Primer composition and application thereof in preparation of product for detecting drug-related SNP (Single Nucleotide Polymorphism) site of antipsychotic drug

Abstract

The invention discloses a primer composition and application thereof in preparation of a product for detecting drug-related SNP (Single Nucleotide Polymorphism) sites of antipsychotic drugs. According to the invention, a group of primer compositions capable of sensitively and specifically detecting 18 SNP loci related to the administration of antipsychotic drugs are obtained aiming at 18 SNP loci screened according to literature reports, clinical pharmacological genetics, alliance guidelines and the like, and the corresponding kit is constructed by utilizing the primer compositions. By utilizing the primer composition or the kit, the 18 SNP sites can be detected in combination with time-of-flight mass spectrometry, personalized medication of antipsychotic drugs is guided according to the detection conditions of the SNP sites, the conditions of no medication reaction, serious side effects and the like are avoided, and treatment of schizophrenia and other psychoses is facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection. More specifically, it relates to a primer composition and its application in the preparation of a product for detecting SNP sites related to the use of antipsychotic drugs. Background Art

[0002] Schizophrenia is a mental illness characterized by detachment from reality (psychosis), various hallucinations (usually auditory hallucinations), firm false beliefs (delusions), abnormal thinking and behavior, reduced emotional expression, loss of motivation, and impairment of daily functions (including work, social, and self-care abilities). The cause is complex and not fully understood. Antipsychotic drugs are currently the core means for treating mental illnesses such as schizophrenia.

[0003] Clinical practice shows that there are significant individual differences in the efficacy of antipsychotic drugs. Approximately 30% - 50% of patients do not respond to initial drug treatment, and about 20% of patients are forced to discontinue the drug due to severe side effects (such as metabolic syndrome, extrapyramidal reactions). The fundamental cause of these individual differences lies in the functional variations of drug-metabolizing enzymes, transporters, and target proteins caused by patient gene polymorphisms. For example, single nucleotide polymorphism (SNP) sites in key genes such as CYP2D6, CYP3A4, DRD2, and HTR2A have been confirmed to be closely related to the metabolism and efficacy of antipsychotic drugs. Therefore, the individualized medication strategy based on pharmacogenomics (PGx) has become an important direction for improving efficacy and reducing adverse reactions.

[0004] Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a commonly used method for detecting SNP sites, which has the advantages of high throughput, fast analysis speed, and simple result interpretation. Although there have been various reports on the detection of SNP sites related to antipsychotic drug use based on MALDI-TOF MS, most of the detections cover fewer SNP sites, resulting in fewer antipsychotic drugs covered and low detection efficiency. In addition, in some reports, the SNP sites involved lack evidence directly supporting their association with antipsychotic drug reactions or side effects, and they have not been included in the relevant detection sites for guiding antipsychotic drug use, so the detection scientificity is poor. For example, the SNP site g.56002077 T>C of the MC4R gene has no clinical guiding significance for its detection and is likely to bring unnecessary psychological burden to the tested individuals. For the SNP site g.57851097T>C (rs17782313) reported in the literature to be related to antipsychotic drug side effects, there is no multi-site joint detection kit that includes it in the detection range. However, KF Schreyer et al. found that in patients treated only with amisulpride, there was a significant association between the rs17782313 polymorphism and the absolute weight gain and absolute BMI increase in patients, with statistical differences; among them, the absolute weight gain of CC allele carriers was 4.5 times higher than that of TT carriers after 8 weeks of treatment, and that of TC allele carriers was 2.3 times higher than that of TT carriers (Schreyer K F, Leucht S, Heres S, et al. Genetic association of the rs17782313 polymorphism with antipsychotic-induced weight gain[J]. Psychopharmacology, 2023, 240(4).).

[0005] In summary, there is an urgent need for a kit that can sensitively, specifically, scientifically, and accurately detect multiple SNP sites related to antipsychotic drug use in a single-tube reaction system, covering as many antipsychotic drugs as possible, so as to provide more scientific and accurate medication references for the personalized use of antipsychotic drugs. Summary of the Invention

[0006] In view of the deficiencies of existing products for detecting SNP sites related to antipsychotic drug metabolism, response, toxicity, etc. based on time-of-flight mass spectrometry, such as having fewer included SNP sites, covering fewer drugs, and having poor scientificity of covered sites, the present invention provides a primer composition. Using the primer composition of the present invention and combining with time-of-flight mass spectrometry, it is possible to sensitively and accurately detect 18 SNP sites of 10 genes related to the use of antipsychotic drugs, thereby guiding the personalized use of 26 antipsychotic drugs.

