer is SEQ ID No. 45-46, and an extension primer is SEQ ID No. 133; the site rs776746 of CYP3A5 gene, the amplification primer is SEQ ID No 47-48, the extension primer is SEQ ID No 134; the locus rs7997012 of HTR2A gene, the amplification primer is SEQ ID No. 49-50, the extension primer is SEQ ID No. 135.

rs1000940 site of RABEP1 gene, wherein the amplification primer is SEQ ID No. 51-52, and the extension primer is SEQ ID No. 136; the locus rs10042486 of the HTR1A gene has an amplification primer of SEQ ID No. 53-54 and an extension primer of SEQ ID No. 137; the rs1065852 site of the CYP2D6 gene has an amplification primer of SEQ ID No. 55-56 and an extension primer of SEQ ID No. 138; the rs10245483 site of ABCB1 gene, the amplification primer is SEQ ID No. 57-58, and the extension primer is SEQ ID No. 139; the site rs12769205 of CYP2C19 gene, the amplification primer is SEQ ID No 59-60, the extension primer is SEQ ID No 140; an rs130058 locus of the HTR1B gene, wherein an amplification primer is SEQ ID No. 61-62, and an extension primer is SEQ ID No. 141; rs1800497 site of ANKK1 gene, the amplification primer is SEQ ID No. 63-64, the extension primer is SEQ ID No. 142; the site rs2279343 of CYP2B6 gene, the amplification primer is SEQ ID No. 65-66, the extension primer is SEQ ID No. 143, the site rs28371725 of CYP2D6 gene, the amplification primer is SEQ ID No. 67-68, and the extension primer is SEQ ID No. 144; the rs16947 locus of the CYP2D6 gene has an amplification primer of SEQ ID No. 69-70 and an extension primer of SEQ ID No. 145; the rs28399499 locus of CYP2B6 gene, the amplification primer is SEQ ID No. 71-72, the extension primer is SEQ ID No. 146; the rs28399504 locus of CYP2C19 gene, the amplification primer is SEQ ID No. 73-74, the extension primer is SEQ ID No. 147; the rs4148739 locus of ABCB1 gene has the amplification primer of SEQ ID No. 75-76 and the extension primer of SEQ ID No. 148; ZNF385D gene rs4261893 locus, the amplification primer is SEQ ID No 77-78, the extension primer is SEQ ID No 149; the rs61888800 locus of the BDNF gene has an amplification primer of SEQ ID No. 79-80 and an extension primer of SEQ ID No. 150; an rs6295 site of an HTR1A gene, wherein an amplification primer is SEQ ID No. 81-82, and an extension primer is SEQ ID No. 151; an rs6311 locus of an HTR2A gene, wherein an amplification primer is SEQ ID No. 83-84, and an extension primer is SEQ ID No. 152; an rs6318 site of the HTR2C gene, wherein an amplification primer is SEQ ID No. 85-86, and an extension primer is SEQ ID No. 153; the site rs762551 of CYP1A2 gene has the amplification primer of SEQ ID No. 87-88 and the extension primer of SEQ ID No. 154.

rs1045642 site of ABCB1 gene, wherein the amplification primer is SEQ ID No. 89-90, and the extension primer is SEQ ID No. 155; the rs1135840 site of CYP2D6 gene has amplification primer of SEQ ID No. 91-92 and extension primer of SEQ ID No. 156; the rs1135835 site of CYP2D6 gene has amplification primer of SEQ ID No 93-94 and extension primer of SEQ ID No 157; rs1360780 site of FKBP5 gene, wherein the amplification primer is SEQ ID No. 95-96, and the extension primer is SEQ ID No. 158; the site rs1799853 of the CYP2C9 gene has an amplification primer of SEQ ID No. 97-98 and an extension primer of SEQ ID No. 159; the rs2032583 site of ABCB1 gene, the amplification primer is SEQ ID No. 99-100, the extension primer is SEQ ID No. 160; the rs2032582 site of the ABCB1 gene, the amplification primer is SEQ ID No. 101-102, and the extension primer is SEQ ID No. 161; the rs2235040 site of ABCB1 gene has the amplification primer of SEQ ID No. 103-104 and the extension primer of SEQ ID No. 162; the site rs324420 of the FAAH gene, the amplification primer is SEQ ID No. 105-106, and the extension primer is SEQ ID No. 163; the site rs3888190 of the SH2B1 gene, wherein the amplification primer is SEQ ID No. 107-108, and the extension primer is SEQ ID No. 164; the site rs4986893 of the CYP2C19 gene has an amplification primer of SEQ ID No. 109-110 and an extension primer of SEQ ID No. 165.

