CN113584147A - Gene polymorphism detection kit for guiding medication of psychosis - Google Patents

Gene polymorphism detection kit for guiding medication of psychosis Download PDF

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CN113584147A
CN113584147A CN202110666035.9A CN202110666035A CN113584147A CN 113584147 A CN113584147 A CN 113584147A CN 202110666035 A CN202110666035 A CN 202110666035A CN 113584147 A CN113584147 A CN 113584147A
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primer
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郭建成
占闽宁
冯帅升
朱丹丹
孙晓晓
张悦
史健翔
许红恩
刘海芳
薛夏
柳丹华
秦亚平
薛滨雨
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Henan Shenyou Medical Laboratory Co ltd
Zhengzhou University
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Zhengzhou University
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Abstract

The invention discloses a gene polymorphism detection kit for guiding medication of mental diseases, which comprises a specific primer group and a fluorescent probe group for detecting rs1065852 site of CYP2D6 gene, rs1414334 site of HTR2C gene, rs1800497 site of ANKK1 gene, rs762551 site of CYP1A2 gene, rs489693 site of MC4R gene, and rs1799978 site of DRD2 gene. The invention can simultaneously and accurately detect the polymorphism of a plurality of sites of a plurality of genes related to psychotropic drugs by utilizing the primer group and the fluorescent probe group and specifically amplifying the template DNA by using the fluorescent PCR amplification technology; in addition, ARMS primer design is adopted, mismatched bases are introduced to improve the specificity of primer amplification, the sensitivity is high, the minimum 10ng of DNA initial amount can be used for carrying out accurate genotyping on human template DNA; the detection speed is fast, and is efficient: the PCR reaction can be completed in less than 1h, and the cost is low: the required reagent consumables are clinically common reagents, the cost is low, and the clinical popularization is facilitated.

Description

Gene polymorphism detection kit for guiding medication of psychosis
Technical Field
The invention relates to the technical field of biological medicines, in particular to a gene polymorphism detection kit for guiding medication of mental diseases.
Background
At present, the clinical treatment method and means of mental diseases are still mainly drug therapy, schizophrenia is one of the most serious mental diseases, and patients with schizophrenia generally need to take medicines for a long time or even take medicines for life, namely, the patients have high drug dependence and viscosity. Therefore, when the mental drugs are clinically applied to treat and save patients, adverse reactions caused by the mental drugs cannot be ignored. Clinical practice shows that in different stages of metabolism, transportation, receptor and the like of the medicine, when different individuals receive the same medicine for treatment, the treatment effect, the generated toxic and side effect, the tolerance to the medicine and the like of the different individuals have obvious differences. The gene polymorphism is the essential cause of individual differences in drug reactions from person to person. Therefore, the detection of the genotype of the gene related to the psychotropic drugs is beneficial to improving the psychotropic drug treatment effect of many patients, and particularly has important significance for comprehensively measuring the drug efficacy and adverse reactions of the drugs and realizing individual medication for the patients with pharmacokinetic abnormality.
At present, the detection of genes related to psychotropic drugs mainly comprises a PCR-Sanger sequencing method, a fluorescence quantitative PCR method, a gene chip method and the like, and the traditional conventional Sanger sequencing method is a recognized gold standard for detecting genotyping, but has the factors of long detection period, low detection flux, high cost and the like, and limits the rapid and accurate screening of the genes in a clinical laboratory, so the method is not suitable for the detection of multiple genes and multiple sites; the gene chip method limits the rapid and accurate screening in clinical laboratories due to factors such as higher cost, complex operation and the like; the fluorescence quantitative PCR method has the advantages of high sensitivity, accurate typing, simple and quick operation and the like, and is widely applied to clinical genotyping detection at present.
Disclosure of Invention
Therefore, based on the above background, the present invention provides a gene polymorphism detection kit for guiding medication of psychosis, which can simultaneously and accurately detect polymorphisms at multiple sites of multiple genes related to psychosis drugs, and can effectively shorten detection time and reduce cost, so that medication of psychosis can be better guided, and personalized medication can be better realized.
The technical scheme provided by the invention is as follows:
a gene polymorphism detection kit for guiding medication of mental diseases comprises a specific primer group and a fluorescent probe group for detecting rs1065852 site of CYP2D6 gene, rs1414334 site of HTR2C gene, rs1800497 site of ANKK1 gene, rs762551 site of CYP1A2 gene, rs489693 site of MC4R gene, and rs1799978 site of DRD2 gene.