[0007] The first object of the present invention is to provide a primer composition.

[0008] The second object of the present invention is to provide the application of the primer composition in the preparation of a product for detecting SNP sites related to the use of antipsychotic drugs.

[0009] The third object of the present invention is to provide a kit containing the primer composition.

[0010] The above objects of the present invention are achieved by the following technical solutions: Based on the "Technical Guidelines for the Detection of Drug-Metabolizing Enzymes and Drug-Action Target Genes (Trial)", the present invention integrates key genes and SNP sites related to antipsychotic drug metabolism, response, and toxicity, that is, related to the use of antipsychotic drugs, retrieved from the Pharmacogenomics Knowledgebase (PharmGKB) (https: / / www.pharmgkb.org / ), dbSNP (https: / / www.ncbi.nlm.nih.gov / snp / ), and the Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines (https: / / cpicpgx.org / ). Considering clinical needs, 18 SNP sites of 10 genes related to the use of 26 antipsychotic drugs are obtained. For the said SNP sites, the present invention obtains a primer composition that can sensitively, specifically, scientifically, and accurately detect the said SNP sites simultaneously in a single-tube reaction system, and can provide scientific and accurate medication references for the personalized use of 26 antipsychotic drugs. Therefore, the present invention requests protection for the said primer composition and its related applications.

[0011] The present invention provides a primer composition, including a multiplex PCR amplification primer set and a single-base extension primer set; wherein, the multiplex PCR amplification primer set contains 36 amplification primers, and the nucleotide sequences of the amplification primers are respectively shown as SEQ ID NO.1 - 36; the single-base extension primer set contains 18 extension primers, and the nucleotide sequences of the extension primers are respectively shown as SEQ ID NO.37 - 54.

[0012] Using the primer composition of the present invention, combined with time-of-flight mass spectrometry, SNP sites related to the use of antipsychotic drugs can be detected. Therefore, the present invention claims the application of the primer composition in the preparation of a product for detecting SNP sites related to the use of antipsychotic drugs.

[0013] Specifically, the SNP sites include rs951439, rs16947, rs1065852, rs1058164, rs1799978, rs3892097, rs28371725, rs1135840, rs1414334, rs3813929, rs17782313, rs489693, rs1800497, rs762551, rs67666821, rs1799732, rs334558, and rs11869731.

[0014] Specifically, the SNP sites are related to the use of perospirone, iloperidone, cariprazine, tetrabenazine, carbamazepine, brexpiprazole, asenapine, haloperidol, fluphenazine, sulpiride, trifluoperazine, zuclopenthixol, pimozide, loxapine, lithium carbonate, olanzapine, quetiapine, risperidone, aripiprazole, lurasidone, ziprasidone, clozapine, paliperidone (papanidone), amisulpride, perphenazine, and chlorpromazine.

[0015] The present invention also provides a kit, which contains the primer composition of the present invention.

[0016] Specifically, the kit is a kit for detecting SNP sites related to the use of antipsychotic drugs; the SNP sites include rs951439, rs16947, rs1065852, rs1058164, rs1799978, rs3892097, rs28371725, rs1135840, rs1414334, rs3813929, rs17782313, rs489693, rs1800497, rs762551, rs67666821, rs1799732, rs334558, and rs11869731.

[0017] Specifically, the kit further contains reagents required for multiplex PCR amplification reaction, SAP reaction, and single-base extension reaction.

[0018] Specifically, the reagents required for the multiplex PCR amplification reaction include PCR reaction buffer and PCR enzyme.

[0019] Specifically, the reagents required for the SAP reaction include SAP reaction buffer and SAP enzyme.

[0020] Specifically, the reagents required for the single-base extension reaction include an extension reaction buffer, an extension reaction termination solution, and an extension enzyme.

[0021] Specifically, the kit also contains reagents required for time-of-flight mass spectrometry detection.

[0022] Optionally, the kit also contains reagents required for extracting sample DNA.

