CN113584151B

CN113584151B - Composite amplification system and kit for antipsychotic individualized medication related genotyping detection

Info

Publication number: CN113584151B
Application number: CN202110810688.XA
Authority: CN
Inventors: 张奇
Original assignee: Beijing Yuewei Gene Technology Co ltd
Current assignee: Beijing Yuewei Gene Technology Co ltd
Priority date: 2021-07-17
Filing date: 2021-07-17
Publication date: 2022-03-18
Anticipated expiration: 2041-07-17
Also published as: CN113584151A

Abstract

The invention discloses an anti-mental individual medication related genotyping composite amplification system and a detection kit, wherein the amplification system and the kit can simultaneously type 13 sites through a tubular reaction, and can realize the direct amplification of blood, DNA and blood card samples; compared with methods such as fluorescence quantification and the like, the method has the advantages of comprehensive sites, simple and convenient operation, high specificity, high sensitivity, strong reliability, low cost and batch detection capability.

Description

Composite amplification system and kit for antipsychotic individualized medication related genotyping detection

Technical Field

The invention belongs to the technical field of clinical molecular detection, and particularly relates to an amplification detection technology combining multiple fluorescence allele specific PCR with capillary electrophoresis.

Background

Disease burden studies conducted worldwide by the world health organization and world banks show that neuropsychiatric disorders account for 10.4% of the total burden of disease, while psychiatric disorders account for 7.4% of the total burden. In 2019, the first main results of the Chinese mental health survey published by the department of medicine of Beijing university are published as "Lancet & psychiatry", the study is the first national epidemiological survey of mental disorders in China, and the results of the survey on 3.2 million people in 31 provinces in China show that the prevalence rate of anxiety disorders is the highest among seven types of mental disorders such as mood disorders, anxiety disorders, schizophrenia and the like, and is 4.98%.

Currently, drug therapy is the main clinical means of mental illness, and it is counted that in FDA approved drug specifications, the first three recommended biomarkers for pharmacogenomic detection are anti-tumor drugs, psychotropic drugs, and cardiovascular drugs, respectively. Moreover, a great deal of research and clinical data has also shown the criticality and necessity of individual pharmaceutical development in the psychiatric department. Therefore, with the intensive pharmacogenomics research, gene polymorphism, in vivo metabolic enzyme and receptor transporter mutation related to the antipsychotic drugs may be important factors influencing the differences of treated individuals, and the gene detection for guiding the clinical treatment of the psychotropic drugs becomes a new means.

The mental drugs mainly play a role by acting on neurotransmitter receptors or related transporters, and the action target sites of most mental drugs are located in the brain, wherein the action target sites comprise serotonin receptors, dopamine receptors, glutamic acid receptors, serotonin transporters and the like. With the intensive research of pharmacogenomics and the research on the relationship between the gene polymorphism of enzymes related to the absorption, distribution, metabolism and clearance process of drugs in vivo (such as cytochrome enzyme P450 superfamily, ABC transporter family and the like) and the protein related to the action mechanism of drugs (such as the gene polymorphism of target protein dopamine receptor, serotonin receptor and serotonin transporter and the like), the gene polymorphism, the in vivo metabolic enzyme and the receptor transporter related to the antipsychotic drugs can be important factors influencing the differences of treated individuals.

At present, methods for detecting gene polymorphism are many, and in recent years, many new SNP typing methods and techniques such as Denaturing High Pressure Liquid Chromatography (DHPLC), primer extension-coupled time of flight mass spectrometry (MALDI-TOF MS), dynamic allele specific hybridization (dynamic allele specific hybridization) and gene chip method, TaqMan probe technique, pyrosequencing technique, etc. have been established in the market. The sensitivity and the flux of the compounds are greatly improved compared with those of the prior method, so that the compounds are more and more widely applied to forensic detection. Although these techniques can accomplish detection of SNPs, they are also somewhat deficient in application. Some detection processes are complicated and require restriction enzyme digestion; some require two PCR amplification reactions: some new technologies have the advantages of high flux, easy automation and the like, but require expensive instruments and equipment; and some techniques have poor repeatability, difficult interpretation of results, few detection sites, low detection flux, long detection period, high detection cost and the like.

Compared with the limitations of the gene polymorphism technologies, the genotyping amplification detection technology combining multiple fluorescence PCR with a capillary electrophoresis platform is established, and the platform not only combines various advantages of the technologies, but also has the characteristics of simple operation, low cost, high flux, short period, good specificity, high sensitivity, easy type judgment and the like. The invention is provided in view of the above.

Disclosure of Invention

The invention aims to find a multiplex amplification system for detecting polymorphism of genes related to anti-mental disease personalized medicine and a using method thereof. The system has the detection characteristics of simple operation, strong specificity, high sensitivity, high flux, strong reliability and low cost. In order to achieve the above purpose, the invention specifically provides the following technical scheme:

the invention firstly provides an anti-mental disease individualized medication related genotyping composite amplification system, which is characterized in that the amplification system comprises a primer combination for amplifying 13 SNP sites, wherein the SNP sites comprise:

the 2137G > A site of the ANKK1 gene;

CYP1A2 gene 5732C > A site;

DRD2 gene 4651A > G site;

the 324497C > G site of the HTR2C gene;

the 57882787C > A site of the MC4R gene;

CYP2D6 x 2rs1135840 gene 1457G > C site;

CYP2D6 × 2rs16947 gene 886C > T site;

CYP2D6 x 3 gene 775delA site;

CYP2D6 x 4 gene 506-1G > a site;

CYP2D6 x 6 gene 454delT site;

CYP2D6 x 10 gene 100C > T site;

CYP2D6 x 14 gene 1758G > a site;

CYP2D6 x 35 gene-1584C > G site.

