A primer combination and its application in preparing a product for detecting SNP sites associated with antipsychotic drug use

Abstract

The present invention discloses a primer composition and its use in the preparation of a product for detecting SNP sites associated with the use of antipsychotic drugs. The present invention is directed to 18 SNP sites associated with the use of antipsychotic drugs, screened according to literature reports and the guidelines of the Clinical Pharmacogenetics Implementation Alliance, and obtains a set of primer compositions capable of sensitively and specifically detecting the 18 SNP sites, and constructs a corresponding kit using the primer composition. Using the primer composition or the kit of the present invention, the 18 SNP sites can be detected in combination with time-of-flight mass spectrometry, and the personalized use of antipsychotic drugs can be guided based on the SNP site detection results, thereby avoiding situations such as drug non-response and severe side effects, which is beneficial to the treatment of mental illnesses such as schizophrenia.

Description

Technical Field

[0001] The present invention belongs to the field of gene detection technology. More specifically, it relates to a primer combination and its application in preparing a product for detecting SNP sites associated with antipsychotic drug use. Background Art

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

[0003] Clinical practice demonstrates significant inter-individual variability in the efficacy of antipsychotic drugs. Approximately 30% to 50% of patients fail to respond to initial treatment, and approximately 20% are forced to discontinue medication due to severe side effects (such as metabolic syndrome and extrapyramidal reactions). The fundamental cause of these inter-individual variability lies in functional variations in drug-metabolizing enzymes, transporters, and target proteins caused by genetic polymorphisms. For example, single nucleotide polymorphisms (SNPs) in key genes such as CYP2D6, CYP3A4, DRD2, and HTR2A have been shown to be closely associated with the metabolism and efficacy of antipsychotic drugs. Therefore, personalized medication strategies based on pharmacogenomics (PGx) have become an important approach to improve efficacy and minimize adverse reactions.

[0004] Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a commonly used method for SNP detection, offering advantages such as high throughput, rapid analysis, and simple result interpretation. Although there have been numerous reports of MALDI-TOF MS-based detection of SNPs associated with antipsychotic use, most assays cover a limited number of SNPs, resulting in limited coverage of antipsychotic drugs and low detection efficiency. Furthermore, some of the SNPs mentioned in these reports lack evidence supporting a direct association with antipsychotic response or side effects, and they have not yet been included in the list of relevant assays used to guide antipsychotic prescribing, making the testing less scientifically sound. For example, testing for the MC4R gene SNP g.56002077 T>C currently has no clinical relevance and can place unnecessary psychological burden on the patient. Furthermore, there are currently no multi-locus assay kits that include the SNP g.57851097 T>C (rs17782313), which has been reported in the literature to be associated with antipsychotic side effects. KF Schreyer et al. found that in patients who received only amisulpride treatment, there was a significant association between the rs17782313 polymorphism and the patients' absolute weight gain and absolute BMI increase, which was statistically significant; among them, the absolute weight gain of CC allele carriers was 4.5 times higher than that of TT carriers after a full 8 weeks of treatment, and the absolute weight gain of TC allele carriers was 2.3 times higher than that of TT carriers (Schreyer KF, 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, thereby providing a more scientific and accurate medication reference for personalized antipsychotic drug use. Summary of the Invention

[0006] The present invention provides a primer combination to address the shortcomings of existing time-of-flight mass spectrometry-based detection of SNPs associated with antipsychotic drug metabolism, response, and toxicity, such as limited inclusion of SNPs, limited drug coverage, and poor scientific validity of the coverage. The primer combination of the present invention, combined with time-of-flight mass spectrometry, enables sensitive and accurate detection of 10 genes and 18 SNPs associated with antipsychotic drug use, thereby guiding personalized medication for 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 a use of the primer combination in preparing a product for detecting SNP sites associated with antipsychotic drug use.