[0023] The present invention also provides a method for detecting 18 SNP sites related to antipsychotic drug use by using the primer composition or the kit, comprising the following steps: S1. Extract genomic DNA of the sample to be tested, and use this as a template to perform a multiplex PCR amplification reaction with the multiplex PCR amplification primer set to obtain a PCR reaction product; S2. Mix the PCR reaction product obtained in S1 with SAP reaction reagents to perform a SAP digestion reaction to obtain a SAP reaction product; S3. Mix the SAP reaction product obtained in S2 with the single-base extension primer set and UEP reaction reagents to perform a UEP single-base extension reaction to obtain a UEP reaction product; S4. Perform time-of-flight mass spectrometry detection and result judgment on the UEP reaction product obtained in S3.

[0024] Optionally, in S1, the sample is human blood or oral swab.

[0025] Specifically, when performing the multiplex PCR amplification reaction in S1, the final concentration of the amplification primers with nucleotide sequences as shown in SEQ ID NO.1 - 36 in the multiplex PCR amplification reaction system is 0.1 - 0.3 μM.

[0026] More specifically, the final concentration of the amplification primers is 0.2 μM.

[0027] Preferably, in the multiplex PCR amplification reaction system, the final concentration of the amplification primer for amplifying the rs334558 site on the gene GSK3B is 0.4 μM, and the final concentration of the remaining amplification primers is 0.2 μM.

[0028] Specifically, in S1, the multiplex PCR amplification reaction system includes the following components: 10 × PCR Buffer with 20 mM MgCl2 0.5 μL, 25 mM MgCl2 0.4 μL, 25 mM dNTP Mix 0.1 μL, amplification primer mixture 1 μL; 5 U / μL PCR Enzyme 0.2 μL; genomic DNA 10 - 50 ng, and ultrapure water (HPLC grade) is added to make up 5 μL.

[0029] Specifically, in S1, the multiplex PCR amplification reaction procedure is as follows: (1) 95°C, 2 min; (2) 95°C, 30 sec; 56°C, 30 sec; 72°C, 60 sec, for a total of 45 cycles; (3) 72°C, 5 min.

[0030] Specifically, in S2, the SAP digestion reaction system includes the following components: 0.17 μL of SAP buffer, 0.3 μL of SAP enzyme, and 1.53 μL of ultrapure water.

[0031] Specifically, in S2, the SAP digestion reaction procedure is: 37°C, 40 min; 85°C, 5 min.

[0032] Specifically, when performing single-base extension reaction in S3, the final concentration of the extension primers with nucleotide sequences shown in SEQ ID NO. 37 - 54 in the single-base extension reaction system is 0.50 - 3.00 µM.

[0033] More specifically, in the single-base extension reaction system, the final concentration of the primer shown in SEQ ID NO.37 is 1.29 - 1.49 μM; the final concentration of the primer shown in SEQ ID NO.38 is 0.62 - 0.82 μM; the final concentration of the primer shown in SEQ ID NO.39 is 1.33 - 1.53 μM; the final concentration of the primer shown in SEQ ID NO.40 is 1.12 - 1.32 μM; the final concentration of the primer shown in SEQ ID NO.41 is 0.56 - 0.86 μM; the final concentration of the primer shown in SEQ ID NO.42 is 0.71 - 0.91 μM; the final concentration of the primer shown in SEQ ID NO.43 is 0.67 - 0.87 μM; the final concentrations of the primers shown in SEQ ID NO.44 and 49 are 2.78 - 2.98 μM; the final concentration of the primer shown in SEQ ID NO.45 is 0.52 - 0.72 μM; the final concentration of the primer shown in SEQ ID NO.46 is 1.30 - 1.50 μM; the final concentration of the primer shown in SEQ ID NO.47 is 1.46 - 1.66 μM; the final concentration of the primer shown in SEQ ID NO.48 is 1.31 - 1.51 μM; the final concentration of the primer shown in SEQ ID NO.50 is 1.81 - 2.01 μM; the final concentration of the primer shown in SEQ ID NO.51 is 1.03 - 1.23 μM; the final concentration of the primer shown in SEQ ID NO.52 is 0.66 - 0.86 μM; the final concentration of the primer shown in SEQ ID NO.53 is 0.50 - 0.70 μM; the final concentration of the primer shown in SEQ ID NO.54 is 1.23 - 1.46 μM. Specifically, in S3, the UEP single-base extension reaction system comprises the following components: 0.62 μL of ultrapure water (HPLC grade), iPLEX ® Buffer Plus 0.2 μL, iPLEX ® Termination mix 0.2 μL, Extend Primer Mix 0.94 μL, iPLEX ® Pro Enzyme 0.04 μL.