The reaction system for PCR is shown in the following table, and the reagents used were obtained from Agena, USA, except for the DNA template, ultrapure water, and primers.

The cycling parameters of the PCR were:

95 ℃, 2 minutes, 1 cycle,

95 deg.C, 10 seconds, 56 deg.C, 10 seconds, 72 deg.C, 30 seconds, 35 cycles,

72 ℃, 5 minutes, 1 cycle,

and preserving at 4 ℃.

Purification of PCR product

To 5. mu.L of the PCR product, reagents shown in the following Table were added, and the enzymatic digestion reaction was carried out using an ABI model 9700 PCR instrument, and the added reagents except ultrapure water were obtained from Agena, USA.

The reaction parameters for the enzymatic digestion were:

at 37 c, 40 minutes, 1 cycle,

at 85 deg.C, 5 minutes, 1 cycle,

and preserving at 4 ℃.

Four.single base extension

To 7. mu.L of the enzyme-digested product, reagents shown in the following Table, all of which were obtained from Agena, USA, except ultrapure water and primers, were added, and an extension reaction was performed using an ABI model 9700 PCR instrument.

The extended cycle parameters were:

at 94 ℃, 30 seconds, 1 cycle,

94 ℃, 5 seconds, 52 ℃, 5 seconds, 80 ℃, 5 seconds, 40 cycles,

72 ℃, 3 minutes, 1 cycle,

and preserving at 4 ℃.

Fifthly, purification of extension product

To 9. mu.L of the extension product, 16. mu.L of ultrapure water was added and mixed, and then 6mg of a resin (resin, Agena, USA) was added and mixed by inverting the mixture upside down for 30 minutes.

Sixth, Mass Spectrometry detection

The purified product was spotted onto a substrate-containing chip (SpectroCHIP, Agena, USA) using a nanoliter spotter (Nanodipen, Agena, USA) in an amount of 10-20nl each; and then detected using a time-of-flight mass spectrometer (Agena, USA).

In addition, wild type control B1-B5 and mutant control C1-C5 were provided at the above positions, respectively. Wherein, the wild control B1-B5 and the mutant control C1-C5 are respectively from artificial plasmids which are sold in the market or deposited in the laboratory.

Seventh, interpretation of results

(1) As shown in a parting effect diagram of fig. 1, D-F are respectively a reaction system 1, a reaction system 2 and a reaction system 3 from top to bottom; circles (original green) from left to right in each row represent sample holes 1-5, 5 clinical samples, and diamonds (original red) represent NTC holes.

And (4) judging the standard:

dark green indicates better data quality (> 85% call rate), light green indicates medium quality data quality (51% -85% call rate), yellow indicates lower data quality (16% -50% call rate), red diamonds indicate NTC (< 15% call rate).

As can be seen from fig. 1, the sample wells of the reaction system 1 and the reaction system 2 of each sample both show dark green color and have good data quality, and the reaction system 3 of each sample respectively shows dark green color and light green color and has good and medium data quality, which indicates that the typing effect is good and the mutual interference of the primers is small.

(2) The extension products generated from the respective genotypes at the 55 genetic polymorphic sites have different molecular weights, each molecular weight corresponds to a respective mass spectrum peak, and when a mass spectrum peak appears at a certain molecular weight, it is judged that a substance (extension primer or product) corresponding to the molecular weight exists.

And (4) judging the standard:

(1) if the mass spectrum peaks corresponding to the wild type and the mutant type do not appear, judging that the experiment fails no matter whether the mass spectrum peak corresponding to the extended primer exists or not;

(2) if only one mass spectrum peak corresponding to the wild type or the mutant type appears, the genotype corresponding to the appeared mass spectrum peak is judged to be homozygous;

(3) and if the mass spectrum peaks corresponding to the wild type or the mutant type appear, judging the hybrid type.