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

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] This invention is based on the "Technical Guidelines for Detection of Drug-Metabolizing Enzymes and Drug Target Genes (Trial Implementation)" and integrates key genes and SNPs associated with antipsychotic drug metabolism, response, and toxicity, specifically those related to antipsychotic use, retrieved from the Pharmacogenomics Database (PharmGKB) (https: / / www.pharmgkb.org / ), dbSNP (https: / / www.ncbi.nlm.nih.gov / snp / ), and the Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines (https: / / cpicpgx.org / ). Taking into account clinical needs, this invention identified 18 SNPs in 10 genes associated with the use of 26 antipsychotic drugs. Targeting these SNPs, the present invention has developed a primer combination that can simultaneously detect these SNPs sensitively, specifically, scientifically, and accurately in a single-tube reaction system. This combination can provide a scientific and accurate reference for personalized medication for these 26 antipsychotic drugs. Therefore, the present invention seeks protection for this primer combination and its related applications.

[0012] The present invention provides a primer composition comprising 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 NOs. 1 to 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 NOs. 37 to 54.

[0013] The primer combination of the present invention can be used in conjunction with time-of-flight mass spectrometry to detect SNP sites associated with antipsychotic medication. Therefore, the present invention seeks protection for the use of the primer combination in the preparation of a product for detecting SNP sites associated with antipsychotic medication.

[0014] Specifically, the SNP sites include rs951439, rs16947, rs1065852, rs1058164, rs1799978, rs3892097, rs28371725, rs1135840, rs1414334, rs3813929, rs17782313, rs489693, rs1800497, rs762551, rs67666821, rs1799732, rs334558 and rs11869731.

[0015] Specifically, the SNP site is associated with the use of perospirone, iloperidone, cariprazine, tetrabenazine, cariprazine, epiriprazole, asenapine, haloperidol, fluphenazine, sulpiride, trifluoperazine, zuclopenthixol, pimozide, loxapine, lithium carbonate, olanzapine, quetiapine, risperidone, aripiprazole, lurasidone, ziprasidone, clozapine, paliperidone (paliperidone), amisulpride, perphenazine and chlorpromazine.

[0016] The present invention also provides a kit containing the primer composition of the present invention.

[0017] 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.

[0018] Specifically, the kit also contains reagents required for multiplex PCR amplification reaction, SAP reaction and single base extension reaction.

[0019] Specifically, the reagents required for the multiplex PCR amplification reaction include PCR reaction buffer and PCR enzyme.

[0020] Specifically, the reagents required for the SAP reaction include SAP reaction buffer and SAP enzyme.

[0021] Specifically, the reagents required for the single-base extension reaction include an extension reaction buffer, an extension reaction termination solution, and an extension enzyme.

[0022] Specifically, the kit also contains reagents required for time-of-flight mass spectrometry detection.

[0023] Optionally, the kit also contains reagents required for extracting sample DNA.

[0024] The present invention also provides a method for detecting the 18 SNP sites associated with antipsychotic drug use using the primer composition or the kit, comprising the following steps:

[0025] S1. Extracting genomic DNA from the sample to be tested, using it as a template, and performing a multiplex PCR amplification reaction using the multiplex PCR amplification primer set to obtain a PCR reaction product;

[0026] S2. The PCR reaction product obtained in S1 is mixed with a SAP reaction reagent to perform a SAP digestion reaction to obtain a SAP reaction product;

[0027] S3. The SAP reaction product obtained in S2 is mixed with the single base extension primer set and the UEP reaction reagent to perform a UEP single base extension reaction to obtain a UEP reaction product;

[0028] S4. Perform time-of-flight mass spectrometry detection on the UEP reaction product obtained in S3 and judge the results.

[0029] Optionally, in S1, the sample is human blood or oral swab.

[0030] Specifically, when performing the multiplex PCR amplification reaction in S1, the final concentration of the amplification primers having nucleotide sequences as shown in SEQ ID NOs. 1 to 36 in the multiplex PCR amplification reaction system is 0.1 to 0.3 µM.

