Digital PCR detection system, kit and method for detecting single nucleotide polymorphism and copy number variation of CYP2D6 gene and application

Abstract

The invention discloses a digital PCR detection system, kit and method for detecting single nucleotide polymorphism and copy number variation of a CYP2D6 gene and application, and belongs to the technical field of molecular biology. The method comprises the following steps: designing three pairs of CYP2D6 and reference gene RPP30 specific primers, matching with four locked nucleic acid modified TaqMan fluorescent probes, constructing a single-tube multiple digital PCR detection system, and synchronously and accurately detecting three single nucleotide polymorphisms of CYP2D6 * 10, CYP2D6 * 36 and CYP2D6 * 41 and CYP2D6 * 5 copy number variation; wherein the RPP30 reference gene can synchronously realize sample amplification quality control and target gene absolute quantitative calibration. The detection system depends on the advantages of high specificity of the LNA probe and high sensitivity of digital PCR, the detection sensitivity reaches 0.02 ng / mu L, the detection period is short, and the result is stable and reliable.

Description

Technical Field

[0001] This application relates to the field of molecular biology technology, and more specifically, to a digital PCR detection system, kit, method, and application for detecting single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) in the CYP2D6 gene. Background Technology

[0002] Cytochrome P450 2D6 (CYP2D6) is an important drug-metabolizing enzyme that participates in the metabolism of approximately 25% of commonly used drugs in clinical practice. Its gene polymorphisms (such as single nucleotide polymorphisms, copy number variations, etc.) can lead to differences in enzyme activity, which in turn can cause individual differences in drug efficacy or enhanced toxic side effects. Therefore, accurate prediction of enzyme activity through CYP2D6 genotyping is of great significance for guiding individualized drug administration in clinical practice and reducing drug risks.

[0003] However, CYP2D6 forms a homologous gene cluster with CYP2D7 and CYP2D8, with a nucleotide sequence similarity >97%. The presence of these homologous pseudogenes poses a significant technical challenge to the accurate detection of the CYP2D6 gene. Currently, the mainstream detection technologies for CYP2D6 single nucleotide polymorphisms and copy number variations include qPCR, multiplex ligation probe amplification (MLPA), sequencing, and conventional digital PCR. However, these technologies generally have shortcomings: First, they target a single variant, making it difficult to simultaneously cover multiple common clinical variant types; second, their quantitative capabilities are insufficient, such as qPCR relying on standard curves for indirect quantification, which cannot achieve absolutely accurate analysis of copy number variations; third, they are costly and complex, such as MLPA requiring specialized enzymes and quality control reagents, sequencing requiring library construction, high-depth sequencing, and complex bioinformatics analysis, and conventional digital PCR lacking highly specific technologies and being susceptible to interference from homologous sequences; fourth, multi-tube testing further increases the cost and complexity of the process, making it difficult to meet the clinical demand for rapid and accurate detection.

[0004] Therefore, there is an urgent need to develop a detection technology that can simultaneously detect multiple key CYP2D6 variants, possesses high specificity, high sensitivity and absolute quantification capabilities, and is cost-effective and easy to operate. Summary of the Invention

[0005] The purpose of this application is to provide a digital PCR detection system, kit, method and application for detecting single nucleotide polymorphisms and copy number variations of the CYP2D6 gene, which has the characteristics and advantages of comprehensive target coverage (simultaneous detection of 3 SNPs + 1 CNV variant), high specificity (LNA probe eliminates homologous gene interference), high sensitivity (0.02ng / μL) and absolute quantification.

[0006] To achieve the above objectives, this application provides a digital PCR detection system for detecting single nucleotide polymorphisms and copy number variations of the CYP2D6 gene. The detection system can simultaneously detect three SNPs—CYP2D6*10, CYP2D6*36, and CYP2D6*41—and copy number variations of CYP2D6*5. It includes three pairs of specific primers and four TaqMan-specific fluorescent probes. The three pairs of specific primers are a CYP2D6*10 specific primer pair, a CYP2D6*41 specific primer pair, and a RPP30 internal reference gene specific primer pair. The four TaqMan-specific fluorescent probes are all LNA-modified, specifically including: a CYP2D6*1 wild-type probe (FAM label), a CYP2D6*10 mutant probe (ROX label), a CYP2D6*41 mutant probe (CY5 label), and an RPP30 internal reference probe (VIC label).

[0007] Furthermore, the specific primer pair for CYP2D6*10 includes the forward primer CYP2D6*10-F as shown in SEQ ID NO. 1 and the reverse primer CYP2D6*10-R as shown in SEQ ID NO. 2. The sequence of the wild-type probe of CYP2D6*1 is shown in SEQ ID NO. 5, and the sequence of the mutant probe of CYP2D6*10 is shown in SEQ ID NO. 6. The specific primer pair for CYP2D6*41 includes the forward primer CYP2D6*41-F as shown in SEQ ID NO. 3 and the reverse primer CYP2D6*41-R as shown in SEQ ID NO. 4. The sequence of the mutant probe of CYP2D6*41 is shown in SEQ ID NO. 7. The specific primer pair for the internal reference gene RPP30 includes the forward primer RPP30-F as shown in SEQ ID NO. 8 and the reverse primer CYP2D6*10-R as shown in SEQ ID NO. 1. The reverse primer RPP30-R shown in SEQ ID NO. 9, and the sequence of the RPP30 internal reference probe are shown in SEQ ID NO. 10.

[0008] Furthermore, the detection system was a 15 μL PCR reaction system, specifically comprising the following components: 3 μL Probe dPCR HiTapMix reagent A, 0.75 μL Probe dPCR HiTapMix reagent B, 0.8 μL 10 μM CYP2D6*10 forward primer, 0.8 μL 10 μM CYP2D6*10 reverse primer, 0.6 μL 10 μM CYP2D6*1 wild-type probe, 0.6 μL 10 μM CYP2D6*10 mutant probe, 0.6 μL 10 μM CYP2D6*41 forward primer, 0.6 μL 10 μM CYP2D6*41 reverse primer, 0.4 μL 10 μM CYP2D6*41 mutant probe, 0.4 μL 10 μM RPP30 forward primer, and 10 μM... 0.4 μL of RPP30 reverse primer and 2 μL of nucleic acid sample, with the remainder being ddH2O, to be brought to 15 μL.

