Primer set for detecting cyp2d6*4 allelic variation, test strip and preparation method and application

Abstract

This invention discloses a primer set, test strip, preparation method, and application for detecting CYP2D6*4 allelic variants, belonging to the field of genotyping technology. The primer set specifically amplifies nucleic acid fragments containing SNP sites of the CYP2D6*4 gene based on RFLP-PCR; the SNP sites of the CYP2D6*4 gene include c.1847G>A. This invention, based on RFLP-PCR allelic genotyping, designs a specific primer set for amplification at key sites of the CYP2D6 gene, enabling rapid and visual genotyping of key CYP2D6*4 mutations. Using this primer set for CYP2D6*4 allelic variant detection has advantages such as high detection sensitivity, good specificity, and satisfactory detection rates for both positive and negative samples, making it suitable for widespread application.

Description

Technical Field

[0001] This invention relates to the field of allele detection technology, specifically a primer set, test strip, preparation method, and application for detecting CYP2D6*4 allele variation. Background Technology

[0002] Although CYP2D6 accounts for only 2%-4% of the total CYP450 enzymes in the liver, it participates in the metabolism of approximately 20% of commonly used drugs, including the opioid analgesic codeine, antidepressants such as paroxetine, antihypertensive drugs such as metoprolol, and the anticancer drug tamoxifen. Currently, more than one hundred CYP2D6 genotypes have been identified. CYP2D6 plays a crucial role in drug metabolism. Antidepressants such as nortriptyline, antipsychotics such as perphenazine and thioridazine, and antiarrhythmic drugs such as metoprolol are primarily metabolized by CYP2D6. CYP2D6 activity affects the blood concentration of these drugs, thus influencing their efficacy and adverse reactions. Unlike most other CYP isoenzymes, CYP2D6 is not sensitive to enzyme induction effects. Different genotypes have varying effects on enzyme activity and drug metabolism.

[0003] CYP2D6 is a highly polymorphic gene, and different allele combinations result in different metabolites. In the population, CYP2D6 activity exhibits a four-stage distribution: strong metabolizer, intermediate metabolizer, weak metabolizer, and hypermetabolizer. The most important mutation site in the CYP2D6*4 allele, c.1847G>A, represents the weak metabolizer of CYP2D6. The metabolic phenotype resulting from the CYP2D6*4 allele reduces CYP2D6 enzyme activity, necessitating a reduced dosage of medication.

[0004] Restriction fragment length polymorphism (RFLP) analysis is a molecular "fingerprint" identification technique that combines PCR amplification, enzyme digestion, and DNA sequence analysis. This method determines the species and genotype of PCR amplified products based on the restriction enzyme digestion patterns, eliminating the need for cumbersome sequencing analysis and significantly shortening the detection cycle while improving efficiency.

[0005] With the promotion of the concept of precision medicine, the application of pharmacogenomics results to guide personalized medicine has become a hot topic in clinical practice. Evaluating the role of pharmacogenomics in clinical practice should focus on its impact on clinical endpoints, with allele-specific identification being crucial. In the study of the CYP2D6 allele, previous researchers have developed various detection methods, including whole-genome sequencing technology, and commercially available kits are also available; however, these methods all have their own limitations.

[0006] Therefore, developing a rapid, simple, accurate CYP2D6*4 allele detection technology that does not require complex instruments is of great practical significance for guiding rational drug use in clinical practice, avoiding adverse drug reactions, and improving treatment efficiency. Summary of the Invention

[0007] The purpose of this invention is to provide a primer set, test strip, preparation method, and application for detecting CYP2D6*4 allele variation, in order to solve the problems mentioned in the background art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A primer set for detecting CYP2D6*4 allele variation is disclosed, which is based on RFLP-PCR to specifically amplify nucleic acid fragments containing SNP sites of the CYP2D6*4 gene; the SNP sites of the CYP2D6*4 gene include c.1847G>A; the primer set includes an upstream primer with nucleotide sequences as shown in SEQ ID NO:5 and a downstream primer with nucleotide sequences as shown in SEQ ID NO:7.

[0009] Furthermore, the 5' end of the upstream primer shown in SEQ ID NO:5 is labeled with biotin; Furthermore, the 5' end of the downstream primer, as shown in SEQ ID NO:7, is labeled with digoxigenin.

[0010] Another object of the present invention is to provide a test strip for detecting CYP2D6*4 allele variants, which is used in conjunction with the above-mentioned primer set for detecting CYP2D6*4 allele variants to perform genotyping detection of the CYP2D6*4 gene; the test strip comprises: Base plate; The sample pad, conjugation pad, nitrocellulose membrane and absorbent pad are sequentially stacked on the base plate; The binding pad is coated with colloidal gold-labeled mouse anti-digoxin antibody. The nitrocellulose membrane has a detection line at one end near the sample pad and a control line at the other end near the absorbent pad; the detection line is coated with streptavidin; and the control line is coated with goat anti-mouse IgG.

