CN111321214B

CN111321214B - Kit for guiding human depression medication and application thereof

Info

Publication number: CN111321214B
Application number: CN202010143778.3A
Authority: CN
Inventors: 何熲; 石忆湘; 顾孝平; 张国伟; 黄成琛; 王保曼; 张越
Original assignee: Shanghai Kangli Medical Laboratory Co ltd
Current assignee: Shanghai Kangli Medical Laboratory Co ltd
Priority date: 2020-03-04
Filing date: 2020-03-04
Publication date: 2021-01-15
Anticipated expiration: 2040-03-04
Also published as: CN111321214A

Abstract

The invention provides a kit for guiding human depression medication and application thereof, wherein the kit can simultaneously classify 43 gene loci, the kit comprises 43 pairs of amplification primers for amplifying 43 gene segments, the 43 gene loci are shown in a table, and the sequences of the 43 pairs of amplification primers are shown in SEQ ID No. 1-SEQ ID No. 86. On the basis of researching a large sample, the invention provides a brand-new site combination to realize accurate guidance and timely feedback on treatment of depression from the perspective of gene and heredity; and genotyping is carried out based on the time-of-flight mass spectrometry technology, and medium-throughput sequencing is carried out on some genes and variable sites which are selected carefully and most related to depression medication, so that the detection time and the cost can be greatly reduced, and a highly reliable detection result is still obtained, thereby having very important clinical application value.

Description

Kit for guiding human depression medication and application thereof

Technical Field

The invention relates to the field of kits, and particularly relates to a kit for guiding a human to take a medicament for depression and application of the kit.

Background

Depression is one of the major mental disorders worldwide, and has a greater impact on health than chronic diseases such as diabetes. Studies have shown that about 20% of patients with depression cannot be effectively treated, and about 50% of patients experience long-term illness or recurrence of depression, which seriously affects the life of the patients.

Medication guidance for depression treatment involves two main links: detection of genetic variation and interpretation of results.

And (3) gene variation detection: the main means of gene detection at present comprises mutation detection based on a PCR technology and mutation detection based on a next-generation sequencing technology. The detection based on the PCR technology also comprises a fluorescent quantitative PCR technology and PCR-based first-generation sequencing, the fluorescent quantitative PCR detection of gene variation needs to synthesize a fluorescent probe according to variation sites in advance, the process is complex, the flow is complicated, and the cost is high; and the two methods can only detect one mutation site at the same time, have low efficiency and are not multi-site systematic screening. The mutation site detection based on the second-generation sequencing technology needs expensive second-generation sequencing instruments and professional biological information personnel to carry out data analysis, and has the problems of high cost and insufficient accuracy due to the limitation of the prior art; although the deep analysis can be carried out on the genome of a sample, a large number of generated results lack effective biological and medical annotations, and the utilization value is limited, so that data redundancy and resource waste are caused; on the other hand, in the lateral range of large sample detection, a single sample is subjected to DNA library construction, sequencing and data analysis for a period of 1 week to tens of days, and the detection efficiency is not even as high as that of the fluorescent quantitative PCR-based technology.

Interpretation of the results: for the interpretation of the gene variation result, the accurate correspondence to the change of metabolism, response and toxicity of the drug in human body is the key for determining the pharmacogenomics to the treatment guidance of the depression drug, and relates to screening effective gene variation sites according to evidence of evidence-based medicine and adopting a normalized variation interpretation strategy. The similar detection products in the current market mainly have two problems, one is that the evidence grade of evidence is not standard, the clinical significance of the gene variation detection result is not explained in a unified way, and the efficacy of the detection product in the process of guiding the accurate medication of the depression is difficult to be determined; secondly, the medicine covers the incomplete treatment of the depression in China, and does not provide corresponding medication guidance for the newly marketed anti-depression medicine, so that the effect of the detection product is greatly reduced. The PharmGKB organization, based on published studies, classifies the clinical evidence of genetic factors affecting drug therapy into four grades, 1-4, with higher grades providing greater evidence reliability.

Disclosure of Invention

The invention aims to provide a kit for guiding the medication of human depression and application thereof, thereby solving the problems of high cost, insufficient accuracy, low detection efficiency, incomplete coverage of domestic depression treatment medicines and incapability of providing comprehensive medication guidance in the prior art.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to a first aspect of the present invention, there is provided a kit for guiding administration of human depression, which can simultaneously type 43 gene loci, the kit comprising 43 pairs of amplification primers for amplifying 43 gene fragments, wherein the sequences of the 43 gene loci and the 43 pairs of amplification primers are specifically shown below, and are all written in 5 '-3' order, and the primers are synthesized by Life corporation.

1) Locus rs 1800544G → C (wild type G, mutant C, same below)

SEQ ID NO.1 (upstream primer, same as below): ACGTTGGATGCCTGCTGGGAGTTGGCCAT

SEQ ID NO.2 (downstream primer, same below): : ACGTTGGATGTTCTCCCAAGATCCAGCTTC

2) Site rs 12720461C → T

SEQ ID NO.3:ACGTTGGATGTAGAACCTGGAAGCTAGTGG

SEQ ID NO.4:ACGTTGGATGAGAAGTCAAGAGCTGGGTAG

3) Locus rs5030655 delA (wild type is A, position A in mutant is lost)

SEQ ID NO.5:ACGTTGGATGTTTGTGCCCTTCTGCCCATC

SEQ ID NO.6:ACGTTGGATGAACTTGGGCCTGGGCAAGAA

4) Locus rs 16947G → A

SEQ ID NO.7:ACGTTGGATGTGTAGGATCATGAGCAGGAG

SEQ ID NO.8:ACGTTGGATGAGAGCAGCTTCAATGATGAG

5) Site rs 2279343A → G

SEQ ID NO.9:ACGTTGGATGCTTTTTCCATGTGGAGCAGG

SEQ ID NO.10:ACGTTGGATGGTGGAGAAGCACCGTGAAAC

6) Locus rs 28371725C → T

SEQ ID NO.11:ACGTTGGATGTCCCAGCAAAGTTCATGGGC

SEQ ID NO.12:ACGTTGGATGTGATCCTACATCCGGATGTG

7)rs1080985 G→C

SEQ ID NO.13:ACGTTGGATGTCACCCCAGGAATTCAAGAC

SEQ ID NO.14:ACGTTGGATGCCCAATCCCAGCTAATTTTG

8) Site rs 2032582A → C/T

SEQ ID NO.15:ACGTTGGATGAGTAAGCAGTAGGGAGTAAC

SEQ ID NO.16:ACGTTGGATGGTCTGGACAAGCACTGAAAG

9) Locus rs67666821 insT (mutant is wild type with a T base inserted at the position)