[0009] Furthermore, the PCR reaction procedure for the detection system is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s; 62℃ extension for 30 s; 40 cycles.

[0010] This application also provides a digital PCR detection kit for detecting single nucleotide polymorphisms and copy number variations in the CYP2D6 gene, the kit comprising a digital PCR detection system.

[0011] This application also provides a method for detecting single nucleotide polymorphisms and copy number variations of the CYP2D6 gene using a digital PCR detection system, comprising the following steps: Genomic DNA was extracted from the sample to be tested, and the DNA concentration and purity were determined for later use. According to the 15μL PCR reaction system ratio, the nucleic acid sample, specific primers, TaqMan specific fluorescent probe and PCR reaction reagents are mixed to obtain the PCR reaction solution; The PCR reaction solution was placed in a digital PCR instrument and amplified using the PCR reaction program. Simultaneously, fluorescence signals were collected using four channels: FAM, VIC, ROX, and CY5. Using the amplification signal of the VIC channel internal reference gene RPP30 as quality control, and assuming the quality control is qualified, the copy number ratio of the target gene to the internal reference gene under each fluorescence channel is calculated. Based on the copy number ratio, the single nucleotide polymorphisms of CYP2D6*10, CYP2D6*36 and CYP2D6*41 and the copy number variation type of CYP2D6*5 are determined.

[0012] Furthermore, the sample to be tested is a blood sample. The steps for extracting genomic DNA from the sample and determining the DNA concentration and purity include: extracting genomic DNA using column extraction or an automated extractor, dissolving the extracted DNA in elution buffer, and determining the concentration and purity using a UV spectrophotometer.

[0013] Furthermore, the criteria for judgment are as follows: (1) Logic for determining CYP2D6*5 (copy number deletion): Since CYP2D6*5 is a whole-gene deletion variant, the determination is made by the ratio of the (FAM+ROX) channel (representing the total copy number of the CYP2D6 gene) to the VIC channel (internal reference RPP30, representing the total copy number of diploid genes). The logic is that when CYP2D6 is deleted (gene number decreases), its copy number will be lower than the normal diploid level. Therefore, a stable copy number of another gene, RPP30, is needed for comparison to determine whether copy number deletion has occurred. That is, the role of the RPP30 gene is to determine the deletion of *5. If a gene representing the total copy number of genes is not used for comparison, it is impossible to determine whether the CYP2D6 gene has been deleted.

[0014] CYP2D6*5 / CYP2D6*5 (homozygous deletion): Only the VIC fluorescent channel has a signal, (FAM+ROX) / VIC=0, indicating that the CYP2D6 gene is completely deleted. Therefore, the FAM (*1 wild type) and ROX (*10 mutant) channels have no signal, and only the internal reference VIC has a signal, with a ratio of 0. CYP2D6*5 / CYP2D6*1 (heterozygous deletion): ROX / FAM and VIC channels show fluorescent signals, (FAM+ROX) / VIC=0.5, indicating that there is only 1 copy of the CYP2D6 gene (normally there are 2 copies). Therefore, FAM / ROX shows a signal but the total amount is halved, and the ratio to VIC is 0.5. Among them, ROX and FAM are mutually exclusive, that is, either ROX or FAM is 0. (2) Regarding the absence of CYP2D6*5, the interpretation criteria for CYP2D6*10, CYP2D6*41, and CYP2D6*36 genotypes are as follows: CYP2D6*5 not missing: FAM, ROX, VIC and CY5 (if CYP2D6*41 mutation occurs) all have fluorescent signals, (FAM+ROX) / VIC=1, indicating that there are 2 normal copies of the CYP2D6 gene, the total amount of FAM / ROX signal matches the internal reference VIC, and the ratio is 1.

[0015] (2.1) Logic for determining CYP2D6*10 (single nucleotide polymorphism): CYP2D6*10 is a single base mutation (c.188C>T), which is determined by the ratio of ROX channel (*10 mutant) to FAM channel (*1 wild type) (i.e. mutation rate).

[0016] CYP2D6*1 / CYP2D6*1 (homozygous wild type): FAM and VIC channels show fluorescent signals, mutation rate = ROX / VIC = 0, indicating no *10 mutation, ROX channel shows no signal, mutation rate is 0; CYP2D6*1 / CYP2D6*10 (heterozygous mutation): mutation rate = ROX / (FAM+ROX) = 0.5, indicating that one copy is wild-type and one copy is mutant, the ROX and FAM signal amounts are equal, and the mutation rate is 0.5; CYP2D6*10 / CYP2D6*10 (homozygous mutation): mutation rate = ROX / VIC = 1, indicating that both copies are mutant, only ROX has a signal, and the mutation rate is 1.

[0017] (2.2) Logic for determining CYP2D6*41 (single nucleotide polymorphism): CYP2D6*41 is a single base mutation (c.2850C>T), and is determined by the ratio of CY5 channel (*41 mutant) to VIC channel.

[0018] CYP2D6*1 / CYP2D6*41 heterozygous mutation: mutation rate = CY5 / VIC = 0.5, indicating that 1 copy is a mutant, the CY5 signal is half of the internal reference, and the ratio is 0.5; CYP2D6*41 / CYP2D6*41 (homozygous mutation): mutation rate = CY5 / VIC = 1, indicating that both copies are mutant, the CY5 signal matches the internal reference, and the ratio is 1; (2.3) Logic for determining CYP2D6*10-*36 (recombinant variant): CYP2D6*10-36 is a variant of recombination between * and CYP2D7. The *36 site has a *10 mutation that leads to an increase in copy number. The ratio of ROX to FAM is used for determination.

[0019] CYP2D6*1 / *10-*36 mutation: ROX / FAM=2, *10 mutation rate=ROX / (FAM+ROX)=0.66, which means 1 wild-type copy (FAM), 2 *10 copies (ROX), one of which is a *36 copy, and the mutation rate is 0.66; CYP2D6*10-*36 / *10-*36: ROX / VIC=2, indicating two *10 copies and two *36 copies. The ROX signal is twice that of the internal reference, with a ratio of 2. The *10 and *36 copies have undergone tandem recombination, which is not a mutation.

[0020] The judgment criteria of this application combine the characteristics of different fluorescent channels corresponding to different targets with the numerical pattern of copy number / mutation rate to accurately distinguish various variant genotypes of CYP2D6.