[0011] Another object of the present invention is to provide a method for preparing the above-mentioned test strip for detecting CYP2D6*4 allele variation, comprising the following steps: Preparation of digoxin antibody-gold nanoparticle complex; A nitrocellulose membrane was provided, and it was coated with streptavidin as a test line and goat anti-mouse IgG as a control line. The digoxin antibody-gold nanoparticle complex was coated onto the binding pad; On the base plate, the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are assembled in sequence to make the test strip.

[0012] Another object of the present invention is to provide the application of the above-mentioned primer set for detecting CYP2D6*4 allele variation or the above-mentioned test strip for detecting CYP2D6*4 allele variation in the preparation of a CYP2D6*4 allele detection kit.

[0013] Another objective of this invention is to provide a CYP2D6*4 allele detection kit, comprising the aforementioned primer set for detecting CYP2D6*4 allele variations.

[0014] Furthermore, the CYP2D6*4 allele detection kit also includes the test strips for detecting the CYP2D6*4 allele variation.

[0015] Furthermore, the CYP2D6*4 allele detection kit also includes a PCR reaction mixture, BstN I restriction endonuclease, enzyme digestion buffer, positive control reagent, and negative control reagent.

[0016] This invention utilizes an RFLP-PCR-based allele detection method, designing a specific primer set to amplify key sites in the CYP2D6 gene. This enables rapid and visualized detection of the critical CYP2D6*4 mutation (c.1847G>A). When used for CYP2D6*4 allele variation detection, this primer set demonstrates high sensitivity, good specificity, and satisfactory detection rates for both positive and negative samples, making it suitable for widespread application. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the structure of a test strip for detecting CYP2D6*4 allele variation provided in an embodiment of the present invention.

[0018] Figure 2 The figure shows the experimental results for the optimal pH for preparing digoxin antibody-gold nanoparticle complexes; from left to right, the figures show the addition of 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 μL of 0.2 M K2CO3 solution.

[0019] Figure 3 The figure shows the experimental results of the optimal antibody input amount for preparing digoxin antibody-gold nanoparticle complexes under optimal pH conditions; from left to right in the figure, the amounts of 0, 5, 10, 15, 20, and 25 μg of mouse anti-digoxin antibody are shown.

[0020] Figure 4This is a physical image of a concentrated complex solution of digoxin antibody-gold nanoparticles.

[0021] Figure 5 The image shows the results of RFLP-PCR primer screening for specific CYP2D6*4. From left to right, wells 1 through 6 are shown. Well 1 is the DL-500 DNA Marker; well 2 shows the amplification results of SEQ ID NO:1 and SEQ ID NO:2 in genomic DNA; well 3 shows the amplification results of SEQ ID NO:3 and SEQ ID NO:4 in genomic DNA; well 4 shows the amplification results of SEQ ID NO:5 and SEQ ID NO:6 in genomic DNA; well 5 shows the amplification results of SEQ ID NO:5 and SEQ ID NO:7 in genomic DNA; and well 6 shows the amplification results of SEQ ID NO:5 and SEQ ID NO:8 in genomic DNA.

[0022] Figure 6 The figure shows the stability test results of RFLP-PCR primers SEQ ID NO:3 and SEQ ID NO:4 for specific CYP2D6*4. From left to right, wells 1 through 5 are shown. Well 1 is the DL-500 DNA Marker; well 2 shows the amplification results of SEQ ID NO:3 and SEQ ID NO:4 in the homozygous wild-type CYP2D6*4 plasmid; well 3 shows the amplification results of SEQ ID NO:3 and SEQ ID NO:4 in the homozygous mutant CYP2D6*4 plasmid; well 4 shows the amplification results of SEQ ID NO:3 and SEQ ID NO:4 in the heterozygous CYP2D6*4 plasmid; and well 5 shows the amplification results of SEQ ID NO:3 and SEQ ID NO:4 in genomic DNA.

[0023] Figure 7 The figure shows the stability test results of RFLP-PCR primers SEQ ID NO:5 and SEQ ID NO:8 for specific CYP2D6*4. From left to right, wells 1 through 5 are shown. Well 1 is the DL-500 DNA Marker; well 2 shows the amplification results of SEQ ID NO:5 and SEQ ID NO:8 in the homozygous wild-type CYP2D6*4 plasmid; well 3 shows the amplification results of SEQ ID NO:5 and SEQ ID NO:8 in the homozygous mutant CYP2D6*4 plasmid; well 4 shows the amplification results of SEQ ID NO:5 and SEQ ID NO:8 in the heterozygous CYP2D6*4 plasmid; and well 5 shows the amplification results of SEQ ID NO:5 and SEQ ID NO:8 in genomic DNA.