Further, the primer group comprises two forward primers and a common reverse primer for detecting the rs1065852 locus of the CYP2D6 gene, two forward primers and a common reverse primer for detecting the rs1414334 locus of the HTR2C gene, two forward primers and a common reverse primer for detecting the rs1800497 locus of the ANKK1 gene, two forward primers and a common reverse primer for detecting the rs762551 locus of the CYP1A2 gene, two forward primers and a common reverse primer for detecting the rs489693 locus of the MC4R gene, two forward primers and a common reverse primer for detecting the rs1799978 locus of the DRD2 gene, and a common reverse primer.
Further, the base sequences of the forward primer and the common reverse primer for detecting the rs1065852 locus of the CYP2D6 gene are as follows:
wild-type forward primer: 5'-GCTGGGCTGCACGCTTCC-3' (SEQ ID NO. 1);
mutant forward primer: 5'-GCTGGGCTGCACGCTTCT-3' (SEQ ID NO. 2);
common reverse primers: 5'-ACCTGGTCGAAGCAGTATGGT-3' (SEQ ID NO. 3);
the base sequences of the forward primer and the common reverse primer for detecting the locus rs1414334 of the HTR2C gene are as follows:
wild-type forward primer: 5'-TCTACAGTGACTTTGCTACCTTC-3' (SEQ ID NO. 4);
mutant forward primer: 5'-TCTACAGTGACTTTGCTACCTTG-3' (SEQ ID NO. 5);
common reverse primers: 5'-CATGAGATTGGTGGAAGTTTGT-3' (SEQ ID NO. 6);
the base sequences of the forward primer and the common reverse primer for detecting the rs1800497 site of the ANKK1 gene are as follows:
wild-type forward primer: 5'-CATCCTCAAAGTGCTGGCCG-3' (SEQ ID NO. 7);
mutant forward primer: 5'-CATCCTCAAAGTGCTGGCCA-3' (SEQ ID NO. 8);
common reverse primers: 5'-GCCCTCTAGGAAGGACATGAT-3' (SEQ ID NO. 9);
the base sequences of the forward primer and the common reverse primer for detecting the site rs762551 of the CYP1A2 gene are as follows:
wild-type forward primer: 5'-GGGTGAGCTCTGTGGACC-3' (SEQ ID NO. 10);
mutant forward primer: 5'-GGGTGAGCTCTGTGGACA-3' (SEQ ID NO. 11);
common reverse primers: 5'-GAGGGTTGAGATGGAGACATTC-3' (SEQ ID NO. 12);
the base sequences of the forward primer and the common reverse primer for detecting the rs489693 site of the MC4R gene are as follows:
wild-type forward primer: 5'-CACGCTGTAAACATTTAACAAGCG-3' (SEQ ID NO. 13);
mutant forward primer: 5'-CACGCTGTAAACATTTAACAAGCT-3' (SEQ ID NO. 14);
common reverse primers: 5'-GAAGTCTGCCTACCGGTCTAAA-3' (SEQ ID NO. 15);
the base sequences of the forward primer and the common reverse primer for detecting the locus rs1799978 of the DRD2 gene are as follows:
wild-type forward primer: 5'-CCACCCACACCCAGAGTGAT-3' (SEQ ID NO. 16);
mutant forward primer: 5'-CCACCCACACCCAGAGTGAC-3' (SEQ ID NO. 17);
common reverse primers: 5'-ACCTCTTCCAACACCTCCTCTC-3' (SEQ ID NO. 18).
Further, the fluorescent probe set comprises a CYP2D6-rs1065852 shared probe, an HTR2C-rs1414334 shared probe, an ANKK1-rs1800497 shared probe, a CYP1A2-rs762551 shared probe, an MC4R-rs489693 shared probe and a DRD2-rs1799978 shared probe;
the base sequence of the CYP2D6-rs1065852 common probe is as follows:
5’-FAM-ACATGCAGCAGGTTGCCCAGC-3’-BHQ1(SEQ ID NO.19)；
the base sequence of the HTR2C-rs1414334 common probe is as follows:
5’-FAM-AGAAGCGAGCTAACATTACTCTGGA-3’-BHQ1(SEQ ID NO.20)；
the base sequence of the ANKK1-rs1800497 common probe is as follows:
5’-FAM-CATCATGTCCTTCCTAGAGGGCAA-3’-BHQ1(SEQ ID NO.21)；
the base sequence of the CYP1A2-rs762551 common probe is as follows:
5’-FAM-TAGTCTTTCTGGTATCCAGCTGGGAG-3’-BHQ1(SEQ ID NO.22)；
the base sequence of the MC4R-rs489693 common probe is as follows:
5’-FAM-GTTGCTAGGAAGTCTGCCTACCGGTC-3’-BHQ1(SEQ ID NO.23)；
the base sequence of the DRD2-rs1799978 common probe is as follows:
5’-FAM-ATCCAGCAAGTCAGGCAAGGAGAGG-3’-BHQ1(SEQ ID NO.24)。
further, the kit also comprises a specific primer group of the internal reference gene CFTR and an internal reference gene probe.