[0021] This application also provides a digital PCR detection system and a digital PCR detection kit for the application of preparing clinical diagnostic reagents related to CYP2D6 genotyping.

[0022] This application also provides an application of a digital PCR detection system and a digital PCR detection kit in guiding personalized precision medicine.

[0023] In summary, this application has the following beneficial effects: 1. High affinity and high specificity Given the serious interference from highly homologous pseudogenes (homology >97%) of CYP2D7 and CYP2D8 and single differential SNP sites in CYP2D6 gene detection, this application employs a locked nucleic acid (LNA) modified TaqMan probe, which has stronger specificity and binding affinity. It can accurately identify and distinguish the target sequence from highly homologous pseudogene sequences and effectively identify single nucleotide polymorphisms, thereby achieving ultra-high specificity for CYP2D6 genotyping in complex genetic backgrounds.

[0024] 2. High sensitivity and absolute quantification This application leverages the single-molecule absolute quantification capability of digital PCR, enabling the accurate determination of CYP2D6*5 copy number deletion and the direct and precise quantification of the frequency of mutant alleles such as CYP2D6*10 in a single reaction using only a nucleic acid sample with a concentration of 0.02 ng / μL. This method overcomes the accuracy limitations caused by the inability of traditional techniques to achieve absolute quantification, providing a more reliable quantitative basis for genotyping.

[0025] 3. High accuracy and good repeatability This application creatively combines LNA locked nucleic acid technology with digital PCR technology, using only three pairs of primers and four specific probes to simultaneously detect four gene polymorphisms—CYP2D6*10, CYP2D6*36, CYP2D6*41, and CYP2D6*5 copy number variations—in a single reaction tube, covering the most common clinically significant gene variant sites in East Asian populations. Methodological validation showed that the amplification efficiency of all primers and probes in this system was stable, with R... 2All values ​​were greater than 0.99; the theoretical copy number and the actual detected copy number also showed a high degree of consistency (R0). 2 The result >0.99 indicates that it has excellent repeatability and quantitative accuracy, and the results are stable and reliable.

[0026] 4. Achieve personalized and precise medication The detection system described in this application achieves accurate classification of patients' metabolic phenotypes through precise typing of key CYP2D6 variants, providing direct and objective genetic evidence for personalized treatment. It can directly guide clinical practice in optimizing dosages and selecting treatment regimens for specific drugs, thereby improving the accuracy of treatment response. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 These are the amplification curves and standard curves proposed in the embodiments of this application, using CYP2D6*1, CYP2D6*10, and CYP2D6*41 plasmids as templates, respectively; wherein, Figure 1 A and Figure 1 In the diagram, D represents the amplification curve and the standard curve using the CYP2D6*1 plasmid as a template, respectively. Figure 1 B and Figure 1 In the diagram, E represents the amplification curve and the standard curve using the CYP2D6*10 plasmid as a template, respectively. Figure 1 C and Figure 1 In the figure, F represents the amplification curve and the standard curve using CYP2D6*41 plasmid as template, respectively.

[0029] Figure 2 This refers to the use of four different internal reference genes as the sum of the CYP2D6 gene copy numbers, as proposed in the embodiments of this application.

[0030] Figure 3 This refers to the probe specificity verification and results proposed in the embodiments of this application; wherein, Figure 3 In this context, A represents the copy number detected in the four fluorescence channels of four typical samples. Figure 3 In this context, "B" indicates the verification of the CYP2D6*1 wild-type probe against the CYP2D6*10 mutant template. Figure 3 In this context, C represents the verification of the CYP2D6*1 wild-type probe with the CYP2D6*1 wild-type template. Figure 3The "D" in the diagram represents the verification of the CYP2D6*10 mutant probe against the CYP2D6*1 wild-type template. Figure 3 In this context, "E" indicates validation of the CYP2D6*10 mutant probe against the CYP2D6*10 mutant template. Figure 3 In this context, F indicates the validation of the CYP2D6*41 mutant probe against the CYP2D6*41 wild-type template. Figure 3 In this context, G represents the verification of the CYP2D6*41 mutant probe with the CYP2D6*41 mutant template.

[0031] Figure 4 This is the sensitivity verification proposed in the embodiments of this application, wherein, Figure 4 In this context, A indicates that CYP2D6*1, CYP2D6*10, and CYP2D6*41 are used as templates, and their values ​​are increased from 10... 3 Copies / μL diluted to 10 0 The number of copies detected at copies / μL Figure 3 In the diagram, BD represents a one-dimensional plot of the FAM, ROX, and CY5 channels at four different concentrations.

[0032] Figure 5 This application's embodiments present a linear relationship between the theoretical copy number obtained by using gradient dilutions of CYP2D6*1, CYP2D6*10, and CYP2D6*41 as templates and the actual detected copy number; wherein, Figure 5 In this context, A represents the linear relationship of CYP2D6*1. Figure 5 In this context, B represents the linear relationship of CYP2D6*10. Figure 5 In this context, C represents the linear relationship of CYP2D6*41.

[0033] Figure 6 This application presents a series of one-dimensional detection graphs of different concentrations at varying dilutions in different channels, along with the linear relationship between concentration and copy number. Figure 6 In this context, A represents channel CY5. Figure 6 In this context, B represents the FAM channel. Figure 6 In this context, C represents the ROX channel. Figure 6 In this context, D represents the VIC channel. Figure 6 In this context, E represents the linear relationship between the four concentration samples diluted tenfold and the copy number detected by the four fluorescence channels.

[0034] Figure 7 These are the detection results and one-dimensional images of four typical samples proposed in the embodiments of this application; among them, Figure 7 In this context, A represents the test results of four typical samples. Figure 7In the diagram, BE represents a one-dimensional plot of four typical samples in the FAM, ROX, VIC, and CY5 channels, respectively.

[0035] Figure 8 These are the detection results and ROC plots of different genotypes of the samples proposed in the embodiments of this application; wherein, Figure 8 In this context, A represents the detection results of different genotypes across all samples. Figure 8 In the diagram, BD represents the ROC plots of CYP2D6*5, CYP2D6*10, and CYP2D6*41, respectively.