[0024] Figure 8 The graph shows the optimal control (C) line and test (T) line coating concentrations for the test strips. From left to right, the concentrations of the test strips are: T line 2.5 mg / mL, C line 0.2 mg / mL; T line 2.5 mg / mL, C line 0.4 mg / mL; T line 2.5 mg / mL, C line 0.6 mg / mL; T line 2.5 mg / mL, C line 0.8 mg / mL; T line 2.5 mg / mL, C line 1.0 mg / mL; T line 2 mg / mL, C line 0.2 mg / mL; T line 2 mg / mL, C line 0.4 mg / mL; T line 2 mg / mL, C line 0.6 mg / mL; T line 2 mg / mL, C line 0.8 mg / mL; T line 2 mg / mL, C line 1.0 mg / mL; T line 1.5 mg / mL, C line 0.2 mg / mL; T line 1.5 mg / mL, C line 0.4 mg / mL. mL; T line 1.5 mg / mL, C line 0.6 mg / mL; T line 1.5 mg / mL, C line 0.8 mg / mL; T line 1.5 mg / mL, C line 1.0 mg / mL; T line 1.0 mg / mL, C line 0.2 mg / mL; T line 1.0 mg / mL, C line 0.4 mg / mL; T line 1.0 mg / mL, C line 0.6 mg / mL; T line 1.0 mg / mL, C line 0.8 mg / mL; T line 1.0 mg / mL, C line 1.0 mg / mL; T line 0.5 mg / mL, C line 0.2 mg / mL; T line 0.5 mg / mL, C line 0.4 mg / mL; T line 0.5 mg / mL, C line 0.6 mg / mL; T line 0.5 mg / mL, C line 0.8 mg / mL; T line 0.5 mg / mL, C line 1.0 mg / mL; a total of 25 test strips.

[0025] Figure 9 This is a graph showing the results of the lowest detection line on the test strip; from left to right in the graph, NTC, 10 9 10 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1 10 0 The results of sample detection for gene copy number per μL.

[0026] Figure 10The images show agarose gel electrophoresis results after enzyme digestion using standard plasmids as templates, and corresponding images of the test strips and the digested agarose gel electrophoresis results. In the images, A shows the agarose gel electrophoresis result of BstNI digestion on the CYP2D6*4 heterozygous type, and the corresponding image of the test strip. Well 1 (left) is the DL-500 DNA Marker; Well 2 (right) is the result of BstNI digestion on the CYP2D6*4 heterozygous type. B shows the agarose gel electrophoresis result of BstNI digestion on the CYP2D6*4 homozygous wild-type, and the corresponding image of the test strip. Well 1 (left) is the DL-500 DNA Marker; Well 2 (right) is the result of BstNI digestion on the CYP2D6*4 homozygous wild-type. C shows the agarose gel electrophoresis image of the homozygous CYP2D6*4 mutant after BstN I digestion and the corresponding image of the test strip. Well 1 (left) is the DL-500 DNA Marker; Well 2 (right) is the CYP2D6*4 homozygous mutant after BstN I digestion.

[0027] Figure 11 The images show the results of actual sample testing. From left to right, the first test strip used a homozygous CYP2D6*4 mutant as a template for PCR amplification, followed by BstN I digestion; this is the positive control 1. The second test strip used a heterozygous CYP2D6*4 mutant as a template for PCR amplification, followed by BstN I digestion; this is the positive control 2. The third test strip used a homozygous wild-type CYP2D6*4 as a template for PCR amplification, followed by BstN I digestion; this is the negative control 1. Test strips four through twelve from left to right represent the results of actual sample testing. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] In one embodiment of the present invention, a primer set for detecting CYP2D6*4 allele variation is provided, which is based on RFLP-PCR to specifically amplify nucleic acid fragments containing single nucleotide polymorphism (SNP) sites of the CYP2D6*4 gene, including three types of common primers: homozygous wild type, homozygous mutant type, and heterozygous type; the SNP site of the CYP2D6*4 gene includes c.1847G>A.

[0030] Specifically, the primer set includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO:5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:7.

[0031] Preferably, the 5' end of the upstream primer, as shown in SEQ ID NO:5, is labeled with biotin; and the 5' end of the downstream primer, as shown in SEQ ID NO:7, is labeled with digoxigenin.

[0032] like Figure 1 As shown, in another embodiment of the present invention, a test strip for detecting CYP2D6*4 allele variants is also provided, which is used in conjunction with the above-mentioned primer set for detecting CYP2D6*4 allele variants to perform genotyping detection of the CYP2D6*4 gene; the test strip is an immunochromatographic colloidal gold test strip, specifically comprising: Base plate; The sample pad, conjugation pad, nitrocellulose membrane and absorbent pad are sequentially stacked on the base plate; The binding pad is coated with colloidal gold-labeled mouse anti-digoxin antibody. The nitrocellulose membrane has a detection line at one end near the sample pad and a control line at the other end near the absorbent pad; the detection line is coated with streptavidin; and the control line is coated with goat anti-mouse IgG.