According to the NTC overall typing result graphs of each reaction system in FIGS. 2-4, NTC has no mass spectrum peak at all corresponding positions of wild type and mutant type, namely representing no interference on background, and the interference among primers is extremely small, thus being capable of accurately reflecting the mutation condition of 55 sites, and having low false positive.

Aiming at different genotypes, the invention has a certain guiding function on medication, and the medication guiding prompt of the invention is illustrated by taking the rs4986893 site and the rs12248560 site of the CYP2C19 gene as an example for guiding the medication of clopidogrel. Similarly, the other genes and the guidance of the loci thereof involved in the invention are used for judging the genotype by using the kit and the detection method of the invention, and then the metabolic capability and the guidance of the dosage are judged by combining the existing research results.

CYP2C19 x 1 is the wild type allele of CYP2C19 gene, which encodes CYP2C19 enzyme activity normally;

CYP2C19 x 3 is rs4986893 site of CYP2C19 gene, and the coded CYP2C19 enzyme activity is reduced;

CYP2C19 x 17 is rs12248560 site of CYP2C19 gene, coded CYP2C19 enzyme activity is enhanced.

As shown in the above table, when the genotype is 1/. multidot.1, it means that neither rs4986893 site nor rs12248560 site has mutation, CYP2C19 enzyme activity is normal, and clopidogrel drug metabolism speed is normal, and for patients with this genotype, standard dosage under medical guidance can be adopted.

When the genotype is 1/17 or 17/17, namely rs12248560 site mutation occurs, the CYP2C19 enzyme activity is enhanced, so that the drug metabolism speed of clopidogrel is accelerated, and for patients with the genotype, proper dosage reduction can be considered.

When the genotype is 1/3 or 3/17 or 3/3, it means that rs4986893 site is mutated, the activity of CYP2C19 enzyme is reduced, the drug metabolism speed to clopidogrel is reduced, and the drug resistance phenomenon is generated.

In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

Sequence listing

<110> Shanghai medicine Minaosurvey medical science & technology Co., Ltd

<120> gene polymorphism detection kit and detection method for guiding medication of depression

<160> 165

<170> SIPOSequenceListing 1.0

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acgttggatg gtggttgtgg agtacgagac 30

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acgttggatg gtaatgttag ctcgcttctg 30

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acgttggatg ggcctcatct acccaataac 30

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acgttggatg tcaaaggaga agactggcga 30

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acgttggatg aaaggagctg tacctcctcg 30

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acgttggatg tcaaaggaga agactggcga 30

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acgttggatg aaaggagctg tacctcctcg 30

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acgttggatg ttctcccaag atccagcttc 30

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acgttggatg gggtaccttg agctagagac 30

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acgttggatg ccatatatcc atctatcgag 30

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acgttggatg gactgtgttg acatcttcgg 30

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acgttggatg atgaaaggac cgaagtaggg 30

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acgttggatg atcgtgcctt cacccaattc 30

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acgttggatg agctcggact acggtcatca 30

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acgttggatg gacccacggc gaggacacc 29

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acgttggatg tcagaggcag gaagttgcg 29

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acgttggatg gcaaaatacc cccaacatac 30

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acgttggatg tgagtaggcc tcttctatcc 30

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acgttggatg gtctgggttc cctaacaact 30

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acgttggatg tgagtaggcc tcttctatcc 30

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acgttggatg gtctgggttc cctaacaact 30

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acgttggatg gttgtctgat agtgggtctc 30

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acgttggatg cagaaggtgt tatcaggtgc 30

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acgttggatg tgagacttgt ccaggtgaac 30

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acgttggatg gagagcatac tcgggacaga 30

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acgttggatg tgatcttggt agtggaatcg 30

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acgttggatg tgatgttccc caggcacttc 30

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acgttggatg ttgtccaaga gaccgttggg 30

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acgttggatg gagcaaggtg gatgcacaaa 30

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acgttggatg ttgtccaaga gaccgttggg 30

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acgttggatg gagcaaggtg gatgcacaaa 30

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acgttggatg ttgtccaaga gaccgttggg 30

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acgttggatg gagcaaggtg gatgcacaaa 30

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acgttggatg ttgtccaaga gaccgttggg 30

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acgttggatg gagcaaggtg gatgcacaaa 30