[0036] Figure 9 This is a schematic diagram of a method for detecting CYP2D6 single nucleotide polymorphism and copy number variation using a digital PCR detection system provided in this application; where A represents the A425 channel, F represents the FAM channel, R represents the ROX channel, V represents the VIC channel, 5 represents the CY5 channel, and 7 represents the CY7 channel. Detailed Implementation

[0037] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0038] CYP2D6 is a key drug-metabolizing enzyme in the cytochrome P450 superfamily. Its encoding gene is located on human chromosome 22q13.1 and consists of 9 exons and 8 introns, primarily distributed in liver tissue. Although this enzyme accounts for only 5% of the total CYP450 enzyme protein in the liver, it participates in the metabolism of approximately 25% of commonly used drugs in clinical practice, covering several core categories including antidepressants, antiarrhythmics, antipsychotics, and analgesics. It is one of the earliest drug-metabolizing enzymes to be confirmed to have genetic polymorphism. Gene polymorphism refers to stable variations in gene sequences within a population, including single nucleotide polymorphisms (SNPs), base insertions / deletions, copy number variations (CNVs, which refer to an increase or decrease in gene copy number compared to normal), and gene recombination. Among these, CYP2D6*10 (c.188C>T single base mutation) and CYP2D6*41 (c.2850C>T variation) are weakened functional alleles, while CYP2D6*5 (whole gene deletion) and CYP2D6*36 (recombination with CYP2D7 sequence) are non-functional alleles. These are all high-frequency variants with significant clinical importance in East Asian populations.

[0039] The enzyme activity scoring system established by authoritative clinical institutions shows that there are significant differences in enzyme activity among different CYP2D6 alleles, which can classify individual metabolic phenotypes into four categories: ultra-rapid metabolizers, normal metabolizers, intermediate metabolizers, and weak metabolizers. These differences in metabolic phenotypes can directly lead to insufficient efficacy or enhanced toxic side effects in clinical drug use. Therefore, accurate prediction of enzyme activity through CYP2D6 genotyping is a crucial prerequisite for guiding the development of individualized clinical dosing regimens and reducing drug risks. However, accurate detection of the CYP2D6 gene faces two major technological challenges: first, homologous gene interference. CYP2D6, CYP2D7, and CYP2D8 form a homologous gene cluster, with a nucleotide sequence similarity exceeding 97%. CYP2D7 and CYP2D8 are pseudogenes (non-functionally expressed), which can easily lead to non-specific binding during the detection process, affecting accuracy. Second, the complexity of variant types necessitates the simultaneous and accurate detection of multiple polymorphic sites and copy number variations, requiring extremely high technical compatibility and overall performance.

[0040] Currently, the mainstream technologies used for detecting CYP2D6 single nucleotide polymorphisms and copy number variations include: (1) quantitative real-time PCR (QPCR), which relies on Ct values ​​and standard curves for indirect quantification. It is simple to operate but cannot achieve absolutely accurate quantification of copy number variations. Moreover, it lacks specificity for recognizing single base mutations and is easily affected by homologous sequences; (2) multiplex ligation probe amplification (MLPA), which can detect copy number variations, but has strict requirements for the quality of template nucleic acids, requires special enzymes and quality control reagents, has high detection costs, and has a complicated experimental process and limited throughput; (3) sequencing technology (including San... (4) While conventional digital PCR technology has absolute quantitative capabilities, it does not integrate high-specificity recognition technology, making it difficult to solve the problem of homologous gene interference. Moreover, most schemes have the problem of single detection target, which cannot simultaneously cover multiple key variant types commonly seen in clinical practice. Some schemes require the use of multi-tube detection mode, which further increases the detection cost and operational complexity. Based on this, this application provides a CYP2D6 gene detection technology that has high specificity (can avoid homologous gene interference), high sensitivity, absolute quantitative capabilities, and can realize multi-target simultaneous detection with controllable cost.

[0041] Specifically, in the first aspect, this application provides a digital PCR detection system for detecting single nucleotide polymorphisms and copy number variations of the CYP2D6 gene. The detection system can simultaneously detect polymorphisms of three genes, CYP2D6*10, CYP2D6*36, and CYP2D6*41, as well as copy number variations of the CYP2D6*5 gene. It includes three pairs of specific primers and four TaqMan specific fluorescent probes. The three pairs of specific primers are CYP2D6*10 specific primer pair, CYP2D6*41 specific primer pair, and RPP30 internal reference gene specific primer pair. The four TaqMan specific fluorescent probes are all LNA modified, specifically including: CYP2D6*1 wild-type probe (FAM label), CYP2D6*10 mutant probe (ROX label), CYP2D6*41 mutant probe (CY5 label), and RPP30 internal reference probe (VIC label). This application designs specific TaqMan probes for CYP2D6*1 wild-type (labeled with FAM fluorescent group) and CYP2D6*10 mutant probes (labeled with ROX fluorescent group) for CYP2D6*10. For the mutant sequence of CYP2D6*41, a specific probe (labeled with CY5 fluorescent group) is designed for detection. Furthermore, RPP30 is used as an internal reference gene, and a VIC-labeled probe is used for detection. This serves to monitor sample extraction and amplification efficiency and represents the total DNA template amount in the reaction system, used for absolute quantification of CYP2D6 copy number. Then, by calculating the ratio of allele copy number to internal reference gene copy number under different fluorescence channels, the copy number variation and mutation rate of different polymorphic alleles are detected and determined.

[0042] In specific embodiments, the specific primer pair for CYP2D6*10 includes the forward primer CYP2D6*10-F as shown in SEQ ID NO. 1 and the reverse primer CYP2D6*10-R as shown in SEQ ID NO. 2. The sequence of the wild-type probe of CYP2D6*1 is shown in SEQ ID NO. 5, and the sequence of the mutant probe of CYP2D6*10 is shown in SEQ ID NO. 6. The specific primer pair for CYP2D6*41 includes the forward primer CYP2D6*41-F as shown in SEQ ID NO. 3 and the reverse primer CYP2D6*41-R as shown in SEQ ID NO. 4. The sequence of the mutant probe of CYP2D6*41 is shown in SEQ ID NO. 7. The specific primer pair for the internal reference gene RPP30 includes the forward primer RPP30-F as shown in SEQ ID NO. 8 and the reverse primer CYP2D6*10-R as shown in SEQ ID NO. 6. The reverse primer RPP30-R shown in NO.9, and the sequence of the RPP30 internal reference probe are shown in SEQ ID NO. 10. The sequences of the aforementioned primers and probes are shown in Table 1.