[0033] In another embodiment of the present invention, a method for preparing the above-mentioned test strip for detecting CYP2D6*4 allele variation is also provided, comprising the following steps: S1. Preparation of digoxigenin antibody-gold nanoparticle complex; The optimal pH for preparing the digoxin antibody-gold nanoparticle complex was determined first: Ten EP tubes were used, and 1 mL of colloidal gold solution was added to each tube. Then, according to a gradient, 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 μL of 0.2 M K₂CO₃ solution were added to each tube, respectively. The mixtures were thoroughly mixed, allowed to stand, and the color change was observed. If no color change was observed, an excess of 12 μg of mouse anti-digoxin antibody was added to the tube, and the mixture was incubated at room temperature with shaking. After incubation, 100 μL of 10% sodium chloride solution was added to the tube, and the mixture was mixed again at room temperature. The color change was then observed. The amount of 0.2 M K₂CO₃ added to the first tube where the color began to stabilize was the optimal pH adjustment amount. Next, the optimal antibody dosage for preparing the digoxin antibody-gold nanoparticle complex was determined: Six EP tubes were taken, and 1 mL of colloidal gold solution was added to each tube. After adjusting to the optimal pH, 0, 5, 10, 15, 20, and 25 μg of mouse anti-digoxin antibody were added to each tube in a gradient at the optimal pH, and the mixture was thoroughly mixed. After mixing, the mixture was allowed to stand. Subsequently, 100 μL of 10% sodium chloride solution was added to each tube, and the mixture was further mixed at room temperature. The color change was observed, and the amount of mouse anti-digoxin antibody added to the first tube where the color began to stabilize was the optimal antibody dosage. Then, add the optimal amount of antibody to 1 mL of colloidal gold solution at the optimal pH and incubate with shaking at room temperature. After incubation, add 100 μL of 10% BSA solution to the tube and incubate with shaking at room temperature again. After incubation, centrifuge at room temperature, remove the supernatant, and leave the colloidal gold concentrate. Add 80-100 μL of colloidal gold reconstitution solution to obtain the digoxin antibody-gold nanoparticle complex.

[0034] S2. Provide a nitrocellulose membrane, and coat it with streptavidin as a detection line and goat anti-mouse IgG as a quality control line; S3. The digoxin antibody-gold nanoparticle complex is coated onto the conjugate pad; specifically, the digoxin antibody-gold nanoparticle complex is spread over a glass cellulose membrane to completely impregnate the membrane, and then dried completely in an oven at 37°C to obtain the conjugate pad. S4. Immerse the sample pad in a sample pad treatment solution with pH=8.5 to obtain the treated sample pad. On a PVC base plate, assemble the sample pad, conjugate pad, nitrocellulose (NC) membrane, and absorbent pad in sequence to form a test strip. The specific order of pasting the test strip is as follows: Attach the treated sample pad to one end of the conjugate pad (the sample pad should be pasted to cover the conjugate pad by 2mm), and attach the nitrocellulose membrane to the other end (covering the nitrocellulose membrane by 2mm); attach the treated sample pad to one end of the PVC base plate; attach the absorbent pad to the nitrocellulose membrane and then to the other end of the PVC base plate.

[0035] In another embodiment of the present invention, a CYP2D6*4 allele detection kit is also provided, comprising the above-mentioned primer set for detecting CYP2D6*4 allele variation and the above-mentioned test strip for detecting CYP2D6*4 allele variation.

[0036] Preferably, the CYP2D6*4 allele detection kit further includes a PCR reaction mixture (PCR-Mix), BstN I restriction endonuclease, enzyme digestion buffer, positive control reagent, and negative control reagent. The enzyme digestion quality control positive and negative control reagents are recombinant DNA plasmids containing all target gene target sequence polymorphic sites; the NTC control reagent is ddH2O.

[0037] In practical applications, the method for detecting mutations at SNP sites of the CYP2D6*4 gene includes the following steps: extracting and synthesizing wild-type and mutant CYP2D6*4 plasmids as standard plasmid templates; and designing specific primers.

[0038] Specifically, the following steps are included: S1. Use the above primer set to amplify the sequence fragment belonging to CYP2D6*4.

[0039] The amplification program and conditions are as follows: 95℃, 3 min; 95℃, 30 s; 60℃, 30 s; 72℃, 1 min; 72℃, 8 min; step 2-step 4: 35 cycles; 4℃, hold; The amplification system using plasmid as a template is as follows: Plasmid DNA template: 0.6 μL; upstream primer: 1 μL; downstream primer: 1 μL; ddH2O: 7.4 μL; PCR Mix: 10 μL; The amplification system using the genome as a template is as follows: Genomic DNA template: 1 μL; upstream primer: 1 μL; downstream primer: 1 μL; ddH2O: 7 μL; PCR Mix: 10 μL.