Further, the specific primer group of the reference gene comprises a CFTR reference gene forward primer CFTR-F and a CFTR reference gene reverse primer CFTR-R;
the CFTR internal reference gene forward primer CFTR-F: 5'-CCCCTTTTGTAGGAAGTCACC-3' (SEQ ID NO. 25);
the CFTR internal reference gene reverse primer CFTR-R: 5'-GCTGGGTGTAGGAGCAGTGT-3' (SEQ ID NO. 26).
Further, the reference gene probe CFTR-P is a fluorescent probe, and the sequence of the fluorescent probe is as follows:
5’-VIC-TATGACCCGGATAACAAGGAGGAACGC-BHQ1-3’(SEQID NO.27)。
further, the method for using the gene polymorphism detection kit for guiding the medication of the psychosis comprises the following steps:
1) taking a specific primer group and a probe group in the kit;
2) extracting the genome DNA of a sample to be detected by adopting a DNA extraction kit;
3) preparing a PCR reaction system: adding a wild type forward primer and a mutant forward primer of a target gene into each site of each sample respectively by two tubes, wherein the other contents comprise a template DNA, a downstream primer, a fluorescent probe, a fluorescent PCR-mix, an internal reference gene primer, an internal reference gene probe and the like, and simultaneously configuring a positive control reaction system and a negative control reaction system of the corresponding site, wherein the positive control is a plasmid containing a target gene fragment, and the negative control is purified dd water;
4) and (3) PCR reaction: amplifying the prepared reaction system on a real-time fluorescent PCR instrument, wherein the PCR amplification conditions are as follows: at 37 ℃ for 2 min; at 95 ℃ for 1 min; (95 ℃, 15 sec; 60 ℃, 30sec)40 cycles, and the fluorescence signal was collected at 60 ℃ in the second step of the PCR cycle, with detection channels: FAM and VIC.
The invention also provides application of the gene polymorphism detection kit for guiding the psychotropic medication, and the application of the gene polymorphism detection kit in preparing a reagent for guiding the psychotropic medication of a human.
Further, the drugs include, but are not limited to, risperidone, olanzapine, clozapine, aripiprazole, chlorpromazine, fluphenazine, thioridazine, trifluoperazine.
By adopting the technical scheme, the method has the following beneficial effects:
the invention detects two forward primers of CYP2D6 gene rs1065852 site, HTR2C gene rs1414334 site, ANKK1 gene rs1800497 site, CYP1A2 gene rs762551 site, MC4R gene rs489693 site, DRD2 gene rs1799978 site, a common reverse primer and a specific probe of fluorescence labeling, utilizes a fluorescence PCR amplification technology to carry out specific amplification on template DNA, utilizes the specific primer and the probe of an internal reference gene to monitor whether a reaction system has abnormal amplification or amplification inhibition, and analyzes a fluorescence PCR amplification curve corresponding to a corresponding primer reaction to obtain polymorphism results of a plurality of sites of genes related to psychotropic drugs, such as CYP2D6 gene rs1065852 site, HTR2C gene rs1414334 site and the like;
the ARMS primer design is adopted, the specificity of primer amplification is improved by introducing mismatched bases, the sensitivity is high, the minimum 10ng of DNA initial amount is obtained, and the accurate genotyping can be carried out on human template DNA only by a small amount of samples;
the detection speed is fast, and is efficient: the PCR reaction can be completed in less than 1 h;
the cost is low: the required reagent consumables are clinically common reagents, the cost is low, and the clinical popularization is facilitated.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the amplification result of a sample with the CYP2D6-rs1065852 locus genotype GG in example 1 of the present invention;
FIG. 2 shows the amplification result of a sample with the CYP2D6-rs1065852 locus genotype GA in example 1 of the present invention;
FIG. 3 shows the result of amplification of a sample having CYP2D6-rs1065852 locus genotype TT according to example 1 of the present invention;
FIG. 4 shows the result of amplifying a sample with genotype GG at ANKK1-rs1800497 locus in example 1;
FIG. 5 shows the result of amplification of a sample whose ANKK1-rs1800497 locus genotype is GA in example 1 of the present invention;
FIG. 6 shows the result of amplifying the sample with AA at the ANKK1-rs1800497 locus genotype in example 1;
FIG. 7 shows the result of amplifying a sample having CYP1A2-rs762551 site genotype CC according to example 1 of the present invention;
FIG. 8 shows the result of amplifying a sample having the CYP1A2-rs762551 locus genotype CA according to example 1 of the present invention;
FIG. 9 shows the result of amplifying a sample having the genotype at CYP1A2-rs762551 site AA in example 1 of the present invention;
FIG. 10 shows the result of amplification of a sample having a CC genotype at MC4R-rs489693 in example 1;
FIG. 11 shows the result of amplifying a sample of which MC4R-rs489693 locus genotype is CA in example 1;
FIG. 12 shows the result of amplifying a sample with the MC4R-rs489693 locus genotype as AA in example 1 of the present invention;
FIG. 13 shows the result of amplification of a sample with CC genotype at HTR2C-rs1414334 site in example 1;
FIG. 14 shows the result of amplifying a sample with the genotype at the HTR2C-rs1414334 site CG according to example 1 of the present invention;
FIG. 15 shows the result of amplification of a sample with genotype GG at HTR2C-rs1414334 site in example 1;
FIG. 16 shows the result of amplification of a sample with the genotype of DRD2-rs1799978 locus TT in example 1 of the present invention;
FIG. 17 shows the result of amplification of a sample having TC as the genotype at DRD2-rs1799978 locus in example 1 of the present invention;
FIG. 18 shows the result of amplification of a sample with CC genotype at DRD2-rs1799978 locus in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious 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.