SEQ ID NO.17:ACGTTGGATGTTAGGAGGACTTCTTCAACC

SEQ ID NO.18:ACGTTGGATGAATTCAGGCTCCACTTACGG

10) Site rs774671100 insA

SEQ ID NO.19:ACGTTGGATGAGCAGTATGGTGTGTTCTGG

SEQ ID NO.20:ACGTTGGATGAACGCTGGGCTGCACGCTA

11) Site rs 1160351A → C

SEQ ID NO.21:ACGTTGGATGAAAGGGAATAACCCAGGAGC

SEQ ID NO.22:ACGTTGGATGGGCTGAGTGTCTGGGATTTC

12) Site rs 4713916A → G

SEQ ID NO.23:ACGTTGGATGTATCTGGCAACCCTAACCTC

SEQ ID NO.24:ACGTTGGATGCCTAACGAGATAGTGAGGAG

13) Locus rs 28399499T → C

SEQ ID NO.25:ACGTTGGATGTATGGCATTTTGGCTCGGTC

SEQ ID NO.26:ACGTTGGATGTCTGTACAGAGAGAGTCTAC

14) Locus rs 5443C → T

SEQ ID NO.27:ACGTTGGATGTCGTAGCCAGCGAATAGTAG

SEQ ID NO.28:ACGTTGGATGTCTCCCACGAGAGCATCATC

15) Locus rs 963468G → A

SEQ ID NO.29:ACGTTGGATGGCAGTCTAGGCAAATATCAC

SEQ ID NO.30:ACGTTGGATGTCAGAGTTTCCTTTGGCACC

16) Locus rs 28371706G → A

SEQ ID NO.31:ACGTTGGATGTGACCCACGGCGAGGACAC

SEQ ID NO.32:ACGTTGGATGAAATCTGTCTCTGTCCCCAC

17) Locus rs 3892097C → T

SEQ ID NO.33:ACGTTGGATGACAAAGCGGGAACTGGGAAG

SEQ ID NO.34:ACGTTGGATGTTGTCCAAGAGACCGTTGGG

18)rs4646425 C→T

SEQ ID NO.35:ACGTTGGATGCTTCCTCCCAATAACACCAG

SEQ ID NO.36:ACGTTGGATGTCTGGTGTCACGTTGCTTCC

19) Site rs 7997012A → G

SEQ ID NO.37:ACGTTGGATGCAGGAAAGAACGCTGAGTTG

SEQ ID NO.38:ACGTTGGATGGGCTTGCATAGGCAAGTGAC

20) Locus rs 201377835C → G

SEQ ID NO.39:ACGTTGGATGAGCTGCAGGCTGAACACGTC

SEQ ID NO.40:ACGTTGGATGTGATCCTGGCTTGACAAGAG

21) Locus rs 1135835T → C

SEQ ID NO.41:ACGTTGGATGTCTTCCTCTTCTTCACCTCC

SEQ ID NO.42:ACGTTGGATGACCAGGAAAGCAAAGACACC

22) Site rs 2228479G → A/C

SEQ ID NO.43:ACGTTGGATGCGTCAATGACATTGTCCAGC

SEQ ID NO.44:ACGTTGGATGTTGTCGGACCTGCTGGTGAG

23) Locus rs 34223104T → C

SEQ ID NO.45:ACGTTGGATGTTTTATCCTGACCCTGCCTC

SEQ ID NO.46:ACGTTGGATGGCTAATGCTCCTGGATGATG

24) Site rs 1135840C → G

SEQ ID NO.47:ACGTTGGATGCCATGGTGTCTTTGCTTTCC

SEQ ID NO.48:ACGTTGGATGACTAGGTACCCCATTCTAGC

25) Site rs 3211371C → T

SEQ ID NO.49:ACGTTGGATGGCAAAATACCCCCAACATAC

SEQ ID NO.50:ACGTTGGATGTGACCCTGGAATCCTTTGAC

26) Locus rs 1065852G → A

SEQ ID NO.51:ACGTTGGATGTGATAGTGGCCATCTTCCTG

SEQ ID NO.52:ACGTTGGATGAGTCCACATGCAGCAGGTTG

27) Site rs 2742417C → G/T

SEQ ID NO.53:ACGTTGGATGTTTCTGCACACTGAAGAGGG

SEQ ID NO.54:ACGTTGGATGGAGTCTACGCACAAATCAAC

28) Locus rs 8192709C → T

SEQ ID NO.55:ACGTTGGATGTCACAGGACTCTTGCTACTC

SEQ ID NO.56:ACGTTGGATGGCAGAAGGTTTCCCAAAAGG

29) Locus rs 5030867T → G

SEQ ID NO.57:ACGTTGGATGTAGTGGTGGCTGACCTGTTC

SEQ ID NO.58:ACGTTGGATGTGTTTCCCAGATGGGCTCAC

30) Locus rs 2069514G → A

SEQ ID NO.59:ACGTTGGATGTGTAATTCCAGCTACTCGGG

SEQ ID NO.60:ACGTTGGATGAGTGCAGTGGTGCGATCTTG

31) Locus rs 2235040C → T

SEQ ID NO.61:ACGTTGGATGGGGTCCAAAATGGCCAATTA

SEQ ID NO.62:ACGTTGGATGCGCCAATGATGCTGCTCAAG

32) Locus rs 3745274G → T

SEQ ID NO.63:ACGTTGGATGTTCAGTCTGTGTCCTTGACC

SEQ ID NO.64:ACGTTGGATGATGGAGCAGATGATGTTGGC

33) Site rs35742686 delT

SEQ ID NO.65:ACGTTGGATGTCTCACCTTCTCCATCTCTG

SEQ ID NO.66:ACGTTGGATGCAGCTGGATGAGCTGCTAAC

34) Site rs 12248560C → T

SEQ ID NO.67:ACGTTGGATGCAAATTTGTGTCTTCTGTTC

SEQ ID NO.68:ACGTTGGATGTGAGCTGAGGTCTTCTGATG

35) Site rs 762551C → A

SEQ ID NO.69:ACGTTGGATGTCTGTGATGCTCAAAGGGTG

SEQ ID NO.70:ACGTTGGATGCAGCTGGATACCAGAAAGAC

36) Site rs 1045642A → G

SEQ ID NO.71:ACGTTGGATGTTGCCTATGGAGACAACAGC

SEQ ID NO.72:ACGTTGGATGAAGGCATGTATGTTGGCCTC

37) Locus rs 5030862C → T

SEQ ID NO.73:ACGTTGGATGTGATAGTGGCCATCTTCCTG

SEQ ID NO.74:ACGTTGGATGAGTCCACATGCAGCAGGTTG

38) Site rs 4986893G → A

SEQ ID NO.75:ACGTTGGATGGACTGTAAGTGGTTTCTCAG

SEQ ID NO.76:ACGTTGGATGAACATCAGGATTGTAAGCAC

39) Locus rs 5030865C → T

SEQ ID NO.77:ACGTTGGATGTTCCCAGTTCCCGCTTTGTG

SEQ ID NO.78:ACGTTGGATGTTTGTGCCGCCTTCGCCAAC

40) Site rs 2069526T → G

SEQ ID NO.79:ACGTTGGATGTAGAACCTGGAAGCTAGTGG

SEQ ID NO.80:ACGTTGGATGTCAAGAGCTGGGTAGCAAAG

41) Locus rs 6295C → G

SEQ ID NO.81:ACGTTGGATGGTCAGTCTCCCAATTATTGC

SEQ ID NO.82:ACGTTGGATGCGAGAACGGAGGTAGCTTTT

42) Locus rs 1081003G → A

SEQ ID NO.83:ACGTTGGATGGGCCTGTTTCATGTCCACGAC

SEQ ID NO.84:ACGTTGGATGTCCATCACAGAAGGTGTGAC

43) Site rs 4244285G → A

SEQ ID NO.85:ACGTTGGATGTCCATCGATTCTTGGTGTTC

SEQ ID NO.86:ACGTTGGATGGCAATAATTTTCCCACTATC

According to the present invention, the kit further preferably comprises 43 extension primers for identifying mutations of the 43 gene fragments, and the sequences of the 43 extension primers designed for the 43 specific gene loci are specifically as follows, all written in 5 '-3' order, and the primers are synthesized by Life corporation.