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acgttggatg taacctctct ggactcctac 30

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acgttggatg gtcacaaggg tttgttgtag 30

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acgttggatg atacctgcca cgctgtaaac 30

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acgttggatg ctgcctaccg gtctaaaatc 30

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acgttggatg tgagctcaac tacgaactcc 30

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acgttggatg tcacaggaaa ggttggttcg 30

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acgttggatg gatgaagggt aatgtggtcc 30

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acgttggatg gagataccca cgtatgtacc 30

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acgttggatg caggaaagaa cgctgagttg 30

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acgttggatg ccttccaaga atcctggatg 30

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acgttggatg tcccaacgtg ctgtgctgta 30

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acgttggatg aaagacaagg gtgacaaggg 30

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acgttggatg cagtaggagc ttttagaaac 30

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acgttggatg gctgatggtt ggagtttctc 30

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acgttggatg ctgtggtttc acccaccatc 30

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acgttggatg atttggtagt gaggcaggt 29

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acgttggatg ttattccctg tgtgagagcc 30

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acgttggatg tagcaggcag ctacaaatgg 30

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acgttggatg ccattgctga aaacgattcc 30

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acgttggatg ggaaaacaga ctagcagagc 30

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acgttggatg gagaggaaca accacagacg 30

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acgttggatg tcctcaatta ttcctccgcc 30

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acgttggatg acacagccat cctcaaagtg 30

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acgttggatg tctaggaagg acatgatgcc 30

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acgttggatg tggagaagca ccgtgaaacc 30

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acgttggatg tccttcctca tgttctctcc 30

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<400> 67

acgttggatg ttctgtcccg agtatgctct 30

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<400> 68

acgttggatg ttacaggatc ctggtcaagc 30

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<400> 69

acgttggatg ttctgtcccg agtatgctct 30

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acgttggatg ttacaggatc ctggtcaagc 30

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acgttggatg tctgtacaga gagagtctac 30

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acgttggatg gggagaaggt cggaaaatc 29

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acgttggatg atagtgggcc taggtgattg 30

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acgttggatg gagcacaagg accacaaaag 30