[0031] More specifically, the final concentration of the amplification primer is 0.2 µM.

[0032] Preferably, in a multiplex PCR amplification reaction system, the GSK3B The final concentration of the amplification primer for the rs334558 site on the DNA was 0.4 μM, and the final concentration of the remaining amplification primers was 0.2 μM.

[0033] Specifically, in S1, the multiplex PCR amplification reaction system includes the following components: 10 × PCR Buffer with 20mM MgCl20.5 μL, 25 mM MgCl20.4 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, ultrapure water (HPLC grade) to 5 μL.

[0034] Specifically, in S1, the multiplex PCR amplification reaction program was: (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.

[0035] Specifically, in S2, the SAP digestion reaction system included the following components: 0.17 μL SAP buffer, 0.3 μL SAP enzyme, and 1.53 μL ultrapure water.

[0036] Specifically, in S2, the SAP digestion reaction procedures were: 37°C, 40 min; 85°C, 5 min.

[0037] Specifically, when performing the single base extension reaction in S3, the final concentration of the extension primers having nucleotide sequences as shown in SEQ ID NOs. 37 to 54 in the single base extension reaction system is 0.50 to 3.00 µM.

[0038] 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 concentration of the primer shown in SEQ ID NO.44 and 49 is 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 set forth in SEQ ID NO.46 is 1.30-1.50 µM; the final concentration of the primer set forth in SEQ ID NO.47 is 1.46-1.66 µM; the final concentration of the primer set forth in SEQ ID NO.48 is 1.31-1.51 µM; the final concentration of the primer set forth in SEQ ID NO.50 is 1.81-2.01 µM; the final concentration of the primer set forth in SEQ ID NO.51 is 1.03-1.23 µM; the final concentration of the primer set forth in SEQ ID NO.52 is 0.66-0.86 µM; the final concentration of the primer set forth in SEQ ID NO.53 is 0.50-0.70 µM; and the final concentration of the primer set forth in SEQ ID NO.54 is 1.23-1.46 µM. Specifically, in S3, the UEP single-base extension reaction system includes 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.

[0039] Specifically, in S3, the UEP reaction procedure included: (1) 95°C, 30 sec; (2) 40 cycles of the following procedure: ① 95°C, 5 sec, ② 52°C, 5 sec, 80°C, 5 sec, 5 cycles; (3) 72°C, 3 min.

[0040] The present invention has the following beneficial effects:

[0041] The present invention is directed to 18 SNP sites related to the use of antipsychotic drugs that have been screened based on literature reports and CPIC guidelines, and a group of primer compositions that can sensitively and specifically detect the 18 SNP sites have been obtained, and a corresponding kit has been constructed using the primer composition. Using the primer composition or the kit of the present invention, the 18 SNP sites 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, and can greatly shorten the detection cycle, reduce detection costs, and is suitable for promotion. After the 18 SNP sites are detected, the personalized use of antipsychotic drugs can be guided based on the SNP site detection results, avoiding situations such as no response to medication and the occurrence of serious side effects, which is beneficial to the treatment of mental illnesses such as schizophrenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The electrophoresis diagram of the PCR amplification products of the primers shown in Table 2; Figure 1 A to C are electrophoresis images of PCR amplification products of primers designed for rs16947, rs1065852, and rs1414334 sites, respectively; M in the figure is BM 1000 Marker; lanes 1 to 3 are PCR amplification products of primer pairs 1 to 3, respectively.

[0043] Figure 2 Figure 1 is the nucleic acid mass spectrometry detection image before and after redesigning the extension primer of the rs1065852 site and the Sanger sequencing image of the rs1065852 site; Figure A is the nucleic acid mass spectrometry detection image before redesigning the extension primer of the rs1065852 site; Figure B is the nucleic acid mass spectrometry detection image after redesigning the extension primer of the rs1065852 site; Figure C is the Sanger sequencing image of the rs1065852 site.