[0040] S2. Take 10 μL of the amplified product and add 1 μL of BstN I restriction endonuclease, 3 μL of enzyme digestion buffer, and 6 μL of ddH2O. Then digest at 60℃ for 1-2 hours.

[0041] S3. Add 20 μL of the enzyme digestion product to 150 μL of ddH2O, mix thoroughly, and then drop the mixture onto the prepared immunochromatographic colloidal gold test strip. Wait 3-5 minutes to allow the sample solution to completely flow through the test strip before observing the results. If both the test line and the control line show color, it indicates the presence of the CYP2D6*4 allele. If the test line does not show color but the control line does, it indicates the absence of the CYP2D6*4 allele.

[0042] This invention, for the first time, combines RFLP-PCR with a test strip for the detection of the CYP2D6 allele, offering a novel approach to traditional RFLP-PCR. The upstream primer in this invention is labeled with biotin at its 5' end, and the downstream primer is labeled with digoxigenin at its 5' end. After PCR, restriction endonuclease digestion is performed, and the results are interpreted using an immunochromatographic test strip, enabling allele identification. Using the kit provided in this invention for site mutation detection, the entire process takes no more than 4 hours. The detection is simple, cost-effective, and saves approximately 45 minutes compared to traditional RFLP-PCR. Furthermore, the solution provided in this invention requires minimal manpower, is easy to operate, and has a relatively open system / platform, making it suitable for various detection environments.

[0043] The following embodiments are implementation examples of the technical solution of the present invention in practical applications, but are not limited thereto. Unless otherwise specified, all materials and reagents involved are purchased from commercial channels; unless otherwise specified, all experimental methods used are conventional methods.

[0044] Example 1: Experiment to determine the optimal pH during the preparation of digoxin antibody-gold nanoparticle complex: Take ten 1.5 mL EP tubes, add 1 mL of colloidal gold solution to each tube, and then add 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 μL of 0.2 M K₂CO₃ solution to each tube according to a gradient. Mix thoroughly and let stand for 10 min to observe the color change. If there is no color change, add an excess of 12 μg of mouse anti-digoxin antibody to the tube, mix well, and incubate at room temperature with shaking for 30 min. After 30 min, add 100 μL of 10% NaCl solution to the tube and mix again at room temperature for 30 min. Observe the color change after 30 min. The amount of 0.2 M K₂CO₃ added to the first tube where the color begins to stabilize is the optimal pH adjustment amount. Specific experimental results are as follows: Figure 2 As shown in the figure, the color of the 7th EP tube from the left becomes more stable and brighter red. Therefore, the optimal pH for K2CO3 is the concentration at which 12 μL of 0.2 M K2CO3 is added.

[0045] Example 2: Experiment to determine the optimal antibody dosage in the preparation of digoxin antibody-gold nanoparticle complex: Take six 1.5 mL EP tubes, add 1 mL of colloidal gold solution to each tube, and adjust to the optimal pH (add 12 μL of 0.2 M K₂CO₃). Then, at the optimal pH, add 0, 5, 10, 15, 20, and 25 μg of mouse anti-digoxin antibody to each tube in a gradient, mixing thoroughly. Let stand for 10 min. After 10 min, add 100 μL of 10% sodium chloride solution to each tube and continue mixing at room temperature for 30 min. Observe the color change after 30 min. The amount of mouse anti-digoxin antibody added to the first tube where the color begins to stabilize is the optimal antibody dosage. (See details below.) Figure 3 As shown, the color of the third EP tube from the left stabilizes, indicating that the third EP tube exhibits the best performance. Therefore, the optimal antibody dosage is the one used when adding 10 μg of mouse anti-digoxin antibody. Additionally, the concentrated reconstituted solution of the digoxin antibody-gold nanoparticle complex is shown below. Figure 4 As shown, the reconstituted digoxin antibody-gold nanoparticle complex is deep red.

[0046] Example 3: RFLP-PCR primer screening experiment for specific CYP2D6*4: Using data from PharmVar, SNP sites were identified in 183 alleles. To avoid non-specific amplification of other alleles, both upstream and downstream primers should not contain any SNP sites during primer design. Therefore, under this constraint, CYP2D6*4 specific amplification primers were designed. Using genomic DNA as a template, PCR amplification was performed under the following conditions and system, using primers SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:5 and SEQ ID NO:7; and SEQ ID NO:5 and SEQ ID NO:8. The specific primer sequences are shown in Table 1.

[0047] Table 1

[0048] The amplification program and conditions are as follows: 95℃, 3 min; 95℃, 30 s; 60℃, 30 s; 72℃, 1 min; 72℃, 8 min; step 2-step 4: 35 cycles; 4℃, hold; The amplification system using the genome as a template is as follows: Genomic DNA template: 1 μL; upstream primer: 1 μL; downstream primer: 1 μL; ddH2O: 7 μL; PCR Mix: 10 μL.