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<400> 75

acgttggatg gctttagtaa tgttgccgtg 30

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<400> 76

acgttggatg tttccatgac ttcagaatgc 30

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<400> 77

acgttggatg gggtatacta ccaggcaaag 30

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<400> 78

acgttggatg ggctcaggta aaagtcaccc 30

<210> 79

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<400> 79

acgttggatg cgtgcgattc tcactcgct 29

<210> 80

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 80

acgttggatg ggccgcttaa agggaacgc 29

<210> 81

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 81

acgttggatg tgtcgtcgtt gttcgtttg 29

<210> 82

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 82

acgttggatg tgatggaaga agaccgagtg 30

<210> 83

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 83

acgttggatg tggacacaaa cactgttggc 30

<210> 84

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 84

acgttggatg aaggtaggta agtggcactg 30

<210> 85

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 85

acgttggatg gcacctaatt ggcctattgg 30

<210> 86

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 86

acgttggatg ccagttttgt accccgtctg 30

<210> 87

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 87

acgttggatg cagcgttcat gttgggaatc 30

<210> 88

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 88

acgttggatg tgatgcgtgt tctgtgcttg 30

<210> 89

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 89

acgttggatg actgcagcat tgctgagaac 30

<210> 90

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 90

acgttggatg cattaggcag tgactcgatg 30

<210> 91

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 91

acgttggatg actaggtacc ccattctagc 30

<210> 92

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 92

acgttggatg ctgcagcact tcagcttctc 30

<210> 93

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 93

acgttggatg actaggtacc ccattctagc 30

<210> 94

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 94

acgttggatg ctgcagcact tcagcttctc 30

<210> 95

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 95

acgttggatg ttctatagct gcaagtcccc 30

<210> 96

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 96

acgttggatg tgccagcagt agcaagtaag 30

<210> 97

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 97

acgttggatg cagtgatatg gagtagggtc 30

<210> 98

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 98

acgttggatg ctgcggaatt ttgggatgg 29

<210> 99

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 99

acgttggatg gagcatagta agcagtaggg 30

<210> 100

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 100

acgttggatg gaaaatgttg tctggacaag c 31

<210> 101

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 101

acgttggatg gagcatagta agcagtaggg 30

<210> 102

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 102

acgttggatg gaaaatgttg tctggacaag c 31

<210> 103

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 103

acgttggatg attagtttca tgctggggtc 30

<210> 104

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 104

acgttggatg accactggag cattgactac 30

<210> 105

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 105

acgttggatg gaacaaaggg accaactgtg 30

<210> 106

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 106

acgttggatg cagagcatac cttgtaggtg 30

<210> 107

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 107

acgttggatg gagactgcgg caatggaaac 30

<210> 108

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 108

acgttggatg cccagctgca ttgaatttcc 30

<210> 109

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 109

acgttggatg aacatcagga ttgtaagcac 30

<210> 110

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 110

acgttggatg atgtacttca gggcttggtc 30

<210> 111

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 111

gttaccttgg caatcttgac 20

<210> 112

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 112

ttccgccgtt ctccccg 17

<210> 113

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 113

tttgtgtctt ctgttctcaa ag 22

<210> 114

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 114

gtcgacagtg actttgctac cct 23

<210> 115

<211> 13

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 115

gatccccggc ctg 13

<210> 116

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 116

tgctctccca cccacaccca gagtaa 26

<210> 117

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 117

gttggccatg cagctc 16

<210> 118

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 118

attttatcct acatctttaa ctaaaaat 28

<210> 119

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 119

cttaacccaa ttccgcacct tcc 23

<210> 120

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 120

accgcccgcc tgtgcccatc a 21

<210> 121

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 121

caaaataccc ccaacatacc agatc 25

<210> 122

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 122

ctctgcaccc tgttat 16

<210> 123

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 123

ggctactcct ggttcag 17

<210> 124

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 124

ttcagttcag tgtctccatc acacccag 28

<210> 125

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 125

ggtcccaggt catcc 15

<210> 126

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 126

ctccaccttc ctcttcca 18

<210> 127

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 127

catctcccac cccca 15

<210> 128

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 128

tctgcccatc acccac 16

<210> 129

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 129

tttgtgccgc cttcgcc 17

<210> 130

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 130

gggcaagaag tcgctggagc ag 22

<210> 131

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 131

gactcctaca ttttcctct 19

<210> 132

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 132

gggtccacgc tgtaaacatt taacaaac 28

<210> 133

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 133

ggctctacag taatgacttt aactc 25

<210> 134

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 134

ggggaaagag ctcttttgtc tttca 25

<210> 135

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 135

ggcaagtgac aaatattgt 19

<210> 136

<211> 14

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 136

cggatggcac gcac 14

<210> 137

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 137

gtccattatc cccaaacta 19

<210> 138

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 138

aatggcagtg gcagggggcc tggtg 25

<210> 139

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 139

agagaatgaa tattattcag cat 23

<210> 140

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 140

tcctgggatc tccctcct 18

<210> 141

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 141

cgctgaaact agaggtca 18

<210> 142

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 142

cagccatcct caaagtgctg gtc 23

<210> 143

<211> 14

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 143

ccccagcgcc ccca 14

<210> 144

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 144

cccgcctgta ccctt 15

<210> 145

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 145

agcttcaatg atgagaacct g 21

<210> 146

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 146

ggccaatcac ctgttca 17

<210> 147

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 147

ggaccacaaa aggatcca 18

<210> 148

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 148

gttcaattac atattcctat ctct 24

<210> 149

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 149

aaaacaatgt cactgcatgt t 21

<210> 150

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 150

agctgcgggc gtcctgtcca agg 23

<210> 151

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 151

agaccgagtg tgtcttc 17

<210> 152

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 152

cagtgctgtg agtgtc 16

<210> 153

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 153

ctgggctcac agaaatatca 20

<210> 154

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 154

actaagctcc atctaccatg cgtcctg 27

<210> 155

<211> 14

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 155

tttgctgccc tcac 14

<210> 156

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 156

tggtgtcttt gctttcctgg tga 23

<210> 157

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 157

ccgcttctcg gtgccc 16

<210> 158

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 158

gctttcacat aagcaaagtt a 21

<210> 159

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 159

gaggagcatt gaggac 16

<210> 160

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 160

tgattaatta agtagagtaa agtattc 27

<210> 161

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 161

agataagaaa gaactagaag gt 22

<210> 162

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 162

gcctcctttc tactggt 17

<210> 163

<211> 13

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 163

aggccctgcc ttg 13

<210> 164

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 164

cccccagttc ttccc 15

<210> 165

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 165

tggccttacc tggat 15

Claims

1. A gene polymorphism detection kit for guiding medication of depression is characterized by comprising: an amplification primer and an extension primer for detecting 55 gene polymorphism sites for guiding the medication of depression are divided into three reaction systems,