SEQ ID NO.87：TTGGCCATGCAGCTC

SEQ ID NO.88：AGGGGTCCTGAGTTC

SEQ ID NO.89：CGCCTCCTCGGTCACCC

SEQ ID NO.90：CTCAGCCACCACTATGC

SEQ ID NO.91：TGGTGTCGATGAGGTCC

SEQ ID NO.92：GGGCCCGCCTGTACCCTT

SEQ ID NO.93：GGACAACTTGGAAGAACC

SEQ ID NO.94：GTTTGACTCACCTTCCCAG

SEQ ID NO.95：GACTTCTTCAACCAGAAAAA

SEQ ID NO.96：AGGAAAGTCCACATGCAGCA

SEQ ID NO.97：GCTAGAGAGACCAAGAAACA

SEQ ID NO.98：CGGACTCCTACATTTTCCTCT

SEQ ID NO.99：CCACAGGCCAATCACCTGTTCA

SEQ ID NO.100：GAAACCTGAGGGAGAAGGCCAC

SEQ ID NO.101：CAAAATATCACATCATCAACAGA

SEQ ID NO.102：AAGGCCGCCCGCCTGTGCCCATCA

SEQ ID NO.103：CCCTGACCCGCATCTCCCACCCCCA

SEQ ID NO.104：TGGTCCAATTTCAAAGAAGGAAAAG

SEQ ID NO.105：TTTGCCATTATCTTCAAAGACTTAATT

SEQ ID NO.106：GAAAACGTCCCCGAAGCGGCGCCGCAA

SEQ ID NO.107：CCCCCAGCACTTCAGCTTCTCGGTGCCC

SEQ ID NO.108：ATCATGAGGATGACGGCCGTCTCCAGCA

SEQ ID NO.109：CTCTGCACCCTGTTAT

SEQ ID NO.110：CTTTGCTTTCCTGGTGA

SEQ ID NO.111：CCCCCAACATACCAGATC

SEQ ID NO.112：CGCTGGGCTGCACGCTAC

SEQ ID NO.113：TGTGGCAGGCAGGCTTCG

SEQ ID NO.114：ACTGCTACTCCTGGTTCAG

SEQ ID NO.115：CCTCCTGCTCATGATCCTAC

SEQ ID NO.116：GCATGACAATTGCTTGAATC

SEQ ID NO.117：AAATGGCCAATTAAGACAAA

SEQ ID NO.118：CGGAACCCACCTTCCTCTTCCA

SEQ ID NO.119：GTGTGCTGGGTCCCAGGTCATCC

SEQ ID NO.120：CCCTCGTGTCTTCTGTTCTCAAAG

SEQ ID NO.121：GGTTAAAAGGGTGAGCTCTGTGGGC

SEQ ID NO.122：AGACTGGTGGTGTCACAGGAAGAGAT

SEQ ID NO.123：CCCCTGCCACTGCCC

SEQ ID NO.124：CTTGGCCTTACCTGGAT

SEQ ID NO.125：ACTGCTGCCCATCACCCAC

SEQ ID NO.126：GGGTGGCTAGGTGTAGGGG

SEQ ID NO.127：TCGACGACCGAGTGTGTCTTC

SEQ ID NO.128：GTTGGGAACGCGGCCC

SEQ ID NO.129：TTGTTAAGTAATTTGTTATGGGTTCC

Preferably, the reaction system of the PCR amplification of the kit is as follows:

preferably, the kit further comprises an SAP reaction system, which is as follows:

SAP buffer 0.17. mu.L

SAP enzyme 0.5U

The volume of pure water is made up to 2 μ L.

According to a preferred embodiment of the present invention, the amplification primers SEQ ID No.1 to SEQ ID No.2, SEQ ID No.9 to SEQ ID No.10 have a molar concentration of 0.4 to 0.6. mu.M, SEQ ID No.3 to SEQ ID No.8, SEQ ID No.11 to SEQ ID No.18, SEQ ID No.21 to SEQ ID No.50, SEQ ID No.53 to SEQ ID No.58, SEQ ID No.61 to SEQ ID No.68, SEQ ID No.71 to SEQ ID No.76, SEQ ID No.79 to SEQ ID No.80 have a molar concentration of 0.2 to 0.4. mu.M, SEQ ID No.19 to SEQ ID No.20, SEQ ID No.51 to SEQ ID No.52, SEQ ID No.59 to SEQ ID No.60, SEQ ID No.81 to SEQ ID No.82 have a molar concentration of 0.4 to 0.8. mu.69, SEQ ID No.86 to SEQ ID No.70, and SEQ ID No.85 to SEQ ID No.85 are present in the reaction system, the molar concentration of SEQ ID No. 77-SEQ ID No.78 in the reaction system is 0.8-1.4 mu M, and the molar concentration of SEQ ID No. 83-SEQ ID No.84 in the reaction system is 0.6-1.2 mu M.

It should be understood that any kit based on MassArray has less extension primers, but the importance of the extension primers is less than that of the amplification primers, the kit provided by the invention has the main invention point of selection of gene loci and design of the amplification primers, and the secondary invention point of design of the extension primers, so that the most accurate guidance and the most timely feedback on the treatment of depression diseases are finally realized from the genetic and heredity perspectives.

The invention adopts an Agena Bioscience MassARRAY DNA mass spectrum gene analysis system, which has the following characteristics:

the accuracy is high, the molecular weight of the substance to be detected is directly detected, and the accuracy is over 99.9%; the failure of PCR experiment or the existence of three-allele gene can be detected;

1) the sensitivity is high, and any pmol-level substance can be detected in a detection window;

2) the flux is high, multiple detection of 384 samples can be simultaneously completed on one chip, each reaction hole can realize up to 30 times of reaction, and tens of thousands of genotype analyses can be carried out at most each time;

3) the method is flexible, the number and the position of the samples can be freely selected on one chip, and meanwhile, the pairing of the samples and the SNP sites can be freely selected;

4) the quality control is strict, the mass spectrometry technology is 'one-tube operation', namely, a reaction system is always reacted in one test tube in the biochemical experiment process, and the human error caused by multiple transfers is avoided;

5) the method is simple to operate, and completely changes the disadvantages of high price, long time consumption, complex operation and the like of the traditional sequencing technology in gene detection.

The process of the invention comprises links of specific primer amplification, SAP reaction, single base extension, mass spectrum detection and the like, and a schematic diagram is shown in figure 1.

According to a second aspect of the invention, there is also provided the use of a kit as described above in the manufacture of a reagent for use in guiding depression in a human.

According to the kit provided by the invention, a flight time mass spectrometry technology is selected to design a conventional primer aiming at a variant gene locus, a multi-locus primer combination can work simultaneously and accurately detect the situation of locus variation, a 384-pore plate is adopted for experiment, dozens of variant loci of 384 samples can be detected simultaneously, the period from sample preparation to result generation only needs 2 days, the cost does not exceed 1000 RMB, and the accuracy, the low cost and the high flux are considered.

According to evidence-based medical evidence grade specifications established by PharmGKB, the invention integrates the information of the variation sites in the PharmGKB database and the information of other variation sites which are publicly researched, screens 43 gene variation sites according to the reliability of the evidence grade, and provides clinical prompts of the drugs in the aspects of absorption, metabolism, response, toxicity and the like in vivo. The sites cover 21 commonly used anti-depression drugs used in clinic, and the practical value of clinical medication guidance is greatly improved.