Further, the primer sequence is shown as SEQ ID NO.1-39, and specifically comprises the following steps:

the 2137G > A site of the ANKK1 gene:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-TGCCCTCTAGGAAGGACATGATG-3' (SEQ ID NO. 3);

CYP1A2 gene 5732C > A site:

forward wild type primer: 5' -CTCCATCTACCATGCGTCCTAG-3’(SEQ ID NO.4)，

Forward mutant primer:

reverse common primer: 5'-GGACTCTTTGGTACAATACCCAGCATG-3' (SEQ ID NO. 6);

DRD2 gene 4651A > G site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-CGGACCTCTTCCAACACCTCCTCT-3' (SEQ ID NO. 9);

HTR2C gene 324497C > G site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-GGCTAGAGACTCAATCTGGGGAATTTG-3' (SEQ ID NO. 12);

MC4R Gene 57882787C > A site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-AGGATGTATGAGCTCTACCCTGTGGT-3' (SEQ ID NO. 15);

CYP2D6 x 2(rs1135840) gene 1457G > C site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-CGTACCCCTGTCTCAAATGCGGCCA-3' (SEQ ID NO. 18);

CYP2D6 × 2(rs16947) gene 886C > T site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-CCTGCACTGTTTCCCAGATGGGCT-3' (SEQ ID NO. 21);

CYP2D6 x 3 gene 775delA site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-CTCTGGGCAAGGAGAGAGGGTGGAG-3' (SEQ ID NO. 24);

CYP2D6 x 4 gene 506-1G > site a:

forward wild type primer:

forward mutant primer: 5' -CCCCTTACCCGCATCTCCCACCCCTAA-3’(SEQ ID NO.26)，

Reverse common primer: 5'-TATGCAAATCCTGCTCTTCCGAGGCCC-3' (SEQ ID NO. 27);

CYP2D6 × 6 gene 454delT site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-GGGTGGTGGATGGTGGGGCTA-3' (SEQ ID NO. 30);

CYP2D6 x 10 gene 100C > T site:

forward wild type primer: 5' -GGCAGTGGCAGGGGGCCTGGTCG-3’(SEQ ID NO.31)，

Forward mutant primer:

reverse common primer: 5'-CCCAATGGGCAGTGAGGCAGCCAT-3' (SEQ ID NO. 33);

CYP2D6 × 14 gene 1758G > a site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-GGGTGGTGGATGGTGGGGCTA-3' (SEQ ID NO. 36);

CYP2D6 x 35 gene-1584C > G site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-GTCCAGAGCTCGGCAGCTGCCCTCCCA-3' (SEQ ID NO. 39).

Further, the amplification system also comprises the following primers:

for sex reference sites:

a forward primer: 5'-CCCTGGGCTCTGTAAAGAATAG-3' (SEQ ID NO.40),

reverse primer: 5'-ATCAGAGCTTAAACTGGGAAGCTG-3' (SEQ ID NO. 41);

for reference gene site 1:

a forward primer: 5'-GTGGTGTCCCAGATAATCTGTAC-3' (SEQ ID NO.42),

reverse primer: 5'-GGTGAATAACTCCAAATACTCC-3' (SEQ ID NO. 43);

for reference gene site 2:

a forward primer: 5'-AGGCTCTAGCAGCAGCTCATG-3' (SEQ ID NO.44),

reverse primer: 5'-CTGGAAATGACACTGCTACAACTC-3' (SEQ ID NO. 45).

Further, the system is a multiplex fluorescent allele-specific PCR system.

Further, the primer is added with a modification or a normal base is replaced by a modified base, wherein the modification is a fluorescent group modification, a phosphorylation modification, a thiophosphorylation modification, a locked nucleic acid modification or a peptide nucleic acid modification.

Further, the 13 SNP sites and the 3 control gene sites are respectively marked by two colors of fluorescent labels, the same fluorescent label is regarded as the same group, and the two groups are respectively: a first group of MC4R gene g.57882787C > A site, CYP2D6 x 2 gene c.886C > T site, CYP2D6 x 35 gene c. -1584C > G site, CYP1A2 gene g.5732C > A site, CYP2D6 x 4 gene c.506-1G > A site, HTR2C gene g.324497C > G site, D5S818 site; a second group of Amel locus, C.2137G > A locus of ANKK1 gene, C.100C > T locus of CYP2D6 x 10 gene, G.4651A > G locus of DRD2 gene, C.454delT locus of CYP2D6 x 6 gene, C.505G > A locus of CYP2D6 x 14 gene, C.1457G > C locus of CYP2D6 x 2 gene, C.775delA locus of CYP2D6 3 gene, and Th01 locus.