[0034] Specifically, in S3, the UEP reaction program comprises: (1) 95°C, 30 sec; (2) 40 cycles of the following program: ① 95°C, 5 sec, ② 52°C, 5 sec, 80°C, 5 sec, for 5 cycles; (3) 72°C, 3 min.

[0035] The present invention has the following beneficial effects: The present invention targets 18 SNP loci related to antipsychotic drug use screened according to literature reports and CPIC guidelines, etc., and obtains a primer composition capable of sensitively and specifically detecting the 18 SNP loci, and constructs a corresponding kit using the primer composition. Using the primer composition or the kit of the present invention, the 18 SNP loci can be detected by combining time-of-flight mass spectrometry, which has the advantages of high detection sensitivity, good repeatability, intuitive and accurate detection results, etc., and can greatly shorten the detection cycle and reduce the detection cost, and is suitable for popularization. After detecting the 18 SNP loci, the personalized use of antipsychotic drugs can be guided according to the SNP locus detection situation, avoiding situations such as non-response to drug use and serious side effects, which is beneficial to the treatment of mental diseases such as schizophrenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is the electrophoresis pattern of the PCR amplification product of the primer shown in Table 2; Figure 1 A to C in it are the electrophoresis patterns of the PCR amplification products of the primers designed for the rs16947, rs1065852 and rs1414334 loci in sequence; M in the figure is BM 1000 Marker; lanes 1 to 3 are the PCR amplification products of primer pairs 1 to 3 in sequence.

[0037] Figure 2 It is the nucleic acid mass spectrometry detection pattern and the Sanger sequencing pattern of the rs1065852 locus before and after redesigning the extension primer; A in the figure is the nucleic acid mass spectrometry detection pattern before redesigning the extension primer of the rs1065852 locus; B in the figure is the nucleic acid mass spectrometry detection pattern after redesigning the extension primer of the rs1065852 locus; C in the figure is the Sanger sequencing pattern of the rs1065852 locus.

[0038] Figure 3 It is the nucleic acid mass spectrometry detection pattern of the 18 SNP loci in an EDTA-anticoagulated venous whole blood sample.

[0039] Figure 4 It is the nucleic acid mass spectrometry detection pattern of the rs1058164 locus in an EDTA-anticoagulated venous whole blood sample.

[0040] Figure 5 It is the nucleic acid mass spectrometry detection pattern of the rs1065852 locus in an EDTA-anticoagulated venous whole blood sample.

[0041] Figure 6 It is the nucleic acid mass spectrometry detection pattern and the Sanger sequencing pattern of the rs1065852 locus in clinical sample 1.

[0042] Figure 7Nucleic acid mass spectrometry detection graph for the detection limit test of clinical sample 1. Detailed implementation manners

[0043] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0044] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0045] Example 1 Obtaining of primer composition The present invention aims at 18 SNP loci related to the use of antipsychotics, and through screening and optimization, a primer composition capable of simultaneously detecting the SNP loci in a single-tube reaction system is obtained.

[0046] 1. Selection of SNP loci The SNP loci related to the use of antipsychotics in the present invention are based on the "Technical Guidelines for the Detection of Drug-Metabolizing Enzymes and Drug-Acting Target Genes (Trial)", and at the same time, the key genes and SNP loci related to antipsychotic metabolism, response and toxicity retrieved from the PharmGKB and dbSNP databases and the CPIC guidelines, that is, related to antipsychotic use, are integrated, and the clinical needs are comprehensively considered. The 18 SNP loci related to the use of antipsychotics involve 10 genes and 26 types of antipsychotics. The relationship between the SNP loci and antipsychotics is shown in Table 1.

[0047] Table 1 26 types of antipsychotics and their related genes and SNP loci for drug use

[0048] 2. Obtaining of primer composition After selecting the SNP loci to be detected, the present invention uses the nucleotide sequences of the genes where the SNP loci are located to design multiple sets of PCR amplification primers and single-base extension primers required for the detection based on the MALDI-TOF MS technology. Since the present invention wants to simultaneously detect the selected 18 SNP loci in a single-tube reaction system and also needs to consider the specificity and sensitivity of the detection, the designed primers also need to be screened, adjusted and optimized. The screening process of the PCR amplification primers and single-base extension primers for the rs16947, rs1065852 and rs1414334 loci is provided as an example.