wherein, the reaction system 1 comprises the following amplification primers and extension primers of gene polymorphic sites: rs1049353 site of CNR1 gene, wherein the amplification primer is SEQ ID No. 1-2, and the extension primer is SEQ ID No. 111; the rs1057868 locus of the POR gene, the amplification primer is SEQ ID No. 3-4, and the extension primer is SEQ ID No. 112; the site rs12248560 of CYP2C19 gene, the amplification primer is SEQ ID No. 5-6, the extension primer is SEQ ID No. 113; an rs1414334 site of the HTR2C gene, wherein an amplification primer is SEQ ID No. 7-8, and an extension primer is SEQ ID No. 114; locus rs1799732 of DRD2 gene, wherein the amplification primer is SEQ ID No. 9-10, and the extension primer is SEQ ID No. 115; the locus rs1799978 of DRD2 gene, the amplification primer is SEQ ID No. 11-12, the extension primer is SEQ ID No. 116; the site rs1800544 of the ADRA2A gene has an amplification primer of SEQ ID No. 13-14 and an extension primer of SEQ ID No. 117; the rs1902023 locus of UGT2B15 gene, the amplification primer is SEQ ID No. 15-16, the extension primer is SEQ ID No. 118; rs1954787 site of GRIK4 gene, wherein the amplification primer is SEQ ID No. 17-18, and the extension primer is SEQ ID No. 119; the rs28371706 site of CYP2D6 gene, the amplification primer is SEQ ID No. 19-20, the extension primer is SEQ ID No. 120; the site rs3211371 of CYP2B6 gene, the amplification primer is SEQ ID No 21-22, the extension primer is SEQ ID No 121; the site rs34223104 of CYP2B6 gene has the amplification primer of SEQ ID No. 23-24 and the extension primer of SEQ ID No. 122; the site rs8192709 of the CYP2B6 gene, the amplification primer is SEQ ID No. 25-26, and the extension primer is SEQ ID No. 123; the rs35599367 site of the CYP3A4 gene has an amplification primer of SEQ ID No. 27-28 and an extension primer of SEQ ID No. 124; the site rs35742686 of CYP2D6 gene, the amplification primer is SEQ ID No. 29-30, the extension primer is SEQ ID No. 125; the site rs3745274 of CYP2B6 gene has the amplification primer of SEQ ID No. 31-32 and the extension primer of SEQ ID No. 126; the site rs3892097 of the CYP2D6 gene has an amplification primer of SEQ ID No. 33-34 and an extension primer of SEQ ID No. 127; the rs5030865 locus of the CYP2D6 gene has an amplification primer of SEQ ID No. 35-36 and an extension primer of SEQ ID No. 128; the rs1135824 site of CYP2D6 gene has amplification primer of SEQ ID No. 37-38 and extension primer of SEQ ID No. 129; the rs5030655 site of the CYP2D6 gene has an amplification primer of SEQ ID No. 39-40 and an extension primer of SEQ ID No. 130; rs4713916 locus of FKBP5 gene, wherein the amplification primer is SEQ ID No. 41-42, and the extension primer is SEQ ID No. 131; rs489693 locus of MC4R gene, wherein the amplification primer is SEQ ID No 43-44, and the extension primer is SEQ ID No 132; an rs6313 locus of an HTR2A gene, wherein an amplification primer is SEQ ID No. 45-46, and an extension primer is SEQ ID No. 133; the site rs776746 of CYP3A5 gene, the amplification primer is SEQ ID No 47-48, the extension primer is SEQ ID No 134; an rs7997012 site of HTR2A gene, wherein an amplification primer is SEQ ID No. 49-50, and an extension primer is SEQ ID No. 135;