In the description of the present invention, "the first feature" and "the second feature" may include one or more of the features. Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature.
The invention is further described below with reference to the accompanying drawings.
Example 1: a gene polymorphism detection kit for guiding medication of mental diseases comprises rs1065852 site of CYP2D6 gene, rs1414334 site of HTR2C gene, rs1800497 site of ANKK1 gene, rs762551 site of CYP1A2 gene, and rs489693 site of MC4R gene
The primer group comprises two forward primers and a common reverse primer for detecting rs1065852 locus of CYP2D6 gene, two forward primers and a common reverse primer for detecting rs1414334 locus of HTR2C gene, two forward primers and a common reverse primer for detecting rs1800497 locus of ANKK1 gene, two forward primers and a common reverse primer for detecting rs762551 locus of CYP1A2 gene, two forward primers and a common reverse primer for detecting rs489693 locus of MC4R gene, two forward primers and a common reverse primer for detecting rs1799978 locus of DRD2 gene and a common reverse primer.
The base sequences of the forward primer and the common reverse primer for detecting the rs1065852 locus of the CYP2D6 gene are as follows:
wild-type forward primer: 5'-GCTGGGCTGCACGCTTCC-3' (SEQ ID NO. 1);
mutant forward primer: 5'-GCTGGGCTGCACGCTTCT-3' (SEQ ID NO. 2);
common reverse primers: 5'-ACCTGGTCGAAGCAGTATGGT-3' (SEQ ID NO. 3);
the base sequences of the forward primer and the common reverse primer for detecting the locus rs1414334 of the HTR2C gene are as follows:
wild-type forward primer: 5'-TCTACAGTGACTTTGCTACCTTC-3' (SEQ ID NO. 4);
mutant forward primer: 5'-TCTACAGTGACTTTGCTACCTTG-3' (SEQ ID NO. 5);
common reverse primers: 5'-CATGAGATTGGTGGAAGTTTGT-3' (SEQ ID NO. 6);
the base sequences of the forward primer and the common reverse primer for detecting the rs1800497 site of the ANKK1 gene are as follows:
wild-type forward primer: 5'-CATCCTCAAAGTGCTGGCCG-3' (SEQ ID NO. 7);
mutant forward primer: 5'-CATCCTCAAAGTGCTGGCCA-3' (SEQ ID NO. 8);
common reverse primers: 5'-GCCCTCTAGGAAGGACATGAT-3' (SEQ ID NO. 9);
the base sequences of the forward primer and the common reverse primer for detecting the site rs762551 of the CYP1A2 gene are as follows:
wild-type forward primer: 5'-GGGTGAGCTCTGTGGACC-3' (SEQ ID NO. 10);
mutant forward primer: 5'-GGGTGAGCTCTGTGGACA-3' (SEQ ID NO. 11);
common reverse primers: 5'-GAGGGTTGAGATGGAGACATTC-3' (SEQ ID NO. 12);
the base sequences of the forward primer and the common reverse primer for detecting the rs489693 site of the MC4R gene are as follows:
wild-type forward primer: 5'-CACGCTGTAAACATTTAACAAGCG-3' (SEQ ID NO. 13);
mutant forward primer: 5'-CACGCTGTAAACATTTAACAAGCT-3' (SEQ ID NO. 14);
common reverse primers: 5'-GAAGTCTGCCTACCGGTCTAAA-3' (SEQ ID NO. 15);
the base sequences of the forward primer and the common reverse primer for detecting the locus rs1799978 of the DRD2 gene are as follows:
wild-type forward primer: 5'-CCACCCACACCCAGAGTGAT-3' (SEQ ID NO. 16);
mutant forward primer: 5'-CCACCCACACCCAGAGTGAC-3' (SEQ ID NO. 17);
common reverse primers: 5'-ACCTCTTCCAACACCTCCTCTC-3' (SEQ ID NO. 18).