[0043] Table 1 Primer and probe sequences

[0044] In Table 1, "+" indicates LNA modification, MT represents mutant, WT represents wild type, F represents the front primer, R represents the back primer, and P represents the probe. The bolded positions in the sequence indicate that the base was modified with LNA.

[0045] This application also provides a digital PCR detection kit for detecting single nucleotide polymorphisms and copy number variations in the CYP2D6 gene, the kit comprising a digital PCR detection system.

[0046] This application also provides a method for detecting single nucleotide polymorphisms and copy number variations of the CYP2D6 gene using a digital PCR detection system, comprising the following steps: S1. Extract genomic DNA from the sample to be tested, determine the DNA concentration and purity, and then set it aside for later use.

[0047] In a specific implementation, the sample to be tested is a blood sample. The steps of extracting genomic DNA from the sample and determining the DNA concentration and purity include: extracting genomic DNA using column extraction or an automated extractor, dissolving the extracted DNA in an elution buffer, and determining the concentration and purity using a UV spectrophotometer.

[0048] S2. According to the 15μL PCR reaction system ratio, mix the nucleic acid sample, specific primers, TaqMan specific fluorescent probe and PCR reaction reagent to obtain the PCR reaction solution.

[0049] S3. Place the PCR reaction solution in a digital PCR instrument and amplify it using the PCR reaction program. At the same time, collect fluorescence signals using the four channels FAM, VIC, ROX and CY5.

[0050] S4. Using the amplification signal of the VIC channel internal reference gene RPP30 as quality control, and assuming the quality control is qualified, calculate the copy number ratio of the target gene to the internal reference gene in each fluorescence channel. Based on the ratio, determine the single nucleotide polymorphisms of CYP2D6*10, CYP2D6*36, and CYP2D6*41, and the copy number variation type of CYP2D6*5. The criteria for determination are as follows: CYP2D6*5 / CYP2D6*5 (homozygous deletion): Signal is present only in the VIC fluorescence channel, (FAM+ROX) / VIC=0; CYP2D6*5 / CYP2D6*1 (heterozygous deletion): Fluorescent signals were present in the ROX / FAM and VIC channels, (FAM+ROX) / VIC=0.5; CYP2D6*5 not missing: FAM, ROX, VIC, and CY5 (if CYP2D6*41 mutation occurs) all show fluorescent signals, (FAM+ROX) / VIC=1; Regarding the absence of CYP2D6*5, the interpretation of CYP2D6*10, CYP2D6*41, and CYP2D6*36 genotypes is as follows: CYP2D6*1 / CYP2D6*1: Only the FAM and VIC channels have fluorescence signals, and the copy numbers of the two fluorescence channels are basically the same; CYP2D6*10 / CYP2D6*1:*10 mutation rate = ROX / (FAM+ROX) = 0.5; CYP2D6*10 / CYP2D6*10:*10 mutation rate = ROX / VIC = 1; CYP2D6*41 / CYP2D6*1:*41 mutation rate = CY5 / VIC = 0.5; CYP2D6*41 / CYP2D6*41: *41 mutation rate = CY5 / VIC = 1; CYP2D6*1 / *10-*36 mutation: ROX / FAM=2, *10 mutation rate=ROX / (FAM+ROX)=0.66; CYP2D6*10-*36 / *10-*36: ROX / VIC=2.

[0051] The principle of the method described above in this application is as follows: Figure 9As shown, during PCR amplification, primers extend under the action of DNA polymerase, and the specific TaqMan probes bound to them are hydrolyzed. Precise detection of target molecules can be achieved by detecting changes in the fluorescence signal labeled by the probes. Using synthesized plasmid standards and real blood samples, the optimized detection system of this application can specifically detect the genotypes of five variant molecules, with a detection limit as low as single copy, exhibiting good sensitivity and repeatability. Figure 9 In the diagram, the red flowchart at the top represents the following steps: collecting clinical blood samples; extracting genomic DNA from the samples using automated equipment; preparing a PCR reaction system containing primers, probes, and templates; dividing the reaction system into numerous droplets using a digital PCR instrument; completing the amplification reaction in the PCR instrument; acquiring multi-channel fluorescence signals and generating a two-dimensional map of droplet distribution; and interpreting the results. Figure 9 The figure on the lower left shows the exon structure of the CYP2D6 gene, as well as the specific binding relationships between different fluorescent probes (FAM / ROX / CY5 / VIC) and the target variant sites (*10, *41) and the internal reference gene (RPP30). Figure 9 The diagram in the middle at the bottom illustrates the amplification principle, which is that after the primer binds to the template, it extends, and the Taq enzyme hydrolyzes the bound probe to release a fluorescent signal. Figure 9 The diagram on the lower right clearly shows the copy number of the detection target corresponding to each fluorescence channel (FAM / ROX / VIC / CY5, etc.), explaining the principle of multi-channel differentiation of different targets.

[0052] The testing system in this application can be verified by the following methods: (1) Design specific primers and TaqMan probes for the target variant sites (CYP2D6*10, *41) and the internal reference gene RPP30.

[0053] (2) Synthesize plasmid standards containing the target variant sequence and serially dilute them to different copy numbers (10). 10 copies / μL~10 0 (copies / μL), of which the plasmid DNA was synthesized by a company in Tianjin.

[0054] (3) Mix the plasmid template to be tested, primers / probes of all target genes / internal reference genes in the same reaction tube according to the optimized ratio, run the amplification program using the specified model digital PCR instrument (Linghang D3200), and collect the four-channel fluorescence signals of FAM, VIC, ROX and CY5 at the same time.

[0055] (4) Determine whether the system only produces a positive signal for the target sequence and does not bind to homologous sequences or other irrelevant sequences, clarify the component ratio and amplification program of the final 15μL reaction system, and confirm the role of the internal reference gene RPP30.

[0056] In a specific implementation, the optimized detection system is a 15 μL PCR reaction system, specifically comprising the following components: 3 μL Probe dPCR HiTapMix reagent A (Beijing Xinyi Biotechnology Co., Ltd.), 0.75 μL Probe dPCR HiTapMix reagent B (Beijing Xinyi Biotechnology Co., Ltd.), 0.8 μL 10 μM CYP2D6*10 forward primer, 0.8 μL 10 μM CYP2D6*10 reverse primer, 0.6 μL 10 μM CYP2D6*1 wild-type probe, 0.6 μL 10 μM CYP2D6*10 mutant probe, 0.6 μL 10 μM CYP2D6*41 forward primer, 0.6 μL 10 μM CYP2D6*41 reverse primer, 0.4 μL 10 μM CYP2D6*41 mutant probe, 0.4 μL 10 μM RPP30 forward primer, and 10 μM... 0.4 μL of RPP30 reverse primer and 2 μL of nucleic acid sample were added, with the remaining volume being ddH2O, brought to a final volume of 15 μL. The PCR reaction program for the detection system was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 62℃ extension for 30 s, for 40 cycles.