[0049] (1) Screening of PCR amplification primers The PCR amplification primers designed for rs16947, rs1065852 and rs1414334 sites in the present invention are shown in Table 2.

[0050] Table 2 PCR amplification primers designed for rs16947, rs1065852 and rs1414334 sites

[0051] Synthesize the primers shown in Table 2, use the quality control product JK (human T-lymphocyte leukemia cell DNA, hereinafter collectively referred to as quality control product JK) as the template for PCR amplification, and perform electrophoresis detection on the amplification products to screen primers with good amplification specificity and high amplification efficiency. The electrophoresis pattern of the PCR amplification products of the primers shown in Table 2 is as Figure 1 shown; Figure 1 A to C in Figure 1 are the electrophoresis patterns of the PCR amplification products of the primers designed for rs16947, rs1065852 and rs1414334 sites in sequence. It can be seen from Figure 1 that for the rs16947 site, although all 3 pairs of primers have target amplification bands, the first pair of primers has non-specific amplification bands, and the brightness of the amplification bands of the second pair of primers is significantly weaker than that of the third group of primers, indicating that the third pair of primers has the highest amplification specificity and efficiency. Select the third pair of primers for subsequent experiments for the rs16947 site. Combining the

[0052] shown results, select the first pair of primers for subsequent experiments for the rs1065852 site, and select the second pair of primers for the rs1414334 site. During the process of testing single-base extension primers with clinical samples, the present invention found that the extension primers at individual sites had low extension efficiency in the extension system, resulting in difficult interpretation. The corresponding sites were redesigned or the primer sequences were adjusted. For example, when using the extension primer (5'-TGGGCTGCACGCTAC-3') to detect the rs1065852 site, the detection result is as Figure 2 shown in A of Figure 2 . It can be seen from the figure that the genotype of the rs1065852 site is GA, but the peak of the G base is extremely low, and it is easy to be judged as a heterozygous peak during interpretation, indicating that the extension effect of this extension primer is poor. For the rs1065852 site, the present invention redesigned the single-base extension primer (5'-GCTGGGCTGCACGCTAC-3'), detected the same template, and the detection result is as Figure 2in C). Therefore, the extended primer sequence of rs1065852 locus was finally adjusted to 5’-GCTGGGCTGCACGCTAC-3’.

[0053] After adjustment and optimization, the present invention finally obtained a primer composition capable of simultaneously detecting the SNP loci in a single-tube reaction system, including a multiplex PCR amplification primer set and a single-base extension primer set; wherein, the multiplex PCR amplification primer set contains 36 amplification primers, as shown in Table 3; the single-base extension primer set contains 18 extension primers (UEP extension primers), as shown in Table 4.

[0054] Table 3 Multiplex PCR Amplification Primer Set

[0055] Table 4 Single-Base Extension Primer Set

[0056] Note: The lowercase letter bases at the 5’ end of the primers in the table are used to increase the relative molecular mass of the extension primers for mass spectrometry to distinguish each locus.

[0057] Example 2 Construction of Detection Kit and Detection Method Based on the primer composition obtained in Example 1, the present invention also constructed a detection kit and a detection method that can be used to detect the SNP loci.

[0058] In addition to the primer composition, the kit further contains the following components: (1) Reagents required for multiplex PCR amplification reaction: including PCR reaction buffer and PCR enzyme; (2) Reagents required for SAP reaction: including SAP reaction buffer and SAP enzyme; (3) Reagents required for single-base extension reaction: including extension reaction buffer, extension reaction termination solution and extension enzyme; (4) Reagents required for time-of-flight mass spectrometry detection: specifically, reagents required for nucleic acid matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection.

[0059] The SNP loci and genotypes detected by each primer in the primer composition are shown in Table 5.

[0060] Table 5 Genotypes Corresponding to SNP Loci, Their Amplification Primers and Extension Primers

[0061] The detection method includes the following steps: S1. Extract the genomic DNA of the sample to be tested. Using this as a template, perform a multiplex PCR amplification reaction with the multiplex PCR amplification primer set to obtain a PCR reaction product; S2. Mix the PCR reaction product obtained in S1 with the SAP reaction reagent and perform a SAP digestion reaction to obtain a SAP reaction product; S3. Mix the SAP reaction product obtained in S2 with the single-base extension primer set and the UEP reaction reagent and perform a UEP single-base extension reaction to obtain a UEP reaction product; S4. Perform a time-of-flight mass spectrometry detection and result judgment on the UEP reaction product obtained in S3.