The fluorescent probe set comprises a CYP2D6-rs1065852 common probe, an HTR2C-rs1414334 common probe, an ANKK1-rs1800497 common probe, a CYP1A2-rs762551 common probe, an MC4R-rs489693 common probe and a DRD2-rs1799978 common probe;
the base sequence of the CYP2D6-rs1065852 common probe is as follows:
5’-FAM-ACATGCAGCAGGTTGCCCAGC-3’-BHQ1(SEQ ID NO.19)；
the base sequence of the HTR2C-rs1414334 common probe is as follows:
5’-FAM-AGAAGCGAGCTAACATTACTCTGGA-3’-BHQ1(SEQ ID NO.20)；
the base sequence of the ANKK1-rs1800497 common probe is as follows:
5’-FAM-CATCATGTCCTTCCTAGAGGGCAA-3’-BHQ1(SEQ ID NO.21)；
the base sequence of the CYP1A2-rs762551 common probe is as follows:
5’-FAM-TAGTCTTTCTGGTATCCAGCTGGGAG-3’-BHQ1(SEQ ID NO.22)；
the base sequence of the MC4R-rs489693 common probe is as follows:
5’-FAM-GTTGCTAGGAAGTCTGCCTACCGGTC-3’-BHQ1(SEQ ID NO.23)；
the base sequence of the DRD2-rs1799978 common probe is as follows:
5’-FAM-ATCCAGCAAGTCAGGCAAGGAGAGG-3’-BHQ1(SEQ ID NO.24)。
the kit also comprises a specific primer group of the reference gene CFTR and a reference gene probe.
The specific primer group of the internal reference gene comprises a CFTR internal reference gene forward primer CFTR-F and a CFTR internal reference gene reverse primer CFTR-R;
the CFTR internal reference gene forward primer CFTR-F: 5'-CCCCTTTTGTAGGAAGTCACC-3' (SEQ ID NO. 25);
the CFTR internal reference gene reverse primer CFTR-R: 5'-GCTGGGTGTAGGAGCAGTGT-3' (SEQ ID NO. 26).
The reference gene probe CFTR-P is a fluorescent probe, and the sequence of the fluorescent probe is as follows:
5’-VIC-TATGACCCGGATAACAAGGAGGAACGC-BHQ1-3’(SEQID NO.27)。
the specific operation process of the detection of the kit of the invention is as follows:
1) sample DNA extraction and dilution
It is recommended to use commercial DNA extraction kit (such as Qiagen or Tiangen whole blood DNA extraction kit) to extract human sample genome DNA, the sample type is recommended to use peripheral blood sample, the extracted DNA needs to be measured by ultraviolet spectrophotometer for concentration and purity, the OD260/OD280 value should be in the range of 1.8-2.0, the sample DNA quality is unqualified and cannot be used for detection, and after extraction is completed, the sample is diluted to 10-30 ng/mul according to DNA concentration information for PCR detection.
2) Reaction system preparation (see table 1):
each gene locus is respectively provided with two reaction systems, wild type and mutant upstream primers are respectively added into the two reaction systems, and other contents comprise template DNA, a downstream primer, a fluorescent probe, fluorescent PCR-mix, an internal reference gene primer, an internal reference gene probe and the like; and simultaneously configuring a positive control reaction system and a negative control reaction system of corresponding sites, wherein the positive control is a plasmid containing a target gene fragment, and the negative control is purified dd water.
Table 1: preparation of PCR reaction System
Figure BDA0003117461770000071
Remarking: the reagents in the system are provided by Novonz, and can also be obtained from public channels.
3) And (3) PCR reaction:
amplifying the prepared reaction system on a real-time fluorescent PCR instrument ABI 7500, wherein the PCR amplification conditions are as follows: at 37 ℃ for 2 min; at 95 ℃ for 1 min; (95 ℃, 15 sec; 60 ℃, 30sec)40 cycles, and the fluorescence signal was collected at 60 ℃ in the second step of the PCR cycle, with detection channels: FAM and VIC.
The detection results are shown in fig. 1 to fig. 18, and it can be seen from fig. 1 that in the sample, the CFTR of the internal reference gene is normally amplified, the FAM channel in the rs1065852 wild-type reaction system is normally amplified, and the FAM channel in the rs1065852 mutant-type reaction system is not amplified, indicating that the genotype of the sample is homozygous for GG.
From fig. 2, it can be seen that in the sample, the internal reference genes CFTR are normally amplified, the FAM channel in the rs1065852 wild-type reaction system is normally amplified, and the FAM channel in the rs1065852 mutant-type reaction system is normally amplified, indicating that the genotype of the sample is GA heterozygous.