Further, the 13 SNP sites and the 3 human genome internal reference genes are respectively marked by two colors of fluorescent labels, and the same fluorescent labels are regarded as the same group (as shown in Table 1).

TABLE 1 list of test sites

Note: wherein, the sequence of the two groups of sites from top to bottom is the sequence of fragment arrangement from small to large in the actual detection result graph.

Further, the first group is FAM fluorescent labels, and the second group is HEX fluorescent labels.

Further, the fluorescent markers of the 13 SNP sites are positioned at the 5 'end of the common primer, and the fluorescent marker of the reference gene is positioned at the 5' end of the forward primer.

Further, in the above system, the forward wild-type primer: normal phase mutant primer: reverse common primer 1: 1: 1.5 mixing in proportion; internal reference forward primer: reverse primer 1: mixing at a ratio of 1.

Further, the detection is carried out by capillary electrophoresis, and the amplification is carried out by multiplex fluorescence allele specific PCR amplification.

The invention also provides a kit for detecting the polymorphism of the gene related to the antipsychotic personalized medicine, which is characterized by comprising any one of the amplification systems.

Further, the kit also comprises the following components: hot start DNA Taq enzyme, UDG enzyme, 2 XPCR amplification buffer, quality control product, internal standard ROX500 and the like.

The detection kit provided by the invention adopts a method of combining multiple allele-specific PCR (ASPCR) with quantitative fluorescence PCR (QF-PCR) to amplify target sites, and detects amplified products through capillary electrophoresis to complete the detection of target site typing. Three primers are arranged for each detection site, and a specific primer with different lengths and a fluorescence-labeled downstream primer are respectively arranged for two types of typing. Each specific primer can only be combined with a DNA template of a corresponding genotype and amplified. After PCR amplification and capillary electrophoresis detection are completed, whether a specific genotype exists at a specific site of the sample can be judged through the existence of specific fluorescent markers and amplification products with specific lengths.

The invention also provides application of the composite amplification system in preparing an antipsychotic personalized medicine related gene polymorphism detection kit.

The invention also provides a method for detecting the polymorphism of the gene related to the antipsychotic personalized medicine, which is characterized by comprising the step of amplifying a sample by using the multiplex amplification system; the method specifically comprises the following steps: PCR amplification, detection of amplification products by a genetic analyzer, data analysis, judgment of detection results and the like.

The method is used for detecting the product based on an ABI generation sequencing platform, and has the characteristics of high detection sensitivity, high detection resolution, high flux, simplicity, easiness in operation, convenience in automation, professional software for interpreting the result and the like; meanwhile, the invention also has other characteristics: the coverage sites are comprehensive, including 13 SNP sites related to antipsychotic personalized medication, and an internal reference site for sex determination, sample identification and pollution prevention is added, and a UDG-dUTP anti-pollution measure is also added in the system; in addition, the kit of the invention supports direct amplification of blood and blood cards without any treatment, and eliminates the step of extracting DNA.

Compared with the prior art, the invention has the following advantages:

compared with other detection methods on the market, the detection technical method greatly reduces the operation intensity, can realize the result within 3 hours, greatly reduces the cost, covers a plurality of detection sites, provides more patient gene information for clinicians, provides more complete personalized medication reference for psychopaths and benefits patients.

1) The kit disclosed by the invention has comprehensive coverage sites, comprises 13 SNP sites related to antipsychotic personalized medication, realizes specific detection of the same system of multiple SNP sites through multiple optimization of the sites, primers and the system, has extremely high detection limit and sensitivity, can finish detection of a sample under the condition of insufficient DNA (deoxyribonucleic acid) quantity, and has no influence on a detection result;

2) the system is combined with an ABI generation sequencing platform to detect products, and has the characteristics of high detection sensitivity, high resolution, high flux, simplicity, easiness in operation, convenience in automation, professional software for interpreting results and the like;

3) the invention also adds an internal reference site for determining gender, identifying a sample and preventing pollution, and also adds a UDG-dUTP anti-pollution measure into the system;

4) the detection system or the kit established through optimization can support the direct amplification of blood and blood cards without any treatment, and the step of extracting DNA is omitted;

5) compared with other detection methods on the market, the detection technical method greatly reduces the operation intensity, can realize the result within 3 hours, greatly reduces the cost, covers a plurality of detection sites, provides more patient gene information for clinicians, provides more perfect personalized medication reference for depression patients, benefits the patients and is industrially popularized and applied at present.

Drawings

FIG. 1 screening and identification of rs1065852 site primer combinations (fragment analysis results on the left and corresponding sequencing results on the right)

FIG. 2 shows the screening results of the primer combinations at partial sites (top: combination 1; middle: combination 2; bottom: combination 3);

FIG. 3 shows the results of amplification with 0.5ng (top 1), 1.0ng (middle 2), 2.0ng (middle 3) and 5.0ng (bottom 4) as templates in the detection system;

FIG. 4 is a diagram showing the results of amplification of clinical samples by the kit and detection by capillary electrophoresis.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The following terms or definitions are provided only to aid in understanding the present invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.

Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present invention are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.