[0057] The technical solutions described above in this application will be explained in detail below with reference to specific embodiments.

[0058] Example 1 This embodiment aims to construct and validate a digital PCR detection system for detecting single nucleotide polymorphisms and copy number variations in the CYP2D6 gene, and to clarify its standardized clinical application process, ultimately providing a reliable genotyping basis for personalized medication guidance. Specifically, it includes the following steps: Step 1. Synthesize plasmids, extract and dilute DNA samples A positive plasmid standard was constructed by inserting a sequence designed based on the CYP2D6 genomic reference sequence from NCBI into the PUC57 vector, with the Amp+ gene selected as the resistance gene. The extracted plasmid standard was diluted 10-fold with TE buffer to a final concentration of 10. 0 Store in copies / μL at -20℃. The plasmid was synthesized by a company in Tianjin.

[0059] Genomic DNA was extracted from blood using the Tiangen Blood Genomic DNA Extraction Kit (column extraction method) or the Xinbaiji Automated Extractor, following the instructions in the user manual. The extracted DNA was dissolved in 200 μL of elution buffer, and the concentration and purity of the extracted nucleic acid were determined using a NanodropOne UV spectrophotometer. The qPCR amplification curves using CYP2D6*1 plasmid, CYP2D6*10 plasmid, and CYP2D6*41 plasmid as templates, respectively, were compared with the standard curve as shown below. Figure 1 As shown.

[0060] Step 2. Design primers and probes Primer and probe sequences are detailed in Table 1. Each primer and fluorescent probe was diluted to 10 μmol / L with TE buffer and stored at -20℃. Samples were loaded using the optimized primer-probe concentration ratio. All primers and probes were synthesized by a company in Shanghai.

[0061] Step 3. Sample testing To ensure the accuracy of the results when using the Navigator D3200 digital PCR instrument, it is recommended that the sample loading concentration be above 10 ng / μL. Add the primers and probes for the target gene and the internal reference gene RPP30 to one tube at the same time, and use the FAM channel (CYP2D6*1), VIC channel (RPP30), ROX channel (CYP2D6*10), and CY5 channel (CYP2D6*41) for four-channel fluorescence acquisition.

[0062] The parameters were set as follows: 94℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s; 62℃ extension for 30 s, for a total of 40 cycles. Amplification was performed using the Probe dPCR HiTapMix kit (catalog number 23027, Xinyi Biotechnology).

[0063] The amplification system consisted of: 3 μL Probe dPCR HiTapMix reagent A, 0.75 μL Probe dPCR HiTapMix reagent B, 0.8 μL 10 μM CYP2D6*10 forward primer, 0.8 μL 10 μM CYP2D6*10 reverse primer, 0.6 μL 10 μM CYP2D6*1 wild-type probe, 0.6 μL 10 μM CYP2D6*10 mutant probe, 0.6 μL 10 μM CYP2D6*41 forward primer, 0.6 μL 10 μM CYP2D6*41 reverse primer, 0.4 μL 10 μM CYP2D6*41 mutant probe, 0.4 μL 10 μM RPP30 forward primer, and 10 μM... 0.4 μL of RPP30 reverse primer and 2 μL of nucleic acid sample, with the remainder being ddH2O, to be brought to 15 μL.

[0064] Step 4. Results Analysis As a quality control, the internal reference gene must exhibit an amplification curve or positive droplet. Under the premise that an amplification curve or positive droplet (VIC) is observed for the internal reference gene, the copy number of positive droplets from specific probes (FAM, ROX, and CY5) is checked. The results of different internal reference genes used for CYP2D6 copy number quantification are compared, for example... Figure 2As shown in the diagram. In this application, if the copy number of a positive droplet or the fluorescence signal reaches or exceeds the positive threshold, it indicates that the specific primers and probes for the target fragment have bound to the DNA template, and that the primers can extend successfully, with the base sequence in the primer and probe coverage region being complementary to the template sequence. Then, the copy number of all fluorescence channels is calculated using the method described in this application.

[0065] This embodiment uses 40 blood samples collected from the Xi'an Regional Medical Testing Center as an example. Genomic DNA was extracted, and the nucleic acid samples were tested using the established digital PCR method. ddH2O was used as a negative control template. The consistency analysis of the detection method results in this application with the clinical sample results (verified by qPCR and first-generation sequencing) was performed.

[0066] Specifically, the results include the following: (1) such as Figure 1 As shown, plasmid standards corresponding to CYP2D6*1, CYP2D6*10, and CYP2D6*41 were first prepared. Each plasmid was then added from a high concentration (10... 6 From copies / μL) to low concentrations (10) 0 The plasmid templates were serially diluted 10-fold (copies / μL) to obtain multiple concentration gradients. Using the primers and probes designed in this application, qPCR amplification was performed on each concentration gradient of the plasmid template, and fluorescence signals were collected to obtain... Figure 1 The amplification curves for A, B, and C were obtained. Then, the logarithm of plasmid template concentration was plotted on the x-axis and the Ct value of qPCR amplification was plotted on the y-axis to obtain the desired result. Figure 1 The standard curves for D, E, and F.

[0067] Depend on Figure 1 The amplification curves show that clear S-shaped amplification curves were observed for plasmid templates of different concentration gradients, indicating that the primers and probes could effectively bind to the template and complete amplification, with no obvious non-specific amplification. Figure 1 As can be seen from the standard curve, the R value of the standard curve... 2 All values ​​were greater than 0.99, indicating a very strong linear relationship between concentration and Ct value, and stable quantitative results. The primers and probes designed in this application both have good amplification efficiency.