[0044] Figure 3 This is a nucleic acid mass spectrometry detection diagram of the 18 SNP sites in EDTA anticoagulated venous whole blood samples.

[0045] Figure 4 This is the nucleic acid mass spectrometry detection diagram of the rs1058164 site in EDTA anticoagulated venous whole blood sample.

[0046] Figure 5 This is the nucleic acid mass spectrometry detection diagram of the rs1065852 site in EDTA anticoagulated venous whole blood sample.

[0047] Figure 6 This is the nucleic acid mass spectrometry detection image and Sanger sequencing image of the rs1065852 site in clinical sample 1.

[0048] Figure 7This is the nucleic acid mass spectrometry detection diagram of the detection limit test of clinical sample 1. DETAILED DESCRIPTION

[0049] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0050] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0051] Example 1 Obtaining a primer composition

[0052] The present invention targets 18 SNP sites related to the use of antipsychotic drugs, and obtains a primer combination that can simultaneously detect the SNP sites in a single-tube reaction system through screening and optimization.

[0053] 1. Selection of SNP sites

[0054] The SNPs associated with antipsychotic medication described in this paper are based on the "Guidelines for the Detection of Drug Metabolizing Enzymes and Drug Target Genes (Trial Implementation)." They also integrate key genes and SNPs related to antipsychotic metabolism, response, and toxicity, identified using the PharmGKB and dbSNP databases, as well as the CPIC guidelines, taking into account clinical needs. The 18 SNPs associated with antipsychotic medication involve 10 genes and 26 types / classes of antipsychotic drugs. The relationship between these SNPs and antipsychotic drugs is shown in Table 1.

[0055] Table 1 26 types of antipsychotic drugs and their associated genes and SNPs

[0056]

[0057] 2. Obtaining primer composition

[0058] After selecting the SNP sites to be detected, the present invention utilizes the nucleotide sequence of the gene in which the SNP sites are located and designs multiple sets of PCR amplification primers and single-base extension primers required for detection based on MALDI-TOF MS technology. Because the present invention aims to simultaneously detect the selected 18 SNP sites in a single-tube reaction system, it is necessary to balance the specificity and sensitivity of the detection. Therefore, the designed primers are screened, adjusted, and optimized. As an example, the screening process of PCR amplification primers and single-base extension primers for rs16947, rs1065852, and rs1414334 sites is provided.

[0059] (1) Screening of PCR amplification primers

[0060] The PCR amplification primers designed for the rs16947, rs1065852 and rs1414334 sites of the present invention are shown in Table 2.

[0061] Table 2 PCR primers designed for rs16947, rs1065852, and rs1414334 loci

[0062]

[0063] The primers shown in Table 2 were synthesized and PCR amplification was performed using the quality control product JK (human T lymphocyte leukemia cell DNA, hereinafter referred to as quality control product JK) as a template. The amplified products were detected by electrophoresis to screen primers with good amplification specificity and high amplification efficiency. The electrophoresis diagram of the PCR amplification products of the primers shown in Table 2 is shown in FIG. Figure 1 As shown; Figure 1 A to C in the figure are the electrophoresis images of PCR amplification products of primers designed for rs16947, rs1065852 and rs1414334 sites respectively. Figure 1 It can be seen that for the rs16947 site, although all three 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 pair of primers, indicating that the amplification specificity and efficiency of the third pair of primers are the highest. For the rs16947 site, the third pair of primers was selected for subsequent experiments. Figure 1 As shown in the results, the first pair of primers was selected for the rs1065852 site for subsequent experiments, and the second pair of primers was selected for the rs1414334 site.