The terms "about" and "substantially" in the present invention denote an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The term "nucleic acid" or "nucleic acid sequence" in the present invention refers to any molecule, preferably polymeric molecule, comprising units of ribonucleic acid, deoxyribonucleic acid, or analogues thereof. The nucleic acid may be single-stranded or double-stranded. The single-stranded nucleic acid may be a nucleic acid that denatures one strand of a double-stranded DNA. Alternatively, the single-stranded nucleic acid may be a single-stranded nucleic acid not derived from any double-stranded DNA.

The present invention will be described more fully and in detail with reference to the following examples.

Example 1: screening and determining of antipsychotic personalized medicine related gene locus

The invention discloses an embodiment of a detection system and a kit for detecting antidepressant personalized medicine genes, and the system and the kit can simultaneously detect 13 gene polymorphic sites.

Screening of action targets influencing the drug effect of the antipsychotic drug is carried out comprehensively according to clinical evidence grades in an expert guideline (trial) for gene detection of drug metabolizing enzymes and drug action targets, CPIC, a diagnosis and treatment guideline, an FDA drug specification and a pharmGKB database which are prepared by the State medical science expert Committee.

In this example, 8 antipsychotics were screened as follows: chlorpromazine, haloperidol, olanzapine, clozapine, thioridazine, quetiapine, risperidone, aripiprazole, amisulpride, ziprasidone. The action targets of the medicines comprise the following:

the detection sites determined according to the gene targets and the correlation grades determined according to PharmGKB are shown in the following table:

further, by integrating the characteristics of the gene sequences of each site, the size arrangement of the sites in the system and the difficulty condition of system optimization, the finally selected action target genes are as follows: ANKK1, MC4R, CYP2D6, CYP1A2, HTR2C, DRD 2.

The final SNP site information determined according to the action target gene is as follows:

example 2: test for detecting performance of antipsychotic personalized medication related gene locus detection system

In this example, primers are preliminarily designed for a selected gene locus related to antipsychotic personalized medication, each specific primer can only be combined with a DNA template of a corresponding genotype and amplified according to the allele specific PCR principle, and the preliminarily designed primer sequences are as follows:

the 2137G > A site of the ANKK1 gene:

forward wild type primer: 5'-AGCCATCCTCAAAGTGCTGGTCG-3' the flow of the air in the air conditioner,

forward mutant primer: 5'-ACACAGCCATCCTCAAAGTGCTGGTCA-3' the flow of the air in the air conditioner,

reverse common primer: 5'-TGCCCTCTAGGAAGGACATGATG-3', respectively;

CYP1A2 gene 5732C > A site:

forward wild type primer: 5'-CTCCATCTACCATGCGTCCTGG-3' the flow of the air in the air conditioner,

forward mutant primer: 5'-TAAGCTCCATCTACCATGCGTCCTGT-3' the flow of the air in the air conditioner,

reverse common primer: 5'-GGACTCTTTGGTACAATACCCAGCATG-3', respectively;

DRD2 gene 4651A > G site:

forward wild type primer: 5'-CGCTCCCACCCACACCCAGAGTAAT-3' the flow of the air in the air conditioner,

forward mutant primer: 5'-ACTGCGCTCCCACCCACACCCAGAGTAAC-3' the flow of the air in the air conditioner,

reverse common primer: 5'-CGGACCTCTTCCAACACCTCCTCT-3', respectively;

HTR2C gene 324497C > G site:

forward wild type primer: 5'-TTATCTACAGTGACTTTGCTACCCTC-3' the flow of the air in the air conditioner,

forward mutant primer: 5'-CAGCTTATCTACAGTGACTTTGCTACCCTG-3' the flow of the air in the air conditioner,

reverse common primer: 5'-GGCTAGAGACTCAATCTGGGGAATTTG-3', respectively;

MC4R Gene 57882787C > A site:

forward wild type primer: 5'-TTAATTCTGTTGTCATTAGTTCCC-3' the flow of the air in the air conditioner,

forward mutant primer: 5'-TGTCTTAATTCTGTTGTCATTAGTTCCA-3' the flow of the air in the air conditioner,

reverse common primer: 5'-AGGATGTATGAGCTCTACCCTGTGGT-3', respectively;

CYP2D6 x 2(rs1135840) gene 1457G > C site:

forward wild type primer: 5'-TGGTGTCTTTGCTTTCCTGGTGAG-3' the flow of the air in the air conditioner,

forward mutant primer: 5'-ACCATGGTGTCTTTGCTTTCCTGGTGAC-3' the flow of the air in the air conditioner,

reverse common primer: 5'-CGTACCCCTGTCTCAAATGCGGCCA-3', respectively;

CYP2D6 × 2(rs16947) gene 886C > T site:

forward wild type primer: 5'-AGCAGCTTCAATGATGAGAACCTGC-3'

Forward mutant primer: 5'-TGAGAGCAGCTTCAATGATGAGAACCTGT-3'

Reverse common primer: 5'-CCTGCACTGTTTCCCAGATGGGCT-3'

CYP2D6 x 3 gene 775delA site:

forward wild type primer: 5'-GCTGGGCTGGGTCCCAGGTCATCCTG-3'

Forward mutant primer: 5'-GGGGGCTGGGCTGGGTCCCAGGTCATCC G-3'