[0049] Electrophoresis was performed using a prepared 2% agarose gel. The loading order after electrophoresis was as follows: Well 1: DL-500 DNA Marker; Well 2: Amplification results of SEQ ID NO:1 and SEQ ID NO:2 in genomic DNA; Well 3: Amplification results of SEQ ID NO:3 and SEQ ID NO:4 in genomic DNA; Well 4: Amplification results of SEQ ID NO:5 and SEQ ID NO:6 in genomic DNA; Well 5: Amplification results of SEQ ID NO:5 and SEQ ID NO:7 in genomic DNA; Well 6: Amplification results of SEQ ID NO:5 and SEQ ID NO:8 in genomic DNA. The experimental results are as follows: Figure 5 As shown, using the plasmid amplification system and genome amplification system in this embodiment, SEQ ID NO:3 and SEQ ID NO:4 were used for re-amplification one month apart. In subsequent experiments, this primer pair showed poor stability and was prone to non-specific bands. The experimental results are as follows. Figure 6 As shown, SEQ ID NO:5 and SEQ ID NO:6 were not selected because the amplified bands were too small to be suitable for enzyme digestion experiments. In this example, SEQ ID NO:5 and SEQ ID NO:8 were used for amplification one month apart in both the plasmid amplification system and the genome amplification system. This also resulted in poor primer stability. For details, please refer to [link to relevant documentation]. Figure 7 The amplification system using plasmids (including homozygous wild-type, homozygous mutant, and heterozygous plasmids obtained by mixing equal volumes and concentrations of the two) as templates is as follows: plasmid DNA template: 0.6 μL; upstream primer: 1 μL; downstream primer: 1 μL; ddH2O: 7.4 μL; PCR Mix: 10 μL; based on the singleness of the band, the correctness of the band size, and the brightness of the band, the primer set of SEQ ID NO:5 and SEQ ID NO:7 in well 5 was selected for Biotin and Dig linkage labeling, which was used as the probe for the downstream test strip experiment.

[0050] Example 4: Experiment simulating optimal C-line and T-line coating concentrations of CYP2D6*4 test strips using plasmid as a template: Using the CYP2D6*4 heterozygous mutant plasmid as an amplification template ensured a positive result on the test strip. Following the amplification conditions and system described in Example 3, PCR amplification was performed using the primer set SEQ ID NO:5 and SEQ ID NO:7 (specifically: 5'-Biotin-CACAAAGCGGGAACTGGGAA-3' and 5'-Dig-GAGGGTCGTCGTACTCGAAG-3') with Biotin and Dig ligation markers. PCR amplification was performed in 25 tubes of 20 μL each. The assembled test strips were streaked according to the following concentration gradients: T line 2.5 mg / mL, 2 mg / mL, 1.5 mg / mL, 1 mg / mL, 0.5 mg / mL; C line 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL. Perform combined streaks as follows: T line 2.5 mg / mL, C line 0.2 mg / mL; T line 2.5 mg / mL, C line 0.4 mg / mL; T line 2.5 mg / mL, C line 0.6 mg / mL; T line 2.5 mg / mL, C line 0.8 mg / mL; T line 2.5 mg / mL, C line 1.0 mg / mL; T line 2 mg / mL, C line 0.2 mg / mL; T line 2 mg / mL, C line 0.4 mg / mL; T line 2 mg / mL, C line 0.6 mg / mL; T line 2 mg / mL, C line 0.8 mg / mL; T line 2 mg / mL, C line 1.0 mg / mL; T line 1.5 mg / mL, C line 0.2 mg / mL; T line 1.5 mg / mL, C line 0.4 mg / mL; T line 1.5 mg / mL, C line 0.6 mg / mL; T line 1.5 mg / mL... L, C line 0.8 mg / mL; T line 1.5 mg / mL, C line 1.0 mg / mL; T line 1.0 mg / mL, C line 0.2 mg / mL; T line 1.0 mg / mL, C line 0.4 mg / mL; T line 1.0 mg / mL, C line 0.6 mg / mL; T line 1.0 mg / mL, C line 0.8 mg / mL; T line 1.0 mg / mL, C line 1.0 mg / mL; T line 0.5 mg / mL, C line 0.2 mg / mL; T line 0.5 mg / mL, C line 0.4 mg / mL; T line 0.5 mg / mL, C line 0.6 mg / mL; T line 0.5 mg / mL, C line 0.8 mg / mL; T line 0.5 mg / mL, C line 1.0 mg / mL; Scratch 25 test strips. Take 10 μL of each amplified PCR product and add 1 μL of BstN. I. Restriction endonuclease, 3 μL of restriction buffer, 6 μL of ddH2O, for restriction digestion. After 1 hour and 45 minutes, remove the digestion product and add 150 μL of ddH2O. Mix well and drop the mixture onto the sample pad of the test strip. Wait 3-5 minutes to observe the results. The results are as follows. Figure 8 As shown, the test strip performs best overall and is the least expensive when the T-line concentration is 1 mg / mL and the C-line concentration is 0.6 mg / mL. Therefore, the T-line concentration of 1 mg / mL and the C-line concentration of 0.6 mg / mL were chosen as the subsequent streaking concentrations.