the reaction system 2 comprises the following amplification primers and extension primers of gene polymorphic sites: rs1000940 site of RABEP1 gene, wherein the amplification primer is SEQ ID No. 51-52, and the extension primer is SEQ ID No. 136; the locus rs10042486 of the HTR1A gene has an amplification primer of SEQ ID No. 53-54 and an extension primer of SEQ ID No. 137; the rs1065852 site of the CYP2D6 gene has an amplification primer of SEQ ID No. 55-56 and an extension primer of SEQ ID No. 138; the rs10245483 site of ABCB1 gene, the amplification primer is SEQ ID No. 57-58, and the extension primer is SEQ ID No. 139; the site rs12769205 of CYP2C19 gene, the amplification primer is SEQ ID No 59-60, the extension primer is SEQ ID No 140; an rs130058 locus of the HTR1B gene, wherein an amplification primer is SEQ ID No. 61-62, and an extension primer is SEQ ID No. 141; rs1800497 site of ANKK1 gene, the amplification primer is SEQ ID No. 63-64, the extension primer is SEQ ID No. 142; the rs2279343 site of CYP2B6 gene, the amplification primer is SEQ ID No. 65-66, the extension primer is SEQ ID No. 143; the site rs28371725 of the CYP2D6 gene, the amplification primer is SEQ ID No. 67-68, the extension primer is SEQ ID No. 144; the rs16947 locus of the CYP2D6 gene has an amplification primer of SEQ ID No. 69-70 and an extension primer of SEQ ID No. 145; the rs28399499 locus of CYP2B6 gene, the amplification primer is SEQ ID No. 71-72, the extension primer is SEQ ID No. 146; the rs28399504 locus of CYP2C19 gene, the amplification primer is SEQ ID No. 73-74, the extension primer is SEQ ID No. 147; the rs4148739 locus of ABCB1 gene has the amplification primer of SEQ ID No. 75-76 and the extension primer of SEQ ID No. 148; ZNF385D gene rs4261893 locus, the amplification primer is SEQ ID No 77-78, the extension primer is SEQ ID No 149; the rs61888800 locus of the BDNF gene has an amplification primer of SEQ ID No. 79-80 and an extension primer of SEQ ID No. 150; an rs6295 site of an HTR1A gene, wherein an amplification primer is SEQ ID No. 81-82, and an extension primer is SEQ ID No. 151; an rs6311 locus of an HTR2A gene, wherein an amplification primer is SEQ ID No. 83-84, and an extension primer is SEQ ID No. 152; an rs6318 site of the HTR2C gene, wherein an amplification primer is SEQ ID No. 85-86, and an extension primer is SEQ ID No. 153; the rs762551 site of CYP1A2 gene, the amplification primer is SEQ ID No. 87-88, the extension primer is SEQ ID No. 154;

the reaction system 3 comprises the following amplification primers and extension primers of gene polymorphic sites: rs1045642 site of ABCB1 gene, wherein the amplification primer is SEQ ID No. 89-90, and the extension primer is SEQ ID No. 155; the rs1135840 site of CYP2D6 gene has amplification primer of SEQ ID No. 91-92 and extension primer of SEQ ID No. 156; the rs1135835 site of CYP2D6 gene has amplification primer of SEQ ID No 93-94 and extension primer of SEQ ID No 157; rs1360780 site of FKBP5 gene, wherein the amplification primer is SEQ ID No. 95-96, and the extension primer is SEQ ID No. 158; the site rs1799853 of the CYP2C9 gene has an amplification primer of SEQ ID No. 97-98 and an extension primer of SEQ ID No. 159; the rs2032583 site of ABCB1 gene, the amplification primer is SEQ ID No. 99-100, the extension primer is SEQ ID No. 160; the rs2032582 site of the ABCB1 gene, the amplification primer is SEQ ID No. 101-102, and the extension primer is SEQ ID No. 161; the rs2235040 site of ABCB1 gene has the amplification primer of SEQ ID No. 103-104 and the extension primer of SEQ ID No. 162; the site rs324420 of the FAAH gene, the amplification primer is SEQ ID No. 105-106, and the extension primer is SEQ ID No. 163; the site rs3888190 of the SH2B1 gene, wherein the amplification primer is SEQ ID No. 107-108, and the extension primer is SEQ ID No. 164; the site rs4986893 of the CYP2C19 gene has an amplification primer of SEQ ID No. 109-110 and an extension primer of SEQ ID No. 165.