Reverse common primer: 5'-CTCTGGGCAAGGAGAGAGGGTGGAG-3'

CYP2D6 x 4 gene 506-1G > site a:

forward wild type primer: 5'-TTACCCGCATCTCCCACCCCCAG-3'

Forward mutant primer: 5'-CCCCTTACCCGCATCTCCCACCCCCAA-3'

Reverse common primer: 5'-TATGCAAATCCTGCTCTTCCGAGGCCC-3'

CYP2D6 × 6 gene 454delT site:

forward wild type primer: 5'-GGCGGCCTCCTCGGTCACCCAC-3'

Forward mutant primer: 5'-GGCAGGCGGCCTCCTCGGTCACCCC-3'

Reverse common primer: 5'-GGGTGGTGGATGGTGGGGCTA-3'

CYP2D6 x 10 gene 100C > T site:

forward wild type primer: 5'-GGCAGTGGCAGGGGGCCTGGTGG-3'

Forward mutant primer: 5'-CCCGGGCAGTGGCAGGGGGCCTGGTGA-3'

Reverse common primer: 5'-CCCAATGGGCAGTGAGGCAGCCAT-3'

CYP2D6 × 14 gene 1758G > a site:

forward wild type primer: 5'-TGCCCTTCTGCCCATCACCCACC-3'

Forward mutant primer: 5'-TTTGTGCCCTTCTGCCCATCACCCACT-3'

Reverse common primer: 5'-GGGTGGTGGATGGTGGGGCTA-3'

CYP2D6 x 35 gene-1584C > G site:

forward wild type primer: 5'-CCCAGCTAATTTTGTATTTTTTGTAGAGACCG-3'

Forward mutant primer: 5'-CAATCCCAGCTAATTTTGTATTTTTTGTAGAGACCC-3'

Reverse common primer: 5'-AAGTCCAGAGCTCGGCAGCTG-3'

In order to avoid mutual amplification among primers, coordinate amplification efficiency, improve the peak pattern of a product and facilitate capillary electrophoresis detection, the invention further performs a series of specific base adjustment or modification on all primers.

The following are exemplary:

taking the rs1065852 site primer screening as an example, the site primer introduces different strength mismatches to the 5 'end and the 3' end of the wild type primer and the mutant type primer respectively, the specific screening and identification results are shown in fig. 1, (three primers of the rs1065852 site are mixed in equal proportion, the wild, heterozygous and mutant samples determined by the gold standard are taken as templates for amplification detection, and the results show correctness and no hetero peaks).

In addition, taking the screening of primer combinations at partial sites in the system construction process as an example to illustrate the optimized screening situation of the primer combinations of the system, since there is a certain difference (the difference includes mismatch, position, modification, etc., and is not limited to the modification) between the primer combinations and the final primer combinations in the screening process of all sites, here, only the amplification results of partial combinations (all of which do not contain primers at four gene sites of MC4R, CYP1a2, DRD2, and HTR 2C) are embodied, so as to embody the process of screening primer combinations, as shown in fig. 2, the statistics of the results are as follows:

according to the statistical results shown in fig. 1 and the above table, the effect of the combination 1 and the combination 2 is not good, except the above-described effect, the combination 1 has a condition of a miscellaneous peak as a whole, and the primer combination 3 has the best effect as a whole, so that the subsequent experiment continues to add the site primer on the basis of the primer combination 3 and carries out a screening experiment.

In summary, after the individual screening of each site primer and the combined screening and optimization of each site primer, it was determined that the improved and optimized final primer sequence adopted in this example is as follows:

the 2137G > A site of the ANKK1 gene:

forward wild type primer:

forward mutant primer:

reverse common primer: 5 '-HEX-TGCCCTCTAGGAAGGACATGATG-3' (SEQ ID NO. 3);

CYP1A2 gene 5732C > A site:

forward wild type primer: 5' -CTCCATCTACCATGCGTCCTAG-3’(SEQ ID NO.4)，

Forward mutant primer:

reverse common primer: 5'-GGACTCTTTGGTACAATACCCAGCATG-3' (SEQ ID NO. 6);

DRD2 gene 4651A > G site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-CGGACCTCTTCCAACACCTCCTCT-3' (SEQ ID NO. 9);

HTR2C gene 324497C > G site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-GGCTAGAGACTCAATCTGGGGAATTTG-3' (SEQ ID NO. 12);

MC4R Gene 57882787C > A site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-AGGATGTATGAGCTCTACCCTGTGGT-3' (SEQ ID NO. 15);

CYP2D6 x 2(rs1135840) gene 1457G > C site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-CGTACCCCTGTCTCAAATGCGGCCA-3' (SEQ ID NO. 18);

CYP2D6 × 2(rs16947) gene 886C > T site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-CCTGCACTGTTTCCCAGATGGGCT-3' (SEQ ID NO. 21);

CYP2D6 x 3 gene 775delA site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-CTCTGGGCAAGGAGAGAGGGTGGAG-3' (SEQ ID NO. 24);