[0051] Example 5: Experiment simulating the lowest detection line of the CYP2D6*4 test strip using plasmid as a template: Using the synthesized homozygous mutant plasmid CYP2D6*4 as an amplification template, ensuring a positive result on the test strip, and following the amplification conditions and system described in Example 3 (using the plasmid as a template), PCR amplification was performed using the primer set SEQ ID NO:5 and SEQ ID NO:7 (specifically: 5'-Biotin-CACAAAGCGGGAACTGGGAA-3' and 5'-Dig-GAGGGTCGTCGTACTCGAAG-3'). Five 20 μL tubes were used for PCR amplification, resulting in a total of 100 μL of PCR product. Simultaneously, one 20 μL tube of NTC sample without amplification template was prepared. The 100 μL of PCR amplification product was purified and diluted according to the copy number formula: copy number = product concentration × 10⁻⁶. -9 ×6.02×10 23 The synthesized plasmid length (bp × 660) yielded a copy number of 3.49 × 10⁻⁶. 9 100 μL of purified product was diluted with a serial number of copies / μL, starting from 10... 9 Dilute to 10 0 Add the test strips sequentially to the sample pad according to the copy number from highest to lowest, wait 3-5 minutes, and observe the results to obtain the limit of detection of the test strip. Results are as follows: Figure 9 As shown, from left to right, the test strips contain NTC and 10 samples respectively. 9 10 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1 10 0 The sample detection result for gene copy number / μL was 3.49×10⁻⁶. 5 The minimum detection limit for nucleic acid capacity of the test strip is reached when the number of gene copies / μL is reached.

[0052] Example 6: Simulated CYP2D6*4 test strip detection experiment using plasmid as template: The synthesized homozygous wild-type plasmid CYP2D6*4, the homozygous mutant plasmid, and the heterozygous mutant plasmid prepared by mixing equal volumes and concentrations of the two synthesized plasmids were used as amplification templates. The primer sequences used for amplification were: the primer set SEQ ID NO:5 and SEQ ID NO:7 with ligation markers for Biotin and Dig (specifically: 5'-Biotin-CACAAAGCGGGAACTGGGAA-3' and 5'-Dig-GAGGGTCGTCGTACTCGAAG-3'). PCR amplification was performed under the following amplification conditions and amplification system.

[0053] The amplification program was as follows: 95℃, 3 min; 95℃, 30 s; 60℃, 30 s; 72℃, 1 min; 72℃, 8 min; step 2-step 4: 35 cycles; 4℃, hold. The amplification system is as follows: Plasmid DNA template: 0.6 μL; upstream primer: 1 μL; downstream primer: 1 μL; ddH2O: 7.4 μL; PCR Mix: 10 μL; After amplification, 10 μL of PCR product was removed, and 1 μL of BstN I restriction endonuclease, 3 μL of digestion buffer, and 6 μL of ddH2O were added for digestion. After digestion for 1 hour and 45 minutes, the digested product was removed and 150 μL of ddH2O was added. After mixing well, the mixture was added to the sample pad of the test strip and the results were observed after 3-5 minutes. Simultaneously, the remaining 10 μL of PCR product was observed by electrophoresis on a 2% agarose gel and compared with the test strip results. The results are as follows: Figure 10 As shown. From Figure 10 As can be seen from A, B, and C, the test results correspond perfectly, indicating that the test was successful.

[0054] Example 7: Actual sample detection experiment of the CYP2D6*4 test strip detection method based on RFLP-PCR: Genomic DNA was used, ensuring the DNA quality (260 / 280) was between 1.8 and 2.0 and the concentration was above 10 ng / μL. Genomic DNA meeting the quality standards was used as a template. PCR amplification was performed using the primer set SEQ ID NO:5 and SEQ ID NO:7 (specifically: 5'-Biotin-CACAAAGCGGGAACTGGGAA-3' and 5'-Dig-GAGGGTCGTCGTACTCGAAG-3') with Biotin and Dig ligation markers, and the following procedure and system. Simultaneously, a PCR amplification system was prepared using a synthesized standard plasmid (including a synthesized CYP2D6*4 homozygous wild-type plasmid, a homozygous mutant plasmid, and a heterozygous mutant plasmid prepared by mixing equal volumes and concentrations of the above two synthesized plasmids) as a quality control template. This system served as a negative control and two positive controls. After enzyme digestion, the samples were loaded onto test strips as quality control for the entire actual sample detection to ensure that the restriction endonuclease performed the correct digestion action.