2. The kit of claim 1, wherein the 5' end of the amplification primer and/or the extension primer comprises a protective base sequence.

3. The kit according to claim 2, wherein the protective base sequence comprises 5 to 10 bases.

4. The kit of claim 3, wherein the protective base sequence is a tag of 10 bp: AGCTTGGAAG are provided.

5. The kit of claim 1, further comprising:

6. The kit of claim 5, further comprising: negative quality control product, positive quality control product.

7. The kit of claim 6, further comprising: resin for purifying extension products, target sheet for sample application and mass spectrum detection and human genome DNA extraction reagent.

8. A detection method based on the kit according to any one of claims 1 to 7, comprising the steps of:

(1) multiplex PCR: using specific amplification primers to simultaneously amplify DNA regions where 55 depression medication-assisted gene polymorphic sites are located in three reaction systems to obtain a PCR product containing 55 gene polymorphic sites, wherein the reaction system 1 comprises the following amplification primers and extension primers of the gene polymorphic sites:

rs1000940 site of RABEP1 gene, wherein the amplification primer is SEQ ID No. 51-52, and the extension primer is SEQ ID No. 136; the locus rs10042486 of the HTR1A gene has an amplification primer of SEQ ID No. 53-54 and an extension primer of SEQ ID No. 137; the rs1065852 site of the CYP2D6 gene has an amplification primer of SEQ ID No. 55-56 and an extension primer of SEQ ID No. 138; the rs10245483 site of ABCB1 gene, the amplification primer is SEQ ID No. 57-58, and the extension primer is SEQ ID No. 139; the site rs12769205 of CYP2C19 gene, the amplification primer is SEQ ID No 59-60, the extension primer is SEQ ID No 140; an rs130058 locus of the HTR1B gene, wherein an amplification primer is SEQ ID No. 61-62, and an extension primer is SEQ ID No. 141; rs1800497 site of ANKK1 gene, the amplification primer is SEQ ID No. 63-64, the extension primer is SEQ ID No. 142; the rs2279343 site of CYP2B6 gene, the amplification primer is SEQ ID No. 65-66, the extension primer is SEQ ID No. 143; the site rs28371725 of the CYP2D6 gene, the amplification primer is SEQ ID No. 67-68, the extension primer is SEQ ID No. 144; the rs16947 locus of the CYP2D6 gene has an amplification primer of SEQ ID No. 69-70 and an extension primer of SEQ ID No. 145; the rs28399499 locus of CYP2B6 gene, the amplification primer is SEQ ID No. 71-72, the extension primer is SEQ ID No. 146; the rs28399504 locus of CYP2C19 gene, the amplification primer is SEQ ID No. 73-74, the extension primer is SEQ ID No. 147; the rs4148739 locus of ABCB1 gene has the amplification primer of SEQ ID No. 75-76 and the extension primer of SEQ ID No. 148; ZNF385D gene rs4261893 locus, the amplification primer is SEQ ID No 77-78, the extension primer is SEQ ID No 149; the rs61888800 locus of the BDNF gene has an amplification primer of SEQ ID No. 79-80 and an extension primer of SEQ ID No. 150; an rs6295 site of an HTR1A gene, wherein an amplification primer is SEQ ID No. 81-82, and an extension primer is SEQ ID No. 151; an rs6311 locus of an HTR2A gene, wherein an amplification primer is SEQ ID No. 83-84, and an extension primer is SEQ ID No. 152; an rs6318 site of the HTR2C gene, wherein an amplification primer is SEQ ID No. 85-86, and an extension primer is SEQ ID No. 153; the rs762551 site of CYP1A2 gene, the amplification primer is SEQ ID No. 87-88, the extension primer is SEQ ID No. 154;

(2) and (3) PCR product purification: respectively purifying PCR products obtained in the step (1) in a protogen system;

(4) and (3) purification of an extension product: purifying the extension product obtained in the step (3) to obtain a high-purity extension product;

(6) and (5) judging the result.

9. Use of the kit of claim 1 for guiding medication for depression.

10. Use of the test method of claim 8 for guiding medication for depression.