It can be seen from fig. 3 that in this sample, the CFTR of the reference gene is normally amplified, the FAM channel in the rs1065852 wild-type reaction system is not amplified, and the FAM channel in the rs1065852 mutant-type reaction system is normally amplified, indicating that the genotype of this sample is AA homozygous.
From fig. 4, it can be seen that in the sample, the CFTR of the reference gene is normally amplified, the FAM channel in the rs1800497 wild-type reaction system is normally amplified, and the FAM channel in the rs1800497 mutant-type reaction system is not amplified, indicating that the genotype of the sample is homozygous for GG.
From fig. 5, it can be seen that in the sample, the internal reference genes CFTR are all normally amplified, the FAM channel in the rs1800497 wild-type reaction system is normally amplified, and the FAM channel in the rs1800497 mutant-type reaction system is normally amplified, indicating that the genotype of the sample is GA heterozygous.
From fig. 6, it can be seen that in the sample, the CFTR of the reference gene is normally amplified, the FAM channel in the rs1800497 wild-type reaction system is not amplified, and the FAM channel in the rs1800497 mutant-type reaction system is normally amplified, indicating that the genotype of the sample is AA homozygous.
From fig. 7, it can be seen that in the sample, the internal reference genes CFTR are all normally amplified, the FAM channel in the rs762551 wild-type reaction system is normally amplified, and the FAM channel in the rs762551 mutant-type reaction system is not amplified, indicating that the genotype of the sample is CC homozygous.
From fig. 8, it can be seen that in the sample, the internal reference genes CFTR are all normally amplified, the FAM channel in the rs762551 wild-type reaction system is normally amplified, and the FAM channel in the rs762551 mutant-type reaction system is normally amplified, indicating that the genotype of the sample is CA heterozygous.
It can be seen from fig. 9 that in this sample, the internal reference genes CFTR are all amplified normally, the FAM channel in the rs762551 wild-type reaction system is not amplified, and the FAM channel in the rs762551 mutant-type reaction system is amplified normally, indicating that the genotype of this sample is AA homozygous.
It can be seen from fig. 10 that in this sample, the CFTR of the reference gene is normally amplified, the FAM channel in the rs489693 wild-type reaction system is normally amplified, and the FAM channel in the rs489693 mutant-type reaction system is not amplified, indicating that the genotype of this sample is CC homozygous.
It can be seen from fig. 11 that in this sample, the internal reference genes CFTR were all amplified normally, FAM channels were amplified normally in the rs489693 wild-type reaction system, and FAM channels were amplified normally in the rs489693 mutant-type reaction system, indicating that the genotype of this sample was CA heterozygous.
It can be seen from fig. 12 that in this sample, the CFTR of the reference gene is normally amplified, the FAM channel in the rs489693 wild-type reaction system is not amplified, and the FAM channel in the rs489693 mutant-type reaction system is normally amplified, indicating that the genotype of this sample is AA homozygous.
It can be seen from fig. 13 that in the sample, the CFTR of the reference gene is normally amplified, the FAM channel in the rs1414334 wild-type reaction system is normally amplified, and the FAM channel in the rs1414334 mutant-type reaction system is not amplified, indicating that the genotype of the sample is CC homozygous.
It can be seen from fig. 14 that in the sample, the internal reference genes CFTR are all amplified normally, the FAM channel in the rs1414334 wild-type reaction system is amplified normally, and the FAM channel in the rs1414334 mutant-type reaction system is amplified normally, indicating that the genotype of the sample is CG heterozygous.
It can be seen from fig. 15 that in the sample, the CFTR of the reference gene is normally amplified, the FAM channel in the rs1414334 wild-type reaction system is not amplified, and the FAM channel in the rs1414334 mutant-type reaction system is normally amplified, indicating that the genotype of the sample is homozygous for GG.
From fig. 16, it can be seen that in the sample, the CFTR of the internal reference gene is normally amplified, the FAM channel in the rs1799978 wild-type reaction system is normally amplified, and the FAM channel in the rs1799978 mutant-type reaction system is not amplified, indicating that the genotype of the sample is TT homozygous.
It can be seen from fig. 17 that in the sample, the internal reference genes CFTR are all amplified normally, the FAM channel in the rs1799978 wild-type reaction system is amplified normally, and the FAM channel in the rs1799978 mutant-type reaction system is amplified normally, indicating that the genotype of the sample is TC heterozygous.
It can be seen from fig. 18 that in this sample, the CFTR of the reference gene is normally amplified, the FAM channel in the rs1799978 wild-type reaction system is not amplified, and the FAM channel in the rs1799978 mutant-type reaction system is normally amplified, indicating that the genotype of this sample is CC homozygous.