The kit provided by the invention comprises 43 groups of primers, and can detect the variation condition of 15 genes related to pharmacogenomics (ABCB1, ADRA2A, CYP1A2, CYP2B6, CYP2C19, CYP2D6, CYP3A4, DRD3, FKBP5, GNB3, HTR1A, HTR2A, MC1R, MDGA2 and SACM 1L). rs7997012 affects citalopram efficacy; rs2032582 affects the fluoxetine efficacy and the drug toxicity of clomipramine, venlafaxine and paroxetine; rs1160351 affects the drug toxicity of venlafaxine, milnacipran and paroxetine; rs4713916 affects the efficacy of citalopram, venlafaxine, mirtazapine, paroxetine; rs5443 affects nortriptyline efficacy; rs7997012 affects citalopram efficacy; rs2228479 affects the efficacy of desipramine; rs2742417 affects bupropion toxicity; rs2235040 affects the therapeutic effects of amitriptyline, citalopram, paroxetine and venlafaxine; rs1045642 affects the curative effects of venlafaxine and agomelatine, and affects the toxicity of nortriptyline drugs; rs6295 affects the therapeutic efficacy of fluoxetine, fluvoxamine, paroxetine, sertraline and milnacipran.

The invention provides a primer combination and a kit for detecting genes influencing the action of depression treatment drugs, which are applied to detecting gene loci influencing the action of human depression treatment drugs in vivo and judging the influence of individual genetic factors on the metabolic level, action effect and toxic and side effects of the depression treatment drugs. The medicine is absorbed, distributed, metabolized, excreted and the like in human body, and related genes participate in each link. For example, the ABCB gene family is responsible for transporting drugs across the membrane of cells, the cytochrome 450 family gene is mainly responsible for the metabolism of the drugs in vivo, and the dopamine receptor family gene influences the action effect of the drugs for treating depression. Different genetic variations alter the action of drugs in the human body, affecting drug metabolism, toxicity and efficacy. Research shows that the gene variation of individuals can change the functions of corresponding genes, so that the medicine has different effects among individuals. According to the in vivo action process of the drug, the invention selects key genes influencing drug metabolism, response and toxicity, designs specific primer combinations for a group of gene loci, adopts the time-of-flight mass spectrometry technology to detect and type the gene loci, predicts the action condition of the drug in individuals by analyzing the genetic variation condition of the individuals, and can provide reference for optimizing a treatment scheme and improving the treatment efficiency.

Up to now, more than 50,000 samples have been tested by Shanghai Kangli medical laboratory and research has been conducted in the fields of mental diseases, including depression, schizophrenia, anxiety, epilepsy, hyperactivity, analgesia, sedation and hypnosis, etc. The invention establishes a database based on Chinese multi-nationality, multi-region and multi-course on the basis of the research of a large sample, develops the kit which has good effect and can be used for clinical medication guidance on the basis of data polymorphism, and provides accurate guidance and timely feedback for the treatment of the depression from the aspects of gene and heredity.

In conclusion, on the basis of researching a large sample, the invention provides a brand-new locus combination, and realizes accurate guidance and timely feedback on treatment of depression from the aspects of gene and heredity; the invention carries out genotyping based on the time-of-flight mass spectrometry technology, and carries out medium-flux sequencing on some genes and variation sites which are selected carefully and are most related to depression medication, so that the time period and the detection cost can be greatly reduced, and a highly reliable detection result is still obtained, thereby ensuring that the technology has practical use value in clinic.

Drawings

FIG. 1 is a schematic workflow diagram of the present invention.

Detailed Description

The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

Details of the kit

1.1 composition of

PCR mixed reaction liquid, Taq enzyme, amplification primer mixed liquid, SAP buffer solution, SAP enzyme, extension primer mixed liquid, iPlex enzyme, single-base extension reaction mixed liquid, deionized water, a positive control product, desalting resin and a mass spectrum chip.

1.2 storage conditions and expiration dates

The reaction reagent and the enzyme are stored at-20 ℃ and the shelf life is 9 months.

1.3 matching instruments

General purpose PCR instrument, DR MassArray analyzer.

1.4 sample requirement

The product is suitable for extracting genome DNA from oral mucosa cells, oral exfoliative cells, blood, tissues and dried blood slices, and requires that the ratio of DNA A260/A280 is between 1.8 and 2.0. Frozen DNA samples should be below-20 ℃ and repeated freeze thawing is avoided.

1.5 Experimental procedures

PCR amplification

a. In PCR I, the reagents were removed from the freezer at-20 ℃ and placed on ice. The amplification primers were removed from 4 ℃, vortexed for 10s, and briefly centrifuged for use.

b. Related reagents are added into the components in the following table at one time, PCR reaction liquid is prepared, and the PCR reaction liquid is subpackaged into a 96-well plate with the volume of 3 mu L per well. After completion, the DNA fragment was transferred to PCR II region through the transfer window.

c. In PCR II area, DNA template was taken out from refrigerator at-20 deg.C, melted on ice (4 deg.C), vortexed for 10s, centrifuged briefly, and a certain amount of DNA was aspirated and diluted to 5 ng/. mu.L for use.

d. Adding 2 muL of 5 ng/muL DNA template into each well of a 96-well plate, covering a tube cover, performing vortex oscillation for 10s, centrifuging briefly, transferring from a PCR II area to a PCR III area through a transfer window, and transferring from the PCR III area to a PCR IV area through the transfer window, wherein a blank control (2 muL ddH) must be set in each experiment₂O), negative control (2 μ L DNA extraction eluate) and positive control.

e. The 96-well plate was placed in the amplification apparatus, and the program was run: pcr, the specific procedure is as follows:

SAP reaction

a. SAP mixtures were prepared in 1.5ml EP tubes according to the following table:

SAP buffer	0.17 μ L of CutSmart buffer (manufacturer NEB)
		SAP enzymes	0.5U (manufacturer NEB)
Water (W)	Make up to 2 mu L

b. Add 2. mu.L of SAP mixture to the wells of a 96-well plate, seal the 96-well plate with a sealing membrane, vortex, and centrifuge for 5s at 4000 rpm.

c. The 96-well plate was transferred to a PCR instrument for the following reactions:

37℃40min→85℃ 5min→4℃

extension reaction

a. And taking out the 96-well plate after the reaction in the previous step, and centrifuging at 2000 rpm for 1 min. An iPLEX extension reaction solution was prepared in a 1.5ml EP tube according to the following table:

b. the iPLEX extension reaction solution, 2. mu.L/well, was added to the 96-well plate at the end of the previous reaction, the 96-well plate was resealed, vortexed, and centrifuged (supra).

c. The 96-well plate was transferred to a PCR instrument and the following program was run:

sample desalting

a. Clean Resin (Resin) was spread flat over 96/15mg crater plate and air dried for at least 10min, changing the Resin from dark yellow to light yellow.

b. To each well of the sample plate, 41. mu.L of water was added, the membrane was sealed, and then centrifuged.

c. Add 15mg of clean Resin (Resin): the sample plate was gently inverted in the air and the hole-to-hole placed on the crater plate with resin placed. The crater plate, along with the sample plate, is then inverted (both plates are not horizontally movable during the process) allowing the resin to drop into the wells. The 96 well plate was sealed and shaken upside down on a rotator for 15 minutes.

