A kit for detecting anesthetic general medicine gene and application thereof

Abstract

This invention relates to the fields of biotechnology and pharmacogenomics detection, and discloses a kit and application for gene detection of drugs used in general anesthesia. The kit contains a primer set for detecting genetic polymorphic loci related to commonly used perioperative anesthetic, analgesic, and muscle relaxant drugs. The primer set includes multiplex PCR amplification primers and single-base extension primers. The kit also includes multiplex PCR amplification reaction solution, SAP reaction solution, SAP enzyme, extension buffer, single-base extension termination mixture, extension reaction enzyme, desalting purification resin, standards, calibrators, deionized water, and instructions for use. This invention uses matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) to genotype SNP loci, obtaining genotype results for 103 genetic polymorphic loci in a single test. This allows for the prediction of individual differences in drug metabolism, drug efficacy, and adverse reaction risk, providing a basis for personalized medication in general anesthesia.

Description

Technical Field

[0001] This invention relates to the field of biotechnology and pharmacogenomics detection, and more specifically, to a kit and application for gene detection of drugs used in general anesthesia. Background Technology

[0002] Anesthesia uses drugs or other methods to temporarily numb all or part of the body, providing conditions such as painlessness, muscle relaxation, absence of unpleasant memories, and proper regulation of stress response for surgery. In clinical practice, anesthesiology departments often use a combination of anesthetic drugs, analgesics, and muscle relaxants to achieve the ideal depth of anesthesia and surgical conditions.

[0003] Significant individual differences exist in the dosage requirements, onset time, duration of action, recovery time, efficacy, and side effects of anesthetic drugs in clinical practice. Besides factors such as gender, age, and liver and kidney function, genetic polymorphisms of drug-metabolizing enzymes, transporters, and drug targets are important genetic factors leading to differences in drug response. Detection of relevant loci can be used to predict an individual's metabolic capacity for specific drugs, efficacy, and risk of adverse reactions, thereby assisting in individualized anesthesia management.

[0004] Existing technologies have disclosed some gene detection kits related to anesthesia or analgesia, but most of them focus on single drugs or a few adverse reaction risk sites. They do not provide comprehensive coverage of commonly used drugs in anesthesia (anesthesia, analgesia, muscle relaxants), and lack systematic integration of sites related to metabolism, efficacy and toxicity, making it difficult to meet the one-time testing needs of the entire anesthesia management process. Summary of the Invention

[0005] In response to the clinical need for combined use of anesthetics, analgesics, and muscle relaxants during the entire course of anesthesia management, and the significant individual differences in these medications, existing detection methods often only cover a single drug or a small number of adverse reaction sites. Alternatively, high-throughput sequencing leads to high costs and long detection cycles, making it difficult to achieve one-time, rapid, and comprehensive genetic polymorphism typing and interpretation in the perioperative setting. The technical problem this invention aims to solve is to provide a primer set and kit for multi-site detection of general anesthesia medications that can be implemented on a MALDI-TOF time-of-flight mass spectrometry platform, along with corresponding detection methods, achieving a balance between comprehensive site coverage, high throughput, accuracy, and clinical feasibility.

[0006] A primer set for gene detection of general anesthesia medications, the primer set comprising: Multiplex PCR amplification primer pairs were used to amplify DNA fragments corresponding to genetic polymorphic sites related to general anesthesia medication, and their sequences are shown in SEQ ID NO.1-206; Single-base extension primers, used to perform single-base extension reactions on the genetic polymorphic sites, have sequences shown in SEQ ID NO.207-309.

[0007] Preferably, the 5' end of the multiplex PCR amplification primer pair contains the tag sequence ACGTTGGATG.

[0008] A kit for gene detection of drugs used in general anesthesia, the kit comprising the primer set and reagents for matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) SNP genotyping, the reagents comprising: multiplex PCR amplification reaction solution, SAP reaction solution, SAP enzyme, extension buffer, single-base extension termination mixture, extension reaction enzyme, desalting purification resin, standards, calibrators, and deionized water.