[0064] (2) Screening of single-base extension primers

[0065] In the process of testing single-base extension primers using clinical samples, the present invention found that the extension primers of individual sites had low extension efficiency in the extension system, resulting in difficulty in interpretation. The primer sequences were redesigned or adjusted for the corresponding sites. For example, when the extension primer (5'-TGGGCTGCACGCTAC-3') was used to detect the rs1065852 site, the test results were as follows: Figure 2 As shown in A in the figure, the genotype of the rs1065852 site is GA, but the peak of the G base is very low and is easily judged as a miscellaneous peak during interpretation, indicating that the extension effect of this extension primer is poor. For the rs1065852 site, the present invention redesigned a single base extension primer (5'-GCTGGGCTGCACGCTAC-3') to detect the same template, and the detection results are as follows Figure 2As shown in B. As can be seen from the figure, the rs1065852 site can be clearly interpreted as GA, and both the G base and the A base can produce good peaks, which is consistent with the Sanger sequencing results ( Figure 2 Therefore, the final extension primer sequence for rs1065852 was adjusted to 5'-GCTGGGCTGCACGCTAC-3'.

[0066] After adjustment and optimization, the present invention ultimately obtained a primer composition capable of simultaneously detecting the SNP sites 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.

[0067] Table 3 Multiplex PCR amplification primer sets

[0068]

[0069] Table 4 Single base extension primer sets

[0070]

[0071] Note: The lowercase bases at the 5' end of the primers in the table are used to increase the relative molecular mass of the extended primers to facilitate the distinction of each site by mass spectrometry.

[0072] Example 2 Construction of detection kit and detection method

[0073] Based on the primer composition obtained in Example 1, the present invention also constructs a detection kit and a detection method that can be used to detect the SNP site.

[0074] In addition to the primer combination, the kit also contains the following components:

[0075] (1) Reagents required for multiplex PCR amplification reaction: including PCR reaction buffer and PCR enzyme;

[0076] (2) Reagents required for SAP reaction: including SAP reaction buffer and SAP enzyme;

[0077] (3) Reagents required for single-base extension reaction: including extension reaction buffer, extension reaction termination solution and extension enzyme;

[0078] (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.

[0079] The SNP sites and genotypes detected by each primer in the primer combination are shown in Table 5.

[0080] Table 5 Genotypes corresponding to SNP sites and their amplification and extension primers

[0081]

[0082] The detection method comprises the following steps:

[0083] S1. Extracting genomic DNA from the sample to be tested, using it as a template, and performing a multiplex PCR amplification reaction using the multiplex PCR amplification primer set to obtain a PCR reaction product;

[0084] S2. The PCR reaction product obtained in S1 is mixed with a SAP reaction reagent to perform a SAP digestion reaction to obtain a SAP reaction product;

[0085] S3. The SAP reaction product obtained in S2 is mixed with the single base extension primer set and the UEP reaction reagent to perform a UEP single base extension reaction to obtain a UEP reaction product;

[0086] S4. Perform time-of-flight mass spectrometry detection on the UEP reaction product obtained in S3 and judge the results.

[0087] This embodiment takes the collected EDTA anticoagulated venous whole blood sample as an example to illustrate the detection method in detail.

[0088] (1) Sample DNA extraction

[0089] A 5 mL EDTA-anticoagulated venous whole blood sample was collected, and genomic DNA was extracted using a Hybribio Nucleic Acid Extraction Kit (model DR-4801-KZ). The DNA concentration was measured using an ultraviolet spectrophotometer.

[0090] (2) Multiplex PCR amplification reaction

[0091] Multiplex PCR amplification was performed using the genomic DNA extracted in (1) as a template using the Agena PCR Reagent Set amplification kit. The primer mix (Primer Mix) was prepared by mixing the primers (SEQ ID NOs. 1 to 36) shown in Table 3, all at a concentration of 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 primer was 0.2 μM.

[0092] Table 6 Multiplex PCR amplification reaction system

[0093]

[0094] The multiplex PCR amplification reaction program is shown in Table 7.

[0095] Table 7 Multiplex PCR amplification reaction program

[0096]

[0097] To achieve better detection results, the gene GSK3B The concentration of the amplification primer for the rs334558 site on the PCR amplification reaction system was adjusted to 2.0 μM, so that the final concentration in the PCR amplification reaction system was 0.4 μM.