[0062] In this example, the collected EDTA-anticoagulated whole blood sample is taken as an example to illustrate the detection method in detail.

[0063] (1) Sample DNA extraction Collect 5 mL of EDTA-anticoagulated whole blood sample, extract the genomic DNA of the whole blood sample using the Kapu nucleic acid extraction kit (model DR-4801-KZ), and measure the DNA concentration with a UV spectrophotometer.

[0064] (2) Multiplex PCR amplification reaction Using the genomic DNA extracted in (1) as a template, perform a multiplex PCR amplification with the Agena PCR Reagent Set amplification kit; among them, the amplification primer mixture (Primer Mix) is obtained by mixing each amplification primer (SEQ ID NO.1-36) shown in Table 3, and the concentration is 1.0 μM. The multiplex PCR amplification reaction system is shown in Table 6. In the PCR amplification reaction system, the final concentration of each amplification primer is 0.2 μM.

[0065] Table 6 Multiplex PCR amplification reaction system

[0066] The multiplex PCR amplification reaction program is shown in Table 7.

[0067] Table 7 Multiplex PCR amplification reaction program

[0068] To obtain better detection results, adjust the concentration of the amplification primer at the rs334558 locus on the gene GSK3B to 2.0 μM, so that its final concentration in the PCR amplification reaction system is 0.4 μM.

[0069] (3) SAP digestion reaction Use Agena iPLEX ®Perform the SAP digestion reaction using the ProReagent Set kit. Prepare the SAP mixture according to Table 8, and add 2 μL of the SAP mixture to the PCR amplification reaction product obtained in (2), with a total volume of 7 μL.

[0070] Table 8 SAP mixture

[0071] The SAP digestion reaction program is shown in Table 9.

[0072] Table 9 Multiplex PCR reaction program

[0073] (4)Single-base extension reaction Use the Agena iPLEX ® ProReagent Set kit to perform the single-base extension reaction. Prepare the single-base extension reaction solution according to Table 10, and add 2 μL of the single-base extension reaction solution to the SAP digestion reaction product obtained in (3), with a total volume of 9 μL; among them, the extension primer mixture (Extend Primer Mix) is obtained by mixing each single-base extension primer (SEQ ID NO. 37 - 54) shown in Table 4. In the single-base extension reaction solution, the final concentration of each extension primer is as shown in Table 11.

[0074] Table 10 Single-base extension reaction solution

[0075] Table 11 Final concentration of each extension primer in the single-base extension reaction solution

[0076] The single-base extension reaction program is shown in Table 12.

[0077] Table 12 Single-base extension reaction program

[0078] (5)Mass spectrometry detection After the single-base extension reaction is completed, add 16 µL of ultrapure water to each reaction well to make the total volume of the reaction product in each well 25 µL. After mixing evenly, perform time-of-flight mass spectrometry detection.

[0079] Import the edited site Assay information in the Typer software, select the reaction well positions of the test samples to build a plate, import the corresponding sample names, connect the detection chip, place the 384-well plate and the detection chip at the corresponding positions on the mass spectrometer, and start the time-of-flight mass spectrometry detection. The detection results are automatically analyzed using the Typer 4.0 software, and the peak areas of each site are exported to an Excel file.

[0080] (6)Result interpretation The single-base extension products are analyzed by mass spectrometry to detect the time of flight of the extension products in the vacuum tube, thereby calculating the molecular weight of the extension products. Then, after comparing the molecular weight of the extension products with the preset molecular weight in the software using the analysis software, the peak of the extension product is determined to be wild type, heterozygous mutant, or homozygous mutant. The nucleic acid mass spectrometry detection graphs of the 18 SNP sites in the samples used in this example are as Figure 3 shown. As can be seen from Figure 3 , the detection of the 18 SNP sites can be achieved using the kit and method of the present invention. The relative molecular weights of the extension primers and extension products at each site of the present invention are shown in Table 13. For the convenience of checking against the relative molecular weights in Table 13, as an example, the nucleic acid mass spectrometry detection graphs of the rs1058164 and rs1065852 sites of the present invention are provided in sequence as Figure 4 and Figure 5 shown.