According to the invention, the related genes in the sample can be typed, and the use of the medicine can be personalized and guided by referring to different genotyping and corresponding medication guidance information (see table 2).
Table 2: different genotyping and corresponding drug administration guidance information
Figure BDA0003117461770000091
Example 2: consistency comparison experiment
Randomly selecting 16 clinical samples to genotype the 6 sites by adopting the method (qPCR) of the invention, and simultaneously verifying the results by adopting a first-generation sequencing method, and finding that the coincidence rate of all the sites of the qPCR typing results and the first-generation sequencing results is 100 percent, wherein the qPCR typing results and the first-generation sequencing results are shown in Table 3:
table 3: 16 clinical samples were subjected to site typing and first-generation sequencing result comparison by the method
Figure BDA0003117461770000101
According to the consistency experiment, the invention can accurately and rapidly classify the related genes of the mental drugs, thereby better guiding the individual medication of patients.
The present invention and its embodiments have been described above, and the description is not intended to be limiting, and the drawings are only one embodiment of the present invention, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> Zhengzhou university
Henan Shenyouu Medical Laboratory Co., Ltd.
<120> a gene polymorphism detection kit for guiding medication of mental disease
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Claims (10)

1. A gene polymorphism detection kit for guiding medication of mental diseases is characterized by comprising a specific primer group and a fluorescent probe group for detecting rs1065852 site of CYP2D6 gene, rs1414334 site of HTR2C gene, rs1800497 site of ANKK1 gene, rs762551 site of CYP1A2 gene, rs489693 site of MC4R gene, and rs1799978 site of DRD2 gene.
2. The kit for detecting gene polymorphism for guiding medication of psychosis according to claim 1, wherein the primer set comprises two forward primers and a common reverse primer for detecting rs1065852 site of CYP2D6 gene, two forward primers and a common reverse primer for detecting rs1414334 site of HTR2C gene, two forward primers and a common reverse primer for detecting rs1800497 site of ANKK1 gene, two forward primers and a common reverse primer for detecting rs762551 site of CYP1A2 gene, two forward primers and a common reverse primer for detecting rs489693 site of MC4R gene, two forward primers and a common reverse primer for detecting rs1799978 site of DRD2 gene.
3. The kit for detecting gene polymorphism for guiding medication for mental disease according to claim 2, wherein,
the base sequences of the forward primer and the common reverse primer for detecting the rs1065852 locus of the CYP2D6 gene are as follows:
wild-type forward primer: 5'-GCTGGGCTGCACGCTTCC-3' (SEQ ID NO. 1);
mutant forward primer: 5'-GCTGGGCTGCACGCTTCT-3' (SEQ ID NO. 2);
common reverse primers: 5'-ACCTGGTCGAAGCAGTATGGT-3' (SEQ ID NO. 3);
the base sequences of the forward primer and the common reverse primer for detecting the locus rs1414334 of the HTR2C gene are as follows:
wild-type forward primer: 5'-TCTACAGTGACTTTGCTACCTTC-3' (SEQ ID NO. 4);
mutant forward primer: 5'-TCTACAGTGACTTTGCTACCTTG-3' (SEQ ID NO. 5);
common reverse primers: 5'-CATGAGATTGGTGGAAGTTTGT-3' (SEQ ID NO. 6);
the base sequences of the forward primer and the common reverse primer for detecting the rs1800497 site of the ANKK1 gene are as follows:
wild-type forward primer: 5'-CATCCTCAAAGTGCTGGCCG-3' (SEQ ID NO. 7);
mutant forward primer: 5'-CATCCTCAAAGTGCTGGCCA-3' (SEQ ID NO. 8);
common reverse primers: 5'-GCCCTCTAGGAAGGACATGAT-3' (SEQ ID NO. 9);
the base sequences of the forward primer and the common reverse primer for detecting the site rs762551 of the CYP1A2 gene are as follows:
wild-type forward primer: 5'-GGGTGAGCTCTGTGGACC-3' (SEQ ID NO. 10);
mutant forward primer: 5'-GGGTGAGCTCTGTGGACA-3' (SEQ ID NO. 11);
common reverse primers: 5'-GAGGGTTGAGATGGAGACATTC-3' (SEQ ID NO. 12);
the base sequences of the forward primer and the common reverse primer for detecting the rs489693 site of the MC4R gene are as follows:
wild-type forward primer: 5'-CACGCTGTAAACATTTAACAAGCG-3' (SEQ ID NO. 13);
mutant forward primer: 5'-CACGCTGTAAACATTTAACAAGCT-3' (SEQ ID NO. 14);
common reverse primers: 5'-GAAGTCTGCCTACCGGTCTAAA-3' (SEQ ID NO. 15);
the base sequences of the forward primer and the common reverse primer for detecting the locus rs1799978 of the DRD2 gene are as follows:
wild-type forward primer: 5'-CCACCCACACCCAGAGTGAT-3' (SEQ ID NO. 16);
mutant forward primer: 5'-CCACCCACACCCAGAGTGAC-3' (SEQ ID NO. 17);
common reverse primers: 5'-ACCTCTTCCAACACCTCCTCTC-3' (SEQ ID NO. 18).