[0068] (2) such as Figure 2As shown, using four different internal reference genes as the sum of CYP2D6 gene copy numbers, only RPP30 met the theoretical results: for CYP2D6*5 without deletion, the ratio was around 1; for CYP2D6*5 heterozygous deletion, the ratio was around 0.5; for CYP2D6*41 heterozygous deletion, the ratio was around 0.5; and for CYP2D6*41 without deletion, the ratio was around 1 (+: no deletion; -: deletion). This demonstrates that RPP30 is the optimal internal reference gene for CYP2D6 copy number quantification in this detection system, and can be used as a reference to determine CYP2D6 gene polymorphism and copy number variation.

[0069] (3) Pairing experiments were conducted with different combinations of probes (CYP2D6*1 wild-type probe, CYP2D6*10 mutant probe, CYP2D6*41 mutant probe) and templates (wild-type, mutant, pseudogene). The specificity of probe-template binding was verified by analyzing the droplet signal distribution in digital PCR (genotyping map of A, channel signal map of BG). Figure 3 As shown in Figure A, droplets of different genotypes can be accurately detected. In Figure BG, each probe only produces a specific fluorescent signal with a completely complementary template (e.g., the CYP2D6*1 wild-type probe only matches the CYP2D6*1 wild-type template, and the CYP2D6*10 mutant probe only matches the CYP2D6*10 mutant template). The three sequence-specific fluorescent probes only pair with completely complementary genotypes and do not pair with single-base mutation SNPs or pseudogenes. This indicates that the LNA-modified TaqMan probes in this application have high specificity and solve the interference problem between CYP2D6 and homologous pseudogenes (CYP2D7 / 8).

[0070] (4) such as Figure 4 (The results of the sensitivity verification of the detection system are shown.) The plasmid templates of CYP2D6*1, CYP2D6*10, and CYP2D6*41 were changed from 10... 3 Copies / μL diluted to 10 0 The copy number of template at each concentration was determined using the multiplex digital PCR system described in this application, and the results were obtained. Figure 4 The number of copies of A detected in the middle and Figure 4 One-dimensional plot of droplet signal in BD. A shows that templates with different concentration gradients can be effectively quantified, and BD shows that even template concentrations as low as 10... 0 At the single-copy level, the multiplex digital PCR system of this application can still stably capture positive signals, achieving single-copy level detection.

[0071] (5) such as Figure 5(Verification results of quantitative accuracy and stability of the detection system) As shown, CYP2D6*1, CYP2D6*10 and CYP2D6*41 were serially diluted as templates. A linear relationship was fitted with "theoretical copy number of template" as the x-axis and "actual copy number detected by digital PCR" as the y-axis. At the same time, the background signal (LOB) of blank samples and the detection ability (LOD) of the lowest concentration template were verified. Figure 5 In AC, the linear correlation coefficient (R²) between the theoretical copy number of CYP2D6*1, CYP2D6*10, and CYP2D6*41 and the actual detected value is... 2 All values ​​> 0.996, indicating a very strong linear relationship. The background signal (LOB) of the blank sample < 1, and the lowest concentration template (LOD) can be stably detected, demonstrating excellent accuracy and stability of the detection results. Figure 5 The numerical values ​​show that the detection system of this application has good repeatability, high sensitivity and strong stability, which also demonstrates the technical superiority of this method in quantitative detection and meets the methodological validation standards for clinical molecular diagnostics.

[0072] (6) For example Figure 6 (The results of the sensitivity and dynamic range verification of the detection system) show that the initial concentration of 20 ng / μL sample was diluted tenfold to 2 ng / μL, 0.2 ng / μL and 0.02 ng / μL. The signals of the four concentration samples were detected by four channels: FAM, VIC, ROX and CY5, and a one-dimensional detection map was plotted. Figure 6 (AD in the text); simultaneously, using "sample concentration" as the x-axis and "copy number of each channel" as the y-axis, a linear relationship was fitted ( Figure 6 (E in the diagram). As can be seen from AD, even with template concentrations as low as 0.02 ng / μL, the four channels can still stably detect signals. The linear relationship between the copy number of the four channels and the sample concentration in E is good, proving that the system can cover a dynamic range of 0.02 ng / μL to 20 ng / μL, and can detect low-abundance targets (such as low-copy genes in clinical samples) and accurately quantify high-abundance templates.

[0073] (7) For example Figure 7 (Verification of Clinical Sample Test Results) As shown, four typical clinical samples (including *10-*36, *41 heterozygous mutations, *5 complete deletion, etc.) were selected and tested using the multiplex digital PCR system of this application to obtain the genotyping diagrams of each sample. Figure 7 A) and the one-dimensional signal diagram of the four channels FAM / ROX / VIC / CY5 ( Figure 7 The results were compared with clinical validation results from first-generation sequencing and qPCR. Figure 7 As can be seen from A, it conforms to the theoretical ratio and has 100% consistency with clinical information (through first-generation sequencing and qPCR). Figure 7The BE (Beta-Brain) results show clear differentiation between positive and negative droplets, with no specific amplification. This demonstrates the clinical applicability and accuracy of the detection system proposed in this application. It not only performs excellently with plasmid standards but also accurately types actual clinical samples, and its results are completely consistent with existing clinical validation techniques.

[0074] (8) such as Figure 8 (Verification of Sample Detection Accuracy and Discrimination Efficacy) shows that the results of different CYP2D6 genotypes (*5, *10, 41 related variants) of all tested samples were statistically analyzed, and a graph was drawn to show the correspondence between the detected results and the theoretical values. Figure 8 (A in the text); and simultaneously, for the three variant types of 5, *10, and *41, receiver operating characteristic (ROC) curves were constructed. Figure 8 BD in the curve and calculate the area under the curve (AUC). Figure 8 The result A in the figure shows that the genotype detection results of all samples are consistent with the theoretical value, proving that the detection system of this application has 100% accuracy in detecting all samples and has no erroneous typing. Figure 8 In the BD, the AUC values ​​of the ROC curves corresponding to *5, *10, and *41 are all 1, which means that the detection system has achieved the best ability to distinguish these three types of variants (100% accurate distinction between positive and negative samples). This quantitatively verifies that the system's ability to distinguish different CYP2D6 variant types has reached the gold standard level.

[0075] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. Although preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of this application.

[0076] Finally, it should be noted that in this application, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes the element.