[0081] Table 13 Relative molecular weights of extension primers and extension products at each site

[0082] Example 3 Kit detection performance test 1. Accuracy test Perform Sanger sequencing on the quality control product JK and 6 clinical samples. Additionally, use the method described in Example 2 to detect the quality control product JK and the 6 clinical samples (Sample1 - 6), compare whether the mass spectrometry results are consistent with the Sanger sequencing results, and repeat the measurement 3 times. The 3 results are combined as shown in Table 14.

[0083] Table 14 Accuracy test results

[0084] As can be seen from the results shown in Table 14, the results of the mass spectrometry detection of the SNP sites using the primer composition and method of the present invention are consistent with the detection results of the Sanger sequencing method, with an accuracy of 100%. This indicates that the method has high sensitivity and strong specificity, and can meet the clinical detection requirements for SNP sites related to antipsychotic drug use. As an example, the nucleic acid mass spectrometry detection graph and Sanger sequencing graph of the rs1065852 site in clinical sample 1 are asFigure 6 As shown in Figure 6 it can be seen that the mass spectrometry detection results are consistent with those of Sanger sequencing.

[0085] 2. Limit of detection test Using the quality control product JK and 6 clinical samples used in the accuracy test as templates, the lowest limit of detection of the kit of the present invention was tested. The sample DNA was serially diluted to four gradients of 40 ng / μL, 20 ng / μL, 10 ng / μL, and 5 ng / μL. According to the operation steps provided in Example 2 of the present invention, multiplex PCR amplification reaction, SAP digestion, single-base extension, and mass spectrometry detection analysis were carried out respectively; the sample DNA dosage was 1 μL, and each gradient was detected 3 times; the lowest concentration that could be detected 100% was taken as the lowest detection limit, and then at this concentration, it was detected 5 times, and if all 5 times were detected, then this concentration could be determined as the lowest detection limit of the kit. The lowest limit of detection test results of the kit of the present invention are shown in Table 15, and the lowest detection limit is 5 ng / reaction. The nucleic acid mass spectrometry detection chart of the limit of detection test of clinical sample 1 is as Figure 7 shown.

[0086] Table 15 Limit of detection test results

[0087] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A primer composition, characterized in that, It includes a multiplex PCR amplification primer set and a single-base extension primer set; among them, there are 36 amplification primers in the multiplex PCR amplification primer set, and the nucleotide sequences of the amplification primers are respectively shown as SEQ ID NO.1 to 36; there are 18 extension primers in the single-base extension primer set, and the nucleotide sequences of the extension primers are respectively shown as SEQ ID NO.37 to 54.

2. Use of the primer composition according to claim 1 in the preparation of a product for detecting SNP sites related to antipsychotic drug use, characterized in that, The SNP sites include rs951439, rs16947, rs1065852, rs1058164, rs1799978, rs3892097, rs28371725, rs1135840, rs1414334, rs3813929, rs17782313, rs489693, rs1800497, rs762551, rs67666821, rs1799732, rs334558 and rs11869731.

3. A kit, characterized in that, It contains the primer composition described in claim 1.

4. The kit according to claim 3, wherein The kit is a kit for detecting SNP sites related to antipsychotic drug use; the SNP sites include rs951439, rs16947, rs1065852, rs1058164, rs1799978, rs3892097, rs28371725, rs1135840, rs1414334, rs3813929, rs17782313, rs489693, rs1800497, rs762551, rs67666821, rs1799732, rs334558 and rs11869731.

5. The kit according to claim 3 or 4, characterized in that, The kit also contains reagents required for multiplex PCR amplification reaction, SAP reaction and single-base extension reaction.

6. The kit according to claim 5, characterized in that, The reagents required for the multiplex PCR amplification reaction include PCR reaction buffer and PCR enzyme.

7. The kit according to claim 5, wherein The reagents required for the SAP reaction include SAP reaction buffer and SAP enzyme.

8. The kit according to claim 5, characterized in that, The reagents required for the single-base extension reaction include extension reaction buffer, extension reaction termination solution and extension enzyme.

9. The kit according to claim 3 or 4, characterized in that, The kit also contains reagents required for time-of-flight mass spectrometry detection.

10. The kit according to claim 3 or 4, characterized in that, The kit also contains reagents required for extracting sample DNA.

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