4. The kit for detecting gene polymorphism for guiding medication for mental disease according to claim 1, wherein,
the fluorescent probe set comprises a CYP2D6-rs1065852 common probe, an HTR2C-rs1414334 common probe, an ANKK1-rs1800497 common probe, a CYP1A2-rs762551 common probe, an MC4R-rs489693 common probe and a DRD2-rs1799978 common probe;
the base sequence of the CYP2D6-rs1065852 common probe is as follows:
5’-FAM-ACATGCAGCAGGTTGCCCAGC-3’-BHQ1(SEQ ID NO.19)；
the base sequence of the HTR2C-rs1414334 common probe is as follows:
5’-FAM-AGAAGCGAGCTAACATTACTCTGGA-3’-BHQ1(SEQ ID NO.20)；
the base sequence of the ANKK1-rs1800497 common probe is as follows:
5’-FAM-CATCATGTCCTTCCTAGAGGGCAA-3’-BHQ1(SEQ ID NO.21)；
the base sequence of the CYP1A2-rs762551 common probe is as follows:
5’-FAM-TAGTCTTTCTGGTATCCAGCTGGGAG-3’-BHQ1(SEQ ID NO.22)；
the base sequence of the MC4R-rs489693 common probe is as follows:
5’-FAM-GTTGCTAGGAAGTCTGCCTACCGGTC-3’-BHQ1(SEQ ID NO.23)；
the base sequence of the DRD2-rs1799978 common probe is as follows:
5’-FAM-ATCCAGCAAGTCAGGCAAGGAGAGG-3’-BHQ1(SEQ ID NO.24)。
5. the kit for detecting gene polymorphism for guiding medication of mental disease according to claim 1, characterized in that it further comprises a primer set specific to reference gene CFTR and a probe for reference gene.
6. The kit for detecting gene polymorphism for guiding medication of mental disease according to claim 5, wherein the primer set specific to the reference gene comprises CFTR reference gene forward primer CFTR-F and CFTR reference gene reverse primer CFTR-R;
the CFTR internal reference gene forward primer CFTR-F: 5'-CCCCTTTTGTAGGAAGTCACC-3' (SEQ ID NO. 25);
the CFTR internal reference gene reverse primer CFTR-R: 5'-GCTGGGTGTAGGAGCAGTGT-3' (SEQ ID NO. 26).
7. The kit for detecting gene polymorphism for guiding medication for mental disease according to claim 5, wherein,
the reference gene probe CFTR-P is a fluorescent probe, and the sequence of the fluorescent probe is as follows:
5’-VIC-TATGACCCGGATAACAAGGAGGAACGC-BHQ1-3’(SEQ ID NO.27)。
8. the kit for detecting gene polymorphism for use in medication guidance for mental disease according to any one of claims 1 to 7, characterized in that the method of use is:
1) taking a specific primer group and a probe group in the kit;
2) extracting the genome DNA of a sample to be detected by adopting a DNA extraction kit;
3) preparing a PCR reaction system: preparing the same reaction systems of two tubes for each site, adding wild type and mutation type forward primers into the two reaction systems respectively, and preparing a positive control reaction system and a negative control reaction system of corresponding sites simultaneously, wherein the other contents comprise template DNA, a downstream primer, a fluorescent probe, fluorescent PCR-mix, an internal reference gene primer and an internal reference gene probe, the positive control is a plasmid containing a target gene fragment, and the negative control is purified dd water;
4) and (3) PCR reaction: amplifying the prepared reaction system on a real-time fluorescent PCR instrument, wherein the PCR amplification conditions are as follows: at 37 ℃ for 2 min; at 95 ℃ for 1 min; (95 ℃, 15 sec; 60 ℃, 30sec)40 cycles, and the fluorescence signal was collected at 60 ℃ in the second step of the PCR cycle, with detection channels: FAM and VIC.
9. Use of the gene polymorphism detection kit for guiding medication of mental illness according to any one of claims 1 to 8, characterized in that it is used for preparing a reagent for guiding medication of mental illness of human.
10. The kit for detecting gene polymorphism according to claim 9, wherein the drugs include risperidone, olanzapine, clozapine, aripiprazole, chlorpromazine, fluphenazine, thioridazine, and trifluoperazine.
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Application publication date: 20211102