d. Plates were centrifuged for 5 minutes at 3200g (4000 rpm of standard plate centrifuge).

v. mass spectrometric detection

a. And opening the software of the plate management system, editing an experiment plan file, wherein the experiment plan file comprises the position of the sample, the name of the sample and the used primer, and connecting the mass spectrometer with the established experiment plan file.

b. The Start All icon is clicked, the software is started, and the various indicator lights are checked for normality.

c. Click the "chip tray enter/exit" button to place the chip on the tray and then on the chip deck, record the chip position (1 on the left and 2 on the right). Hands do not touch the surface of the chip; placing the 96 plate at the position marked with MTP1/2, and fixing the 96 plate in the direction of A1 at the lower left corner; when the chip is used for the first time, 75 mu L of calibration standard substance is added into the sample adding slot of the calibration substance, and when the chip is not used for the first time, the calibration standard substance does not need to be added. Then click the 'chip tray enter/exit' button and close the clamp plate.

d. Clicking a 'resin adding/maintaining' button, opening a resin tank, adding resin or supplementing high-pressure sterilized purified water (when an instrument is started for the first time, 28g of resin needs to be added into the resin tank and 16mL of sterilized purified water needs to be added for uniform mixing, when the instrument is used for the first time, 9g of resin is completely poured into the resin tank, 5.2mL of high-pressure sterilized purified water is added, a gun head is used for uniform mixing, when the instrument is not used for the first time, the liquid level needs to be observed, if the liquid level of the water is lower than the resin level, a proper amount of high-pressure sterilized purified water needs to be supplemented, so that the liquid level of the water is higher than the resin level, and after the resin solution is added into the resin tank, the resin solution needs to be used as

e. The program set-up parameters are as follows:

f. after finishing printing the mass spectrum, clicking a button for removing the old chip from the analyzer, returning the chip to a chip deck, then clicking a button for entering/exiting a chip tray, taking out a 96-well plate, sealing a film and storing at-20 +/-5 ℃; the chip is put back into the packaging box and stored in a dehumidifier (the chip is used as soon as possible after being opened, the storage time does not exceed 30 days), the calibration standard sample is recovered and stored at minus 20 plus or minus 5 ℃, then a button for 'chip tray entering/exiting' is pressed, and the clamping plate is closed.

1.6 the results show

And (3) judging the effectiveness: the standard substance can detect the corresponding genotype, the blank control has no signal detection, and the weak positive control can detect the corresponding positive signal, the detection result is valid, otherwise, the detection result is invalid.

1.7 product Performance index

[1] The product can detect 1ng of human genome DNA with the A260/A280 purity ranging from 1.70 to 1.90 at the lowest.

[2] The method is influenced by the quality of sample DNA, and false negative results can occur in low-quality DNA.

Experimental example:

case one: zhang XX, female, 60 years old. Depressed mood, obsessive-compulsive behavior for 30 years, was diagnosed as "obsessive-compulsive attack". Since the onset of illness, patients have poor diet and sleep, and the patients can feel poor in the sleep. Deny the past manifestations of much excitement. The past history, personal history, family history, physical examination and auxiliary examination are not special.

And (3) mental examination: clear consciousness, passive contact, thought retardation, depressed emotion, anxiety, hypovolemia, and lack of self-cognition. Admission diagnosis: major depressive episode without psychotic symptoms.

Prescription before examination:

2018.12.06

fluoxetine 40mg/qd, olanzapine 10mg/qn, clonazepam 2mg/qn, propranolol 10mg/bid, fluvoxamine 250mg/qd, and alprazolam 0.4 mg/qn. As a result: the emotion is low, and there are compelling thinking and behaviors.

2018.12.26

Fluoxetine 40mg/qd, olanzapine 10mg/qn, clonazepam 2mg/qn, propranolol 10mg/bid, fluvoxamine 300mg/qd, and alprenolon 0.4 mg/qn. As a result: difficulty in sleeping at night, low mood, and compulsive thinking and behavior.

2019.01.17

Fluoxetine 20mg/qd, clomipramine 50mg/bid, clonazepam 2mg/qn, olanzapine 10mg/qn, propranolol 10mg/bid, fluvoxamine 300 mg/qd. The emotion is low, and there are compelling thinking and behaviors.

2019.02.21

Fluoxetine 20mg/qd, clomipramine 50mg/bid, olanzapine 5mg/qn, clonazepam 2mg/qn, propranolol 10mg/bid, fluvoxamine 300 mg/qd. As a result: the emotion is low, and there are compelling thinking and behaviors.

2019.03.21

Fluoxetine 40mg/qd, olanzapine 5mg/qn, clonazepam 1mg/qn, propranolol 10mg/bid, fluvoxamine 300 mg/qd. As a result: difficulty in sleeping at night, low mood, and compulsive thinking and behavior.

2019.04.18

Fluoxetine 40mg/qd, olanzapine 5mg/qn, clonazepam 1mg/qn, propranolol 10mg/bid, fluvoxamine 300mg/qd, eszolon 1 mg/qd. As a result: the people have difficulty sleeping at night, still have low mood and are lack of strength, and do not want to exercise.

The following results are obtained by adopting the kit provided by the invention for detection:

note that:

(extensive metabolic) drug metabolism is normal, and blood drug concentrations are within the therapeutic window.

(ultrafast metabolic type) accelerated drug metabolism, and the blood concentration may be lower than the effective therapeutic concentration, resulting in poor therapeutic effect.

(intermediary metabolic type) slowing of drug metabolism, blood drug levels may be higher than safe therapeutic levels (or lack of clear data support).

(slow-metabolizing) drug metabolism is significantly slowed, and blood drug concentrations may be higher than safe treatment concentrations, leading to an increased risk of adverse reactions.

5. The drug response is optimal.

6. The drug response is not optimal.

7. The risk of adverse reactions is low.

8. The risk of adverse reactions is high.

Prescription and follow-up after detection:

the clonazepam, fluvoxamine and estazolam in the original prescription are replaced by oxazepam, mirtazapine and alprazolam, so that the side effect of the medicine does not appear in treatment, and the follow-up is performed after the patient is discharged for 1 month, so that the condition is good.

Case two:

zhao XX, female, Han nationality, 69 years old. Low mood, decreased interest, decreased speech, decreased appetite, and slower response; dysphoria, stress, fear; sleep disorder is present; self-awareness exists and the social function is partially impaired. Depression is diagnosed.

Prescription before detection

Paroxetine 50mg/qd, tandospirone 5 mg/tid.

Results of Gene detection

Note that:

5. The drug response is optimal.

6. The drug response is not optimal.

7. The risk of adverse reactions is low.

8. The risk of adverse reactions is high.

Prescription and results after examination

2019.07.01

Venlafaxine was added at 75mg/qd with gradual cessation of paroxetine. The symptoms of the patient such as tension, fear and hypodynamia are improved to some extent; normal spirit, appetite and sleep, and normal stool and urine. The depressed mood and the expression are better than those of the patients admitted to the hospital, and the physical discomfort such as palpitation, hypodynamia, limb numbness and the like is better.

2019.09.09

The patient is full of spirit, clear in voice, moderate in voice speed, high in treatment satisfaction degree and good in compliance after telephone return visit.