CYP2D6 x 4 gene 506-1G > site a:

forward wild type primer:

forward mutant primer: 5' -CCCCTTACCCGCATCTCCCACCCCTAA-3’(SEQ ID NO.26)，

Reverse common primer: 5'-TATGCAAATCCTGCTCTTCCGAGGCCC-3' (SEQ ID NO. 27);

CYP2D6 × 6 gene 454delT site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-GGGTGGTGGATGGTGGGGCTA-3' (SEQ ID NO. 30);

CYP2D6 x 10 gene 100C > T site:

forward wild type primer: 5' -GGCAGTGGCAGGGGGCCTGGTCG-3’(SEQ ID NO.31)，

Forward mutant primer:

reverse common primer: 5'-CCCAATGGGCAGTGAGGCAGCCAT-3' (SEQ ID NO. 33);

CYP2D6 × 14 gene 1758G > a site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-GGGTGGTGGATGGTGGGGCTA-3' (SEQ ID NO. 36);

CYP2D6 x 35 gene-1584C > G site:

forward wild type primer:

forward mutant primer:

reverse common primer: 5'-GTCCAGAGCTCGGCAGCTGCCCTCCCA-3' (SEQ ID NO. 39);

sex reference site:

a forward primer: 5'-CCCTGGGCTCTGTAAAGAATAG-3' (SEQ ID NO.40),

reverse primer: 5'-ATCAGAGCTTAAACTGGGAAGCTG-3' (SEQ ID NO. 41);

reference gene site 1:

a forward primer: 5'-GTGGTGTCCCAGATAATCTGTAC-3' (SEQ ID NO.42),

reverse primer: 5'-GGTGAATAACTCCAAATACTCC-3' (SEQ ID NO. 43);

reference gene site 2:

a forward primer: 5'-AGGCTCTAGCAGCAGCTCATG-3' (SEQ ID NO.44),

reverse primer: 5'-CTGGAAATGACACTGCTACAACTC-3' (SEQ ID NO. 45).

Wherein, the modification changes in the sequence are annotated as follows:

1. - "single underline indicates that each primer was altered 1 to 3 bases at the 3' end-2 to-5 positions of the primer.

Double underline indicates that the sequence of each primer after the 3' end-15 position of the primer was modified, including adding other sequences to the end and changing a partial base sequence.

3. The common primer of all detection sites is subjected to FAM or HEX fluorescent labeling at the 5' end.

4. The control site forward primers are fluorescently labeled with FAM or HEX at the 5' end.

Example 3: antipsychotic personalized medicine related gene locus detection system detection performance test

In the embodiment, the lowest detection limit of the system is tested, 13 gene polymorphism sites are simultaneously detected, three sites for individual identification are added, and interpretation is carried out based on a capillary electrophoresis platform. The specific experimental steps are as follows:

1. preparation of DNA samples

Genomic DNA was extracted using a Tiangen blood/cell/tissue genomic DNA extraction kit (DP304) using a collected fresh peripheral blood sample, and the concentration and purity of DNA were measured using NanoDrop2000(Thermo), and then the genomic DNA was preserved.

The extracted DNA is diluted by concentration gradients which are respectively 0.5ng/ul, 1.0ng/ul, 2ng/ul and 5 ng/ul.

2. Preparation of PCR amplification System

This system comprises primer combinations determined as in example 2, all performed in Biotechnology engineering (Shanghai) Ltd, and all primers were run as forward wild-type primers searched during the experiment: normal phase mutant primer: reverse common primer 1: 1: mixing at a ratio of 1.5; internal reference forward primer: the internal reference reverse primer is 1: 1 ratio, and 10 Xthe primer mix was prepared.

The system also comprises enzyme and PCR amplification buffer solution, wherein the PCR amplification buffer solution contains dATP, dTTP, dCTP, dGTP, dUTP and Mg²⁺And the like, wherein the enzyme comprises a hot start DNA Taq enzyme and a UDG enzyme.

The PCR amplification system was prepared according to the following table, shaken and mixed, and dispensed in tubes of 19ul per tube, based on the number of samples.

Component name	The dosage of each component (ul) of each 20ul system
		PCR amplification buffer	10
10 × primer mix	2
		DNA polymerase (containing UDG enzyme)	0.5
Form panel	1
		Water (W)	6.5
In total	20

3. Adding templates

1ul of DNA sample of each concentration gradient was added to the corresponding PCR reaction tube, and simultaneously, a quality control (quality control: 1ul of quality control product, negative control: 1ul of nucleic acid-free pure water) was set.

4. PCR amplification

Each reaction tube is placed in a reaction tank of a PCR amplification instrument, and a reaction system is set to be 20 ul.

PCR amplification was performed according to the following reaction procedure:

5. capillary electrophoresis detection of the amplified product

Preparing a sample loading mixed solution mixed with a molecular weight internal standard and formamide: (0.5 mul of molecular weight internal standard +8.5 mul of formamide) x detecting the number of samples, and evenly mixing the samples for 10 to 15 seconds by vortex oscillation; dispensing 9 μ L of formamide and internal standard mixture into each well using a pipette; 1ul of the amplification product was added to the mixture of formamide and internal standard and covered with a plate seal. The detection was performed according to the manual procedure of the genetic analyzer user.

6. Data analysis

The relevant files are imported into GeneMapper software, and original data (. fsa file) of the machine under the detector is input to analyze the data.