[0098] (3) SAP digestion reaction

[0099] Using Agena iPLEX ® The SAP digestion reaction was performed using the ProReagent Set kit. The SAP mixture was prepared according to Table 8 and 2 μL of the SAP mixture was added to the PCR amplification reaction product obtained in (2) to a total volume of 7 μL.

[0100] Table 8 SAP mixture

[0101]

[0102] The SAP digestion reaction procedure is shown in Table 9.

[0103] Table 9 Multiplex PCR reaction program

[0104]

[0105] (4) Single base extension reaction

[0106] Using Agena iPLEX ® The ProReagent Set kit was used to perform the single-base extension reaction. The single-base extension reaction solution was prepared according to Table 10, and 2 μL of the single-base extension reaction solution was added to the SAP digestion reaction product obtained in (3), with a total volume of 9 μL. The extension primer mixture (Extend Primer Mix) was obtained by mixing the single-base extension primers (SEQ ID NO. 37 to 54) shown in Table 4. The final concentration of each extension primer in the single-base extension reaction solution was as shown in Table 11.

[0107] Table 10 Single base extension reaction solution

[0108]

[0109] Table 11 Final concentration of each extension primer in the single base extension reaction solution

[0110]

[0111] The single base extension reaction procedure is shown in Table 12.

[0112] Table 12 Single base extension reaction procedure

[0113]

[0114] (5) Mass spectrometry detection

[0115] After the single-base extension reaction is completed, 16 μL of ultrapure water is added to each reaction well to make the total volume of the reaction product in each well 25 μL. After mixing evenly, time-of-flight mass spectrometry detection is performed.

[0116] Import the edited assay information into Typer software, select the reaction well locations for the test samples, create a plate, import the corresponding sample names, connect the detection chip, place the 384-well plate and detection chip in the corresponding positions on the mass spectrometer, and begin time-of-flight mass spectrometry. The test results are automatically analyzed using Typer 4.0 software, and the peak area of ​​each site is exported to an Excel file.

[0117] (6) Interpretation of results

[0118] The single base extension product is analyzed by mass spectrometry to detect the flight time of the extension product in the vacuum tube, thereby calculating the molecular weight of the extension product. The molecular weight of the extension product is then compared with the preset molecular weight in the software by the analysis software to determine whether the peak of the extension product is wild type, heterozygous mutant or homozygous mutant. The nucleic acid mass spectrometry detection diagram of the 18 SNP sites in the sample used in this example is shown in Figure 2. Figure 3 As shown. Figure 3 It can be seen that the detection of the 18 SNP sites can be achieved using the kit and method of the present invention. The relative molecular masses of the extension primers and extension products of each site of the present invention are shown in Table 13. In order to facilitate the verification with the relative molecular masses in Table 13, as an example, the present invention also provides the nucleic acid mass spectrometry detection diagrams of rs1058164 and rs1065852 sites, as shown in Table 13. Figure 4 and Figure 5 shown.

[0119] Table 13 Relative molecular weights of extension primers and extension products at each site

[0120]

[0121] Example 3 Kit Detection Performance Test

[0122] 1. Accuracy test

[0123] Sanger sequencing was performed on the quality control product JK and six clinical samples. The quality control product JK and the six clinical samples (Samples 1-6) were also tested using the method described in Example 2. The mass spectrometry results were compared to confirm consistency with the Sanger sequencing results. The assay was repeated three times, and the combined results are shown in Table 14.

[0124] Table 14 Accuracy test results

[0125]

[0126] As shown in Table 14, the results of mass spectrometry detection of the SNP site using the primer combination and method of the present invention are consistent with the detection results of the Sanger sequencing method, with an accuracy of 100%, indicating that this method has high sensitivity and strong specificity, and can meet the clinical detection needs of SNP sites related to antipsychotic drugs. As an example, the nucleic acid mass spectrometry detection graph and Sanger sequencing graph of the rs1065852 site in clinical sample 1 are shown in Figure 14. Figure 6 As shown. Figure 6 It can be seen that the results of mass spectrometry detection and Sanger sequencing are consistent.