The detection result corresponding to the case is as follows:

1. citalopram

2. Escitalopram

3. Fluvoxamine

4. Fluoxetine

5. Paroxetine

6. Sertraline

7. Duloxetine

8. Venlafaxine

9. Mirtazapine

10. Amitriptyline

11. Chloropropiozine

12. Desipramine

13. Medicine for treating multiple anxiety

14. Imipramine

15. Nortriptyline

16. Trimipramine

17. Trazodone

18. Agomelatine

19. Bupropion derivatives

20. Milnacipran

Serial number	Detection of genes	Detection site	The result of the detection
				1	MDGA2	A>C	AA
2	HTR1A	C>G	CG
				3	ADRA2A	G>C	CG

21. Vothiostatin

Note 1: o genotype interacts with the drug, but the patient's genotype is normally expressed

Note 2: ● genotype interacts with drug and the patient's genotype is shown to be altered

3, injecting a [ metabolic gene ]1, namely CYP1A 2-I; CYP2B 6; CYP2C 19-I; CYP2D 6; CYP3A 4;

[ responsive genes ]

6:ABCB1-I；7:ADRA2A；8:CYP2C19-II；9:DRD3；10:FKBP5；11:GNB3；12:HTR1A；13:HTR2A；14:MC1R；15:SLC6A4；

[ virulence genes ]16 ABCB 1-II; ABCB 1-III; 18: ABCB 1-I; 19 CYP1A 2-II; 20: MDGA 2; 21: SACM 1L; 22 SLC6a 4.

Interpretation of the test results:

drugs with normal drug metabolism, toxicity and response are classified in the first column, and the selection of the drugs in this column can improve the treatment efficiency and reduce the treatment risk.

The above embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and various changes may be made in the above embodiments of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present patent application. The invention has not been described in detail in order to avoid obscuring the invention.

SEQUENCE LISTING

<110> Shanghai Kangli medical laboratory Co., Ltd

<120> kit for guiding medication of human depression and application thereof

<160> 129

<170> PatentIn version 3.5

<210> 1

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<212> DNA

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acgttggatg cctgctggga gttggccat 29

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acgttggatg ttctcccaag atccagcttc 30

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acgttggatg tagaacctgg aagctagtgg 30

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acgttggatg agaagtcaag agctgggtag 30

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acgttggatg tttgtgccct tctgcccatc 30

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acgttggatg aacttgggcc tgggcaagaa 30

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acgttggatg tgtaggatca tgagcaggag 30

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acgttggatg agagcagctt caatgatgag 30

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acgttggatg ctttttccat gtggagcagg 30

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acgttggatg gtggagaagc accgtgaaac 30

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acgttggatg tcccagcaaa gttcatgggc 30

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acgttggatg tgatcctaca tccggatgtg 30

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acgttggatg tcaccccagg aattcaagac 30

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acgttggatg cccaatccca gctaattttg 30

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acgttggatg agtaagcagt agggagtaac 30

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acgttggatg gtctggacaa gcactgaaag 30

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acgttggatg ttaggaggac ttcttcaacc 30

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acgttggatg aattcaggct ccacttacgg 30

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acgttggatg agcagtatgg tgtgttctgg 30

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acgttggatg aacgctgggc tgcacgcta 29

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acgttggatg aaagggaata acccaggagc 30

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acgttggatg ggctgagtgt ctgggatttc 30

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acgttggatg tatctggcaa ccctaacctc 30

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acgttggatg cctaacgaga tagtgaggag 30

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acgttggatg tatggcattt tggctcggtc 30

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acgttggatg tctgtacaga gagagtctac 30

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acgttggatg tcgtagccag cgaatagtag 30

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acgttggatg tctcccacga gagcatcatc 30

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acgttggatg gcagtctagg caaatatcac 30

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acgttggatg tcagagtttc ctttggcacc 30

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acgttggatg tgacccacgg cgaggacac 29

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acgttggatg aaatctgtct ctgtccccac 30

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acgttggatg acaaagcggg aactgggaag 30

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acgttggatg ttgtccaaga gaccgttggg 30

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acgttggatg cttcctccca ataacaccag 30

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acgttggatg tctggtgtca cgttgcttcc 30

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acgttggatg caggaaagaa cgctgagttg 30

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acgttggatg ggcttgcata ggcaagtgac 30

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acgttggatg agctgcaggc tgaacacgtc 30

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acgttggatg tgatcctggc ttgacaagag 30

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acgttggatg tcttcctctt cttcacctcc 30

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acgttggatg accaggaaag caaagacacc 30

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acgttggatg cgtcaatgac attgtccagc 30

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acgttggatg ttgtcggacc tgctggtgag 30

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acgttggatg ttttatcctg accctgcctc 30

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acgttggatg gctaatgctc ctggatgatg 30

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acgttggatg ccatggtgtc tttgctttcc 30

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acgttggatg actaggtacc ccattctagc 30

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acgttggatg gcaaaatacc cccaacatac 30

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acgttggatg tgaccctgga atcctttgac 30

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acgttggatg tgatagtggc catcttcctg 30

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acgttggatg agtccacatg cagcaggttg 30

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acgttggatg tttctgcaca ctgaagaggg 30

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acgttggatg gagtctacgc acaaatcaac 30

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acgttggatg tcacaggact cttgctactc 30

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acgttggatg gcagaaggtt tcccaaaagg 30

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acgttggatg tagtggtggc tgacctgttc 30

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acgttggatg tgtttcccag atgggctcac 30

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acgttggatg tgtaattcca gctactcggg 30

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acgttggatg agtgcagtgg tgcgatcttg 30

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acgttggatg gggtccaaaa tggccaatta 30

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acgttggatg cgccaatgat gctgctcaag 30

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acgttggatg ttcagtctgt gtccttgacc 30

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acgttggatg atggagcaga tgatgttggc 30

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acgttggatg tctcaccttc tccatctctg 30

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acgttggatg cagctggatg agctgctaac 30

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acgttggatg caaatttgtg tcttctgttc 30

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acgttggatg tgagctgagg tcttctgatg 30

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acgttggatg tctgtgatgc tcaaagggtg 30

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acgttggatg cagctggata ccagaaagac 30

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acgttggatg ttgcctatgg agacaacagc 30

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acgttggatg aaggcatgta tgttggcctc 30

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acgttggatg tgatagtggc catcttcctg 30

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acgttggatg agtccacatg cagcaggttg 30

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acgttggatg gactgtaagt ggtttctcag 30

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acgttggatg aacatcagga ttgtaagcac 30

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acgttggatg ttcccagttc ccgctttgtg 30