[0055] The amplification program was as follows: 95℃, 3 min; 95℃, 30 s; 60℃, 30 s; 72℃, 1 min; 72℃, 8 min; step 2-step 4: 35 cycles; 4℃, hold. The amplification system is as follows: Genomic DNA template: 1 μL; upstream primer: 1 μL; downstream primer: 1 μL; ddH2O: 7 μL; PCR Mix: 10 μL; plasmid DNA template: 0.6 μL; upstream primer: 1 μL; downstream primer: 1 μL; ddH2O: 7.4 μL; PCR Mix: 10 μL; After amplification, remove 10 μL of PCR product, add 1 μL of BstN I restriction endonuclease, 3 μL of digestion buffer, and 6 μL of ddH2O for digestion. After 1 hour and 45 minutes of digestion, remove the digested product and add 150 μL of ddH2O. Mix well and drop the mixture onto the sample pad of the test strip. Wait 3-5 minutes to observe the results. The results are as follows. Figure 11As shown, the first test strip from left to right used the homozygous mutant CYP2D6*4 as a template for PCR amplification, and the result after BstN I digestion was detected; this is the positive control 1. The second test strip from left to right used the heterozygous mutant CYP2D6*4 as a template for PCR amplification, and the result after BstN I digestion was detected; this is the positive control 2. The third test strip from left to right used the homozygous wild-type CYP2D6*4 as a template for PCR amplification, and the result after BstN I digestion was detected; this is the negative control 1. The results of positive control 1, positive control 2, and negative control 1 are all correct, proving the effectiveness of the rapid restriction endonuclease. The fourth to twelfth test strips are all actual test samples. The actual test samples were compared with the sequencing results, and the concordance rate in this test was 88.89% (the tenth test strip from left to right showed a very weak positive result, which did not match the sequencing results; the others were consistent). Please see Table 2 for the specific compliance rate.

[0056] Table 2 CYP2D6*4 (c.1847G>A site) Test strip results Sequencing results Compliance rate exist 1 / 9 cases 0 / 9 cases 88.89% Does not exist 8 / 9 cases 9 / 9 cases 88.89% Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A primer set for detecting CYP2D6*4 allele variation, characterized in that, The primer set is based on RFLP-PCR to specifically amplify nucleic acid fragments containing SNP sites of the CYP2D6*4 gene; the SNP sites of the CYP2D6*4 gene include c.1847G>A; the primer set includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO:5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:

7.

2. The primer set for detecting CYP2D6*4 allele variation according to claim 1, characterized in that, The upstream primer of the nucleotide sequence shown in SEQ ID NO:5 is labeled with biotin at its 5' end.

3. The primer set for detecting CYP2D6*4 allele variation according to claim 1, characterized in that, The downstream primer of the nucleotide sequence shown in SEQ ID NO:7 is labeled with digoxigenin at its 5' end.

4. A test strip for detecting CYP2D6*4 allele variation, characterized in that, The test strip is used in conjunction with the primer set for detecting CYP2D6*4 allele variants according to any one of claims 1-3 to perform genotyping of the CYP2D6*4 gene; the test strip comprises: Base plate; The sample pad, conjugation pad, nitrocellulose membrane and absorbent pad are sequentially stacked on the base plate; The binding pad is coated with colloidal gold-labeled mouse anti-digoxin antibody; The nitrocellulose membrane has a detection line at one end near the sample pad and a control line at the other end near the absorbent pad; the detection line is coated with streptavidin; and the control line is coated with goat anti-mouse IgG.

5. A method for preparing a test strip for detecting CYP2D6*4 allele variation as described in claim 4, characterized in that, Includes the following steps: Preparation of digoxin antibody-gold nanoparticle complex; Nitrocellulose membranes are provided, and streptavidin is coated on them as a test line and goat anti-mouse IgG is coated on them as a control line. The digoxin antibody-gold nanoparticle complex was coated onto the binding pad; On the base plate, the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are assembled in sequence to make the test strip.

6. The use of a primer set for detecting CYP2D6*4 allele variants as described in any one of claims 1-3 or a test strip for detecting CYP2D6*4 allele variants as described in claim 4 in the preparation of a CYP2D6*4 genotyping kit.

7. A CYP2D6*4 genotyping detection kit, characterized in that, Includes the primer set for detecting CYP2D6*4 allele variation as described in any one of claims 1-3.

8. The CYP2D6*4 genotyping detection kit according to claim 7, characterized in that, It also includes the test strip for detecting CYP2D6*4 allele variation as described in claim 4.

9. The CYP2D6*4 genotyping detection kit according to claim 7, characterized in that, It also includes PCR reaction mixture, BstN I restriction endonuclease, enzyme digestion buffer, positive control reagent, and negative control reagent.

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