[0127] 2. Detection limit test

[0128] The quality control product JK used in the accuracy test and 6 clinical samples were used as templates to test the minimum detection limit of the kit described in the present invention. The sample DNA was gradient diluted to four gradients of 40 ng / μL, 20 ng / μL, 10 ng / μL and 5 ng / μL, and multiple PCR amplification reaction, SAP digestion, single base extension and mass spectrometry detection analysis were performed respectively according to the operating steps provided in Example 2 of the present invention; the sample DNA amount was 1 μL, and each gradient was tested 3 times; the lowest concentration that could be detected 100% was taken as the minimum detection limit, and then the test was repeated 5 times at this concentration. If all 5 times were detected, it can be determined that this concentration is the minimum detection limit of the kit described in the present invention. The minimum detection limit test results of the kit described in the present invention are shown in Table 15, and the minimum detection limit is 5 ng / reaction. The nucleic acid mass spectrometry detection diagram of the detection limit test of clinical sample 1 is shown in FIG. Figure 7 shown.

[0129] Table 15 Detection limit test results

[0130]

[0131] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection 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; wherein the multiplex PCR amplification primer set contains 36 amplification primers, and the nucleotide sequences of the amplification primers are shown as SEQ ID NOs. 1 to 36, respectively; the single-base extension primer set contains 18 extension primers, and the nucleotide sequences of the extension primers are shown as SEQ ID NOs. 37 to 54, respectively.

2. Use of the primer combination according to claim 1 in preparing a product for detecting SNP sites associated with antipsychotic drug use, characterized in that: The SNP sites are rs951439, rs16947, rs1065852, rs1058164, rs1799978, rs3892097, rs28371725, rs1135840, rs1414334, rs3813929, rs17782313, rs489693, rs1800497, rs762551, rs67666821, rs1 799732, rs334558 and rs11869731; the SNP sites are associated with the use of perospirone, iloperidone, cariprazine, tetrabenazine, cariprazine, epiriprazole, asenapine, haloperidol, fluphenazine, sulpiride, trifluoperazine, zuclopenthixol, pimozide, loxapine, lithium carbonate, olanzapine, quetiapine, risperidone, aripiprazole, lurasidone, ziprasidone, clozapine, paliperidone, amisulpride, perphenazine and chlorpromazine.

3. A kit, characterized in that The kit contains the primer composition according to claim 1, wherein the kit is a kit for detecting SNP sites associated with antipsychotic drug use; the SNP sites are rs951439, rs16947, rs1065852, rs1058164, rs1799978, rs3892097, rs28371725, rs1135840, rs1414334, rs3813929, rs17782313, rs489693, rs1800497, rs762551, rs67666821, rs1799732, rs334558 and rs11869731; the SNP sites are associated with the use of perospirone, iloperidone, cariprazine, tetrabenazine, cariprazine, epiriprazole, asenapine, haloperidol, fluphenazine, sulpiride, trifluoperazine, zuclopenthixol, pimozide, loxapine, lithium carbonate, olanzapine, quetiapine, risperidone, aripiprazole, lurasidone, ziprasidone, clozapine, paliperidone, amisulpride, perphenazine and chlorpromazine.

4. The kit according to claim 3, wherein The kit also contains reagents required for multiplex PCR amplification reaction, SAP reaction and single base extension reaction.

5. The kit according to claim 4, characterized in that The reagents required for the multiplex PCR amplification reaction include PCR reaction buffer and PCR enzyme.

6. The kit according to claim 4, characterized in that The reagents required for the SAP reaction include SAP reaction buffer and SAP enzyme.

7. The kit according to claim 4, characterized in that The reagents required for the single base extension reaction include an extension reaction buffer, an extension reaction termination solution and an extension enzyme.

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

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

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