<210> 78

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<400> 78

acgttggatg tttgtgccgc cttcgccaac 30

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<400> 79

acgttggatg tagaacctgg aagctagtgg 30

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<400> 80

acgttggatg tcaagagctg ggtagcaaag 30

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<400> 81

acgttggatg gtcagtctcc caattattgc 30

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<400> 82

acgttggatg cgagaacgga ggtagctttt 30

<210> 83

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<400> 83

acgttggatg ggcctgtttc atgtccacga c 31

<210> 84

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<400> 84

acgttggatg tccatcacag aaggtgtgac 30

<210> 85

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<400> 85

acgttggatg tccatcgatt cttggtgttc 30

<210> 86

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<400> 86

acgttggatg gcaataattt tcccactatc 30

<210> 87

<211> 15

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<400> 87

ttggccatgc agctc 15

<210> 88

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<400> 88

aggggtcctg agttc 15

<210> 89

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<400> 89

cgcctcctcg gtcaccc 17

<210> 90

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<400> 90

ctcagccacc actatgc 17

<210> 91

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<400> 91

tggtgtcgat gaggtcc 17

<210> 92

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gggcccgcct gtaccctt 18

<210> 93

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ggacaacttg gaagaacc 18

<210> 94

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gtttgactca ccttcccag 19

<210> 95

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<400> 95

gacttcttca accagaaaaa 20

<210> 96

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<400> 96

aggaaagtcc acatgcagca 20

<210> 97

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<400> 97

gctagagaga ccaagaaaca 20

<210> 98

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<400> 98

cggactccta cattttcctc t 21

<210> 99

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<400> 99

ccacaggcca atcacctgtt ca 22

<210> 100

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<400> 100

gaaacctgag ggagaaggcc ac 22

<210> 101

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<400> 101

caaaatatca catcatcaac aga 23

<210> 102

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<400> 102

aaggccgccc gcctgtgccc atca 24

<210> 103

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<400> 103

ccctgacccg catctcccac cccca 25

<210> 104

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<400> 104

tggtccaatt tcaaagaagg aaaag 25

<210> 105

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<400> 105

tttgccatta tcttcaaaga cttaatt 27

<210> 106

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<400> 106

gaaaacgtcc ccgaagcggc gccgcaa 27

<210> 107

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<400> 107

cccccagcac ttcagcttct cggtgccc 28

<210> 108

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<400> 108

atcatgagga tgacggccgt ctccagca 28

<210> 109

<211> 16

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<400> 109

ctctgcaccc tgttat 16

<210> 110

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<400> 110

ctttgctttc ctggtga 17

<210> 111

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<400> 111

cccccaacat accagatc 18

<210> 112

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<400> 112

cgctgggctg cacgctac 18

<210> 113

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<400> 113

tgtggcaggc aggcttcg 18

<210> 114

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<400> 114

actgctactc ctggttcag 19

<210> 115

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<400> 115

cctcctgctc atgatcctac 20

<210> 116

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<400> 116

gcatgacaat tgcttgaatc 20

<210> 117

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<400> 117

aaatggccaa ttaagacaaa 20

<210> 118

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cggaacccac cttcctcttc ca 22

<210> 119

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gtgtgctggg tcccaggtca tcc 23

<210> 120

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<400> 120

ccctcgtgtc ttctgttctc aaag 24

<210> 121

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ggttaaaagg gtgagctctg tgggc 25

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agactggtgg tgtcacagga agagat 26

<210> 123

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<213> Artificial sequence

<400> 123

cccctgccac tgccc 15

<210> 124

<211> 17

<212> DNA

<213> Artificial sequence

<400> 124

cttggcctta cctggat 17

<210> 125

<211> 19

<212> DNA

<213> Artificial sequence

<400> 125

actgctgccc atcacccac 19

<210> 126

<211> 19

<212> DNA

<213> Artificial sequence

<400> 126

gggtggctag gtgtagggg 19

<210> 127

<211> 21

<212> DNA

<213> Artificial sequence

<400> 127

tcgacgaccg agtgtgtctt c 21

<210> 128

<211> 16

<212> DNA

<213> Artificial sequence

<400> 128

gttgggaacg cggccc 16

<210> 129

<211> 26

<212> DNA

<213> Artificial sequence

<400> 129

ttgttaagta atttgttatg ggttcc 26

Claims

1. A kit for detecting gene variation related to medication guidance of human depression treatment drugs is characterized in that the kit can simultaneously type 43 gene loci, the kit comprises 43 pairs of amplification primers for amplifying 43 gene fragments and 43 extension primers for identifying mutation of the 43 gene fragments, and the sequences of the 43 gene loci, the 43 pairs of amplification primers and the 43 extension primers are specifically shown as follows:

gene locus: rs1800544, variant: g > C, upstream primer 5 '-3': SEQ ID NO.1, downstream primer 5 '-3': SEQ ID NO.2, 5 '-3' of the extension primer: SEQ ID No. 87;

gene locus: rs12720461, variant: c > T, upstream primer 5 '-3': SEQ ID NO.3, downstream primer 5 '-3': SEQ ID NO.4, extension primers 5 '-3': SEQ ID No. 88;

gene locus: rs5030655, variant form: delA, upstream primer 5 '-3': SEQ ID NO.5, downstream primer 5 '-3': SEQ ID NO.6, extension primers 5 '-3': SEQ ID No. 89;

gene locus: rs16947, variant: g > A, upstream primer 5 '-3': SEQ ID NO.7, SEQ ID NO.8, extension primers 5 '-3': SEQ ID No. 90;

gene locus: rs2279343, variant: a > G, upstream primer 5 '-3': SEQ ID NO.9, downstream primer 5 '-3': SEQ ID NO.10, extension primers 5 '-3': SEQ ID No. 91;

gene locus: rs28371725, variant: c > T, upstream primer 5 '-3': SEQ ID NO.11, downstream primer 5 '-3': SEQ ID NO.12, extension primers 5 '-3': SEQ ID No. 92;

gene locus: rs1080985, variant: g > C, upstream primer 5 '-3': SEQ ID NO.13, downstream primer 5 '-3': SEQ ID NO.14, extension primers 5 '-3': SEQ ID No. 93;

gene locus: rs2032582, variant form: a is more than C/T, and the upstream primer is 5 '-3': SEQ ID NO.15, downstream primer 5 '-3': SEQ ID NO.16, extension primers 5 '-3': SEQ ID No. 94;

gene locus: rs67666821, variant: insT, upstream primer 5 '-3': SEQ ID NO.17, downstream primer 5 '-3': SEQ ID NO.18, extension primers 5 '-3': SEQ ID No. 95;

gene locus: rs774671100, variant form: insA, upstream primer 5 '-3': SEQ ID NO.19, downstream primer 5 '-3': SEQ ID NO.20, extension primers 5 '-3': SEQ ID No. 96;

gene locus: rs1160351, variant: a > C, upstream primer 5 '-3': SEQ ID NO.21, downstream primer 5 '-3': SEQ ID NO.22, extension primers 5 '-3': SEQ ID No. 97;

gene locus: rs4713916, variant: a > G, upstream primer 5 '-3': SEQ ID NO.23, downstream primer 5 '-3': SEQ ID NO.24, extension primers 5 '-3': SEQ ID number 98;

gene locus: rs28399499, variant: t is more than C, and the upstream primer is 5 '-3': SEQ ID NO.25, downstream primer 5 '-3': SEQ ID NO.26, extension primers 5 '-3': SEQ ID No. 99;

gene locus: rs5443, variant: c > T, upstream primer 5 '-3': SEQ ID NO.27, downstream primer 5 '-3': SEQ ID NO.28, extension primers 5 '-3': SEQ ID No. 100;

gene locus: rs963468, variant: g > A, upstream primer 5 '-3': SEQ ID NO.29, downstream primer 5 '-3': SEQ ID NO.30, extension primers 5 '-3': SEQ ID No. 101;

gene locus: rs28371706, variants: g > A, upstream primer 5 '-3': SEQ ID NO.31, downstream primer 5 '-3': SEQ ID NO.32, extension primers 5 '-3': SEQ ID No. 102;

gene locus: rs3892097, variant: c > T, upstream primer 5 '-3': SEQ ID NO.33, downstream primer 5 '-3': SEQ ID NO.34, extension primers 5 '-3': SEQ ID No. 103;

gene locus: rs4646425, variant: c > T, upstream primer 5 '-3': SEQ ID NO.35, downstream primer 5 '-3': SEQ ID NO.36, extension primers 5 '-3': SEQ ID No. 104;