The results are summarized in the following table:

according to the statistical detection results in the table above, the genotype of each target site can be amplified and detected by the system when four concentration gradient DNAs are used as templates, and the results are consistent, but the overall efficiency of the system is low when 0.5ng of DNAs are used as templates, and the minimum detection limit of the system is finally 1ng (see FIG. 3) in order to ensure the detection rate of the sample after repeated detection.

Therefore, the system has excellent sensitivity, can finish the detection of the sample under the condition of insufficient DNA quantity, has no influence on the detection result, and has the effect beyond the experimental expectation.

Example 4: direct amplification of clinical blood samples and comparison with sequencing results

In this embodiment, the system of the present invention is used to prepare a kit, which is used to directly perform amplification detection on a blood sample, specifically, 1 example of an anticoagulation clinical sample is used as an example to perform amplification detection, and meanwhile, a sequencing method is used to verify the amplification detection result of the kit, so as to formally determine the effectiveness, specificity and accuracy of the primer system and the kit of the present invention.

Detection system

The kit comprises a PCR Master Mix, a quality control product and an internal standard. Wherein the main components of the PCR Master Mix comprise hot start DNA Taq enzyme, UDG enzyme, amplification buffer solution, amplification primers at each site with the proportion determined in example 2, and the like.

Second, detection method

Step 1: PCR amplification reaction

1) PCR premix solution split charging (done in reagent preparation area)

The PCR premix solution (PCR Master Mix) was shaken and mixed, and 3 assays were expected to be performed, each PCR reaction tube being filled with 19 ul.

2) Add template (done in specimen preparation zone)

1ul of blood sample to be detected was added to the corresponding PCR reaction tube, and simultaneously, a quality control (quality control: 1ul of quality control product, negative control: 1ul of nucleic acid-free pure water) was set.

3) PCR amplification (done in the amplification zone)

Each reaction tube was placed in a reaction tank of a PCR amplification apparatus, and a reaction system was set to 20. mu.L.

The PCR machine was set up and PCR amplification was performed according to the reaction program conditions of example 3.

And 2, step 3: detection of amplification products and analysis of data

The procedure was followed in examples 3, 5 and 6.

And 4, step 4: determination of detection result

As shown in the figure, FIG. 4 is a graph of the amplification detection results of clinical samples, in which the wild-type SNP is marked as "Wt" and the mutant type SNP is marked as "Mu". The Amel locus is shown as "XY" for male samples and "X" for female samples.

Thirdly, sequencing by a Sanger method:

according to the finally determined SNP site information, searching the gene sequence of each site, and designing sequencing primers in the regions of at least more than 100bp respectively at the upstream and downstream of the corresponding SNP site (synthesized by Biotechnology engineering (Shanghai) Co., Ltd.). Amplifying fragments of each site of a target sample by using a designed sequencing primer of each site, determining whether an amplification product exists and the size of a target fragment is correct by electrophoresis detection, and then sending the target fragment to a biological engineering (Shanghai) company Limited for Sanger method sequencing.

Fourth, comparison of CE results with sequencing results

And analyzing the obtained clinical sample according to the capillary electrophoresis detection image to obtain the result typing of the amplification detection of the kit. And analyzing the data obtained by sequencing by a sequencing company to obtain the sequencing result typing of each site of the clinical sample.

The results of the site typing comparisons are given in the following table:

TABLE 3 sample test results

Therefore, the kit detection result typing and the sequencing result typing of 13 polymorphic sites of 6 genes of a clinical blood sample can be respectively obtained, and meanwhile, the detection result shows that the detection result of the kit on the clinical blood sample is consistent with the amplification sequencing result. Therefore, the detection result of the kit is reliable in comparison with the gold standard, and the individual medication of the drugs used by the patients is carried out according to the detected genotyping and the corresponding clinical reference information

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

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Claims

1. An anti-psychotic disease personalized medicine related genotyping multiplex amplification system, wherein the amplification system comprises a primer combination for amplifying 13 SNP sites, and the SNP sites comprise:

the 2137G > A site of the ANKK1 gene;

CYP1A2 gene 5732C > A site;

DRD2 gene 4651A > G site;

the 324497C > G site of the HTR2C gene;

the 57882787C > A site of the MC4R gene;

CYP2D6 x 2rs1135840 gene 1457G > C site;

CYP2D6 × 2rs16947 gene 886C > T site;

CYP2D6 x 3 gene 775delA site;

CYP2D6 x 4 gene 506-1G > a site;

CYP2D6 x 6 gene 454delT site;

CYP2D6 x 10 gene 100C > T site;

CYP2D6 x 14 gene 1758G > a site;

CYP2D6 x 35 gene-1584C > G site;

the primer sequence is shown as SEQ ID NO.1-39, and specifically comprises the following steps:

the 2137G > A site of the ANKK1 gene:

forward wild type primer: the amino acid sequence shown in SEQ ID NO.1,

forward mutant primer: the amino acid sequence shown in SEQ ID NO.2,

reverse common primer: SEQ ID No. 3;