[0077] This application uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A digital PCR detection system for detecting single nucleotide polymorphisms and copy number variations in the CYP2D6 gene, characterized in that, The detection system can simultaneously detect three single nucleotide polymorphisms (SNPs) – CYP2D6*10, CYP2D6*36, and CYP2D6*41 – as well as copy number variation of CYP2D6*5, including three pairs of specific primers and four TaqMan specific fluorescent probes. The three pairs of specific primers are the CYP2D6*10 specific primer pair, the CYP2D6*41 specific primer pair, and the internal reference gene RPP30 specific primer pair, respectively. The four TaqMan-specific fluorescent probes were all modified with LNA, specifically including: CYP2D6*1 wild-type probe, CYP2D6*10 mutant probe, CYP2D6*41 mutant probe and RPP30 internal reference probe.

2. The digital PCR detection system for detecting single nucleotide polymorphisms and copy number variations of the CYP2D6 gene according to claim 1, characterized in that, The specific primer pair for CYP2D6*10 includes the forward primer CYP2D6*10-F as shown in SEQ ID NO. 1 and the reverse primer CYP2D6*10-R as shown in SEQ ID NO.

2. The sequence of the wild-type CYP2D6*1 probe is shown in SEQ ID NO. 5, and the sequence of the mutant CYP2D6*10 probe is shown in SEQ ID NO.

6. The specific primer pair for CYP2D6*41 includes the forward primer CYP2D6*41-F as shown in SEQ ID NO. 3 and the reverse primer CYP2D6*41-R as shown in SEQ ID NO.

4. The sequence of the mutant probe of CYP2D6*41 is shown in SEQ ID NO.

7. The specific primer pair for the internal reference gene RPP30 includes the forward primer RPP30-F as shown in SEQ ID NO. 8 and the reverse primer RPP30-R as shown in SEQ ID NO. 9, and the sequence of the RPP30 internal reference probe is shown in SEQ ID NO.

10.

3. The digital PCR detection system for detecting single nucleotide polymorphisms and copy number variations of the CYP2D6 gene according to claim 1, characterized in that, The detection system is a 15 μL PCR reaction system, specifically comprising the following components: 3 μL Probe dPCR HiTapMix reagent A, 0.75 μL Probe dPCR HiTapMix reagent B, 0.8 μL 10 μM CYP2D6*10 forward primer, 0.8 μL 10 μM CYP2D6*10 reverse primer, 0.6 μL 10 μM CYP2D6*1 wild-type probe, 0.6 μL 10 μM CYP2D6*10 mutant probe, 0.6 μL 10 μM CYP2D6*41 forward primer, 0.6 μL 10 μM CYP2D6*41 reverse primer, 0.4 μL 10 μM CYP2D6*41 mutant probe, 0.4 μL 10 μM RPP30 forward primer, and 10 μM... 0.4 μL of RPP30 reverse primer and 2 μL of nucleic acid sample, with the remainder being ddH2O, to be brought to 15 μL.

4. The digital PCR detection system for detecting single nucleotide polymorphisms and copy number variations of the CYP2D6 gene according to claim 1, characterized in that, The PCR reaction procedure for the detection system is as follows: Pre-denaturation at 95℃ for 5 minutes; Denaturation at 95℃ for 10 seconds, extension at 62℃ for 30 seconds, cycled 40 times.

5. A digital PCR detection kit for detecting single nucleotide polymorphisms and copy number variations in the CYP2D6 gene, characterized in that, The kit comprises the digital PCR detection system according to any one of claims 1-4.

6. A method for detecting single nucleotide polymorphisms and copy number variations in the CYP2D6 gene, characterized in that, Based on the digital PCR detection system as described in any one of claims 1-4, the method includes the following steps: Genomic DNA was extracted from the sample to be tested, and the DNA concentration and purity were determined for later use. According to the 15μL PCR reaction system ratio, the nucleic acid sample, specific primers, TaqMan specific fluorescent probe and PCR reaction reagents are mixed to obtain the PCR reaction solution; The PCR reaction solution was placed in a digital PCR instrument and amplified using a PCR reaction program. Simultaneously, fluorescence signals were collected using four channels: FAM, VIC, ROX, and CY5. Using the amplification signal of the VIC channel internal reference gene RPP30 as quality control, and assuming the quality control is qualified, the copy number ratio of the target gene to the internal reference gene in each fluorescence channel is calculated. Based on the copy number ratio, the genotypes of CYP2D6*10, CYP2D6*36 and CYP2D6*41 and the copy number variation type of CYP2D6*5 are determined.

7. The method according to claim 6, characterized in that, The sample to be tested is a blood sample. The steps of extracting genomic DNA from the sample to be tested, determining the DNA concentration and purity, and then using it for later use include: Genomic DNA was extracted using column extraction or an automated extractor, and the extracted DNA was dissolved in elution buffer. The concentration and purity were then determined using a UV spectrophotometer.

8. The method according to claim 6, characterized in that, The criteria for the determination are as follows: CYP2D6*5 / CYP2D6*5: Signal is present only in the VIC fluorescence channel, (FAM+ROX) / VIC=0; CYP2D6*5 / CYP2D6*1: Fluorescent signals are present in the ROX / FAM and VIC channels, (FAM+ROX) / VIC=0.5; CYP2D6*5 is not missing: FAM, ROX, VIC and CY5 all have fluorescent signals, (FAM+ROX) / VIC=1; In the absence of CYP2D6*5 deletion, the criteria for interpreting the CYP2D6*10, CYP2D6*41, and CYP2D6*36 genotypes are as follows: CYP2D6*1 / CYP2D6*1: Only the FAM and VIC channels have fluorescence signals, and the copy number of the two fluorescence channels is the same; CYP2D6*10 / CYP2D6*1:*10 mutation rate = ROX / (FAM+ROX) = 0.5; CYP2D6*10 / CYP2D6*10:*10 mutation rate = ROX / VIC = 1; CYP2D6*41 / CYP2D6*1:*41 mutation rate = CY5 / VIC = 0.5; CYP2D6*41 / CYP2D6*41: *41 mutation rate = CY5 / VIC = 1; CYP2D6*1 / *10-*36 mutation: ROX / FAM=2, *10 mutation rate=ROX / (FAM+ROX)=0.66; CYP2D6*10-*36 / *10-*36: ROX / VIC=2.

9. The application of the digital PCR detection system according to any one of claims 1-4 or the digital PCR detection kit according to claim 5 in the preparation of clinical diagnostic reagents related to CYP2D6 genotyping.

10. The application of the digital PCR detection system according to any one of claims 1-4 or the digital PCR detection kit according to claim 5 in guiding personalized precision medicine.

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