gene locus: rs7997012, variant: a > G, upstream primer 5 '-3': SEQ ID NO.37, downstream primer 5 '-3': SEQ ID NO.38, extension primers 5 '-3': SEQ ID No. 105;

gene locus: rs201377835, variant: c is more than G, and the upstream primer is 5 '-3': SEQ ID NO.39, downstream primer 5 '-3': SEQ ID NO.40, extension primers 5 '-3': SEQ ID No. 106;

gene locus: rs1135835, variant: t is more than C, and the upstream primer is 5 '-3': SEQ ID NO.41, downstream primer 5 '-3': SEQ ID NO.42, extension primers 5 '-3': SEQ ID No. 107;

gene locus: rs2228479, variant: g is more than A/C, and the upstream primer is 5 '-3': SEQ ID NO.43, downstream primer 5 '-3': SEQ ID NO.44, extension primers 5 '-3': SEQ ID No. 108;

gene locus: rs34223104, variant form: t is more than C, and the upstream primer is 5 '-3': SEQ ID NO.45, downstream primer 5 '-3': SEQ ID NO.46, extension primers 5 '-3': SEQ ID No. 109;

gene locus: rs1135840, variant: c is more than G, and the upstream primer is 5 '-3': SEQ ID NO.47, downstream primer 5 '-3': SEQ ID NO.48, 5 '-3' of the extension primer: SEQ ID No. 110;

gene locus: rs3211371, variant: c > T, upstream primer 5 '-3': SEQ ID NO.49, downstream primer 5 '-3': SEQ ID NO.50, extension primers 5 '-3': SEQ ID No. 111;

gene locus: rs1065852, variant: g > A, upstream primer 5 '-3': SEQ ID NO.51, downstream primer 5 '-3': SEQ ID NO.52, extension primers 5 '-3': SEQ ID No. 112;

gene locus: rs2742417, variant: c is more than G/T, and the upstream primer is 5 '-3': SEQ ID NO.53, downstream primer 5 '-3': SEQ ID NO.54, extension primers 5 '-3': SEQ ID No. 113;

gene locus: rs8192709, variant: c > T, upstream primer 5 '-3': SEQ ID NO.55, downstream primer 5 '-3': SEQ ID NO.56, extension primers 5 '-3': SEQ ID No. 114;

gene locus: rs5030867, variant: t is more than G, and the upstream primer is 5 '-3': SEQ ID NO.57, downstream primer 5 '-3': SEQ ID NO.58, extension primers 5 '-3': SEQ ID No. 115;

gene locus: rs2069514, variant: g > A, upstream primer 5 '-3': SEQ ID NO.59, downstream primer 5 '-3': SEQ ID NO.60, 5 '-3' of the extension primer: SEQ ID No. 116;

gene locus: rs2235040, variant form: c > T, upstream primer 5 '-3': SEQ ID NO.61, downstream primer 5 '-3': SEQ ID NO.62, extension primers 5 '-3': SEQ ID No. 117;

gene locus: rs3745274, variant: g > T, upstream primer 5 '-3': SEQ ID NO.63, downstream primer 5 '-3': SEQ ID NO.64, extension primers 5 '-3': SEQ ID No. 118;

gene locus: rs35742686, variant: delT, upstream primer 5 '-3': SEQ ID NO.65, downstream primer 5 '-3': SEQ ID NO.66, extension primers 5 '-3': SEQ ID No. 119;

gene locus: rs12248560, variant: c > T, upstream primer 5 '-3': SEQ ID NO.67, downstream primer 5 '-3': SEQ ID NO.68, extension primers 5 '-3': SEQ ID No. 120;

gene locus: rs762551, variant: c is more than A, and the upstream primer is 5 '-3': SEQ ID NO.69, downstream primer 5 '-3': SEQ ID NO.70, extension primers 5 '-3': SEQ ID No. 121;

gene locus: rs1045642, variant: a > G, upstream primer 5 '-3': SEQ ID NO.71, downstream primer 5 '-3': SEQ ID NO.72, extension primers 5 '-3': SEQ ID No. 122;

gene locus: rs5030862, variant: c > T, upstream primer 5 '-3': SEQ ID NO.73, downstream primer 5 '-3': SEQ ID NO.74, extension primers 5 '-3': SEQ ID No. 123;

gene locus: rs4986893, variant form: g > A, upstream primer 5 '-3': SEQ ID NO.75, downstream primer 5 '-3': SEQ ID NO.76, 5 '-3' of the extension primer: SEQ ID No. 124;

gene locus: rs5030865, variant: c > T, upstream primer 5 '-3': SEQ ID NO.77, downstream primer 5 '-3': SEQ ID NO.78, extension primers 5 '-3': SEQ ID No. 125;

gene locus: rs2069526, variant: t is more than G, and the upstream primer is 5 '-3': SEQ ID NO.79, downstream primer 5 '-3': SEQ ID NO.80, extension primers 5 '-3': SEQ ID No. 126;

gene locus: rs6295, variant: c is more than G, and the upstream primer is 5 '-3': SEQ ID NO.81, downstream primer 5 '-3': SEQ ID NO.82, extension primers 5 '-3': SEQ ID No. 127;

gene locus: rs1081003, variant: g > A, upstream primer 5 '-3': SEQ ID NO.83, downstream primer 5 '-3': SEQ ID NO.84, extension primers 5 '-3': SEQ ID No. 128;

gene locus: rs4244285, variant: g > A, upstream primer 5 '-3': SEQ ID NO.85, downstream primer 5 '-3': SEQ ID NO.86, extension primers 5 '-3': SEQ ID No. 129;

wherein the molar concentration of the amplification primers SEQ ID No. 1-SEQ ID No.2, SEQ ID No. 9-SEQ ID No.10 in the reaction system is 0.4-0.6 mu M, SEQ ID No. 3-SEQ ID No.8, SEQ ID No. 11-SEQ ID No.18, SEQ ID No. 21-SEQ ID No.50, SEQ ID No. 53-SEQ ID No.58, SEQ ID No. 61-SEQ ID No.68, SEQ ID No. 71-SEQ ID No.76, SEQ ID No. 79-SEQ ID No.80 in the reaction system is 0.2-0.4 mu M, SEQ ID No. 19-SEQ ID No.20, SEQ ID No. 51-SEQ ID No.52, SEQ ID No. 59-SEQ ID No.60, SEQ ID No. 81-SEQ ID No.82 in the reaction system is 0.4-0.8 mu M, SEQ ID No. 69-SEQ ID No.70, and SEQ ID No.85 in the reaction system is 0.4-0.6 mu M, the molar concentration of SEQ ID No. 77-SEQ ID No.78 in the reaction system is 0.8-1.4 mu M, and the molar concentration of SEQ ID No. 83-SEQ ID No.84 in the reaction system is 0.6-1.2 mu M.

2. The kit according to claim 1, wherein the reaction system of PCR amplification of the kit is as follows:

PCR buffer 2. mu.L

0.48-0.52 mu L of amplification primer mixed solution

Taq enzyme 1U

DNA template 2. mu.L

Make up to 5 μ L with water.

3. The kit of claim 1, further comprising an SAP reaction system, the SAP reaction system comprising:

SAP buffer 0.17. mu.L

SAP enzyme 0.5U

The volume of pure water is made up to 2 μ L.

4. The kit of claim 1, wherein the kit is for detection using a time-of-flight mass spectrometer.