CYP1A2 gene 5732C > A site:

forward wild type primer: the amino acid sequence shown in SEQ ID NO.4,

forward mutant primer: the amino acid sequence shown in SEQ ID NO.5,

reverse common primer: SEQ ID No. 6;

DRD2 gene 4651A > G site:

forward wild type primer: the amino acid sequence shown in SEQ ID NO.7,

forward mutant primer: the amino acid sequence shown in SEQ ID NO.8,

reverse common primer: SEQ ID No. 9;

HTR2C gene 324497C > G site:

forward wild type primer: the amino acid sequence shown in SEQ ID NO.10,

forward mutant primer: the amino acid sequence shown in SEQ ID NO.11,

reverse common primer: SEQ ID No. 12;

MC4R Gene 57882787C > A site:

forward wild type primer: the amino acid sequence shown in SEQ ID NO.13,

forward mutant primer: the amino acid sequence shown in SEQ ID NO.14,

reverse common primer: SEQ ID No. 15;

CYP2D6 x 2rs1135840 gene 1457G > C site:

forward wild type primer: the amino acid sequence shown in SEQ ID NO.16,

forward mutant primer: the amino acid sequence shown in SEQ ID NO.17,

reverse common primer: SEQ ID No. 18;

CYP2D6 × 2rs16947 gene 886C > T site:

forward wild type primer: the amino acid sequence shown in SEQ ID NO.19,

forward mutant primer: the amino acid sequence shown in SEQ ID NO.20,

reverse common primer: SEQ ID No. 21;

CYP2D6 x 3 gene 775delA site:

forward wild type primer: the amino acid sequence shown in SEQ ID NO.22,

forward mutant primer: the amino acid sequence shown in SEQ ID NO.23,

reverse common primer: SEQ ID No. 24;

CYP2D6 x 4 gene 506-1G > site a:

forward wild type primer: the amino acid sequence shown in SEQ ID NO.25,

forward mutant primer: the amino acid sequence shown in SEQ ID NO.26,

reverse common primer: SEQ ID No. 27;

CYP2D6 × 6 gene 454delT site:

forward wild type primer: the amino acid sequence shown in SEQ ID NO.28,

forward mutant primer: the amino acid sequence shown in SEQ ID NO.29,

reverse common primer: SEQ ID No. 30;

CYP2D6 x 10 gene 100C > T site:

forward wild type primer: the amino acid sequence shown in SEQ ID NO.31,

forward mutant primer: the amino acid sequence shown in SEQ ID NO.32,

reverse common primer: SEQ ID No. 33;

CYP2D6 × 14 gene 1758G > a site:

forward wild type primer: the amino acid sequence shown in SEQ ID NO.34,

forward mutant primer: the amino acid sequence shown in SEQ ID NO.35,

reverse common primer: SEQ ID No. 36;

CYP2D6 x 35 gene-1584C > G site:

forward wild type primer: the amino acid sequence shown in SEQ ID NO.37,

forward mutant primer: the amino acid sequence shown in SEQ ID NO.38,

reverse common primer: SEQ ID No. 39;

the amplification system also comprises the following primers:

for sex reference sites:

a forward primer: the amino acid sequence shown in SEQ ID NO.40,

reverse primer: SEQ ID No. 41;

for reference gene site 1:

a forward primer: the amino acid sequence shown in SEQ ID NO.42,

reverse primer: SEQ ID No. 43;

for reference gene site 2:

a forward primer: the amino acid sequence shown in SEQ ID NO.44,

reverse primer: SEQ ID No. 45;

the system is a multiplex fluorescence allele specific PCR system;

the 13 SNP loci and the 3 internal reference loci are respectively marked by fluorescence of two colors, the same fluorescence is regarded as the same group, and the two groups are respectively: a first group of MC4R gene g.57882787C > A site, CYP2D6 x 2 gene c.886C > T site, CYP2D6 x 35 gene c. -1584C > G site, CYP1A2 gene g.5732C > A site, CYP2D6 x 4 gene c.506-1G > A site, HTR2C gene g.324497C > G site, D5S818 site; a second group of Amel locus, ANKK1 gene c.2137G > A locus, CYP2D6 x 10 gene c.100C > T locus, DRD2 gene g.4651A > G locus, CYP2D6 x 6 gene c.454delT locus, CYP2D6 x 14 gene c.505G > A locus, CYP2D6 x 2 gene c.1457G > C locus, CYP2D6 x 3 gene c.775delA locus, Th01 locus; the first set of fluorescent labels is FAM and the second set of fluorescent labels is HEX.

2. The amplification system according to claim 1, wherein a modification is added to the primer or a normal base is substituted with a modified base, and the modification is a fluorophore modification, a phosphorylation modification, a phosphorothioate modification, a locked nucleic acid modification, or a peptide nucleic acid modification.

3. An antipsychotic personalized medicine related gene polymorphism detection kit, which is characterized by comprising the amplification system of any one of claims 1-2.

4. The test kit according to claim 3, wherein the kit further comprises the following components: hot start DNA Taq enzyme, UDG enzyme and 2 × PCR amplification buffer, quality control and internal standard ROX 500.

5. Use of the multiplex amplification system of any one of claims 1-2 in the preparation of a kit for detecting polymorphisms in genes associated with antipsychotic personalized medicine.