[0009] Preferably, the multiplex PCR amplification reaction solution contains Tris-HCl, MgCl2, dNTPs and thermostable DNA polymerase; the SAP reaction solution contains Tris-HCl and MgCl2; and the single-base extension reaction solution contains a mixture of extension buffer and single-base extension termination solution.

[0010] Preferably, the reference material is a human genomic DNA reference material or a mixture of oligonucleotides containing the target site sequence.

[0011] A method for in vitro detection of genetic polymorphisms related to general anesthesia medications using the aforementioned kit, comprising the following steps: (a) Extracting DNA from the sample to be tested; (b) Perform multiplex PCR amplification on the DNA to obtain the amplification product of the target site; (c) Treat the amplification products with shrimp alkaline phosphatase (SAP); (d) Perform a single-base extension reaction on the SAP-treated product; (e) Desalting and purifying the extended reaction products; (f) The purified product was analyzed by MALDI-TOF time-of-flight mass spectrometry and the typing results were obtained.

[0012] Preferably, the sample to be tested is human peripheral blood.

[0013] Use of the kit in the preparation of a genetic testing product for personalized medication guidance in general anesthesia practice.

[0014] Preferably, the primer set includes 103 genetic polymorphism sites, and the sites are single nucleotide polymorphism (SNP) sites.

[0015] Preferably, the multiplex PCR amplification primer pairs and / or the single-base extension primers are divided into multiple reaction systems according to the mass window and peak shape balance requirements of mass spectrometry typing, preferably into Plex1-Plex5.

[0016] Preferably, the desalting purification reagent is a cation exchange resin, used to remove salt ions from the extended reaction products for mass spectrometry detection.

[0017] Preferably, the typing results can be further analyzed to form explanatory information, which includes at least one or more of the following: metabolic phenotype prediction, drug sensitivity / efficacy related information, and adverse reaction risk information.

[0018] The beneficial effects of this invention are as follows: (1) More comprehensive site coverage: For perioperative anesthesia management, it systematically integrates the genetic polymorphism sites related to four major categories of commonly used drugs: anesthesia, analgesia, sedation and muscle relaxation, and can obtain the typing results of 103 genetic polymorphism sites at one time; (2) More complete assessment dimensions: The site types cover metabolic enzymes, transporters and drug target-related sites, which can be used for comprehensive assessment of metabolic capacity, efficacy sensitivity and adverse reaction risk at the same time; (3) The detection process is high-throughput and fast-turnaround: The process adopts multiplex PCR + SAP processing + single base extension combined with MALDI-TOF mass spectrometry typing, which is suitable for large-scale detection such as 96 wells. (4) Good accuracy and repeatability: In the examples, the selected 10 loci were 100% consistent with the first-generation sequencing control, and the intra-batch and inter-batch genotyping consistency were both 100%; (5) Output results are easy to use in clinical practice: an interpretation rule base can be established based on public databases / guidelines / consensus, and risk warnings and medication advice related to anesthesia general practice can be output to provide auxiliary basis for clinical physicians' decision-making. Attached Figure Description

[0019] Figure 1 This is a complete overview of the Plex1 peak diagram of the anesthesia method of the present invention; Figure 2 This is the rs2032582-AC mass spectrometry detection pattern of the present invention; Figure 3 This is the rs2032582-TC mass spectrometry detection pattern of the present invention; Figure 4 This is the rs2032582-CC mass spectrometry detection pattern of the present invention. Detailed Implementation

[0020] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the subject matter described herein, and changes may be made to the function and arrangement of the elements discussed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the examples. Furthermore, some features described in the examples may be combined in other examples.

[0021] For ease of understanding, the meanings of relevant terms in this manual are as follows: (1) "General medications for anesthesia" refers to anesthetics, analgesics and muscle relaxants and related drugs commonly used in perioperative anesthesia management; (2) "Genetic polymorphism site" preferably refers to single nucleotide polymorphism (SNP) site, but may also include other detectable genetic variation sites related to drug response; (3) "Multiple PCR amplification" refers to the PCR amplification method that simultaneously amplifies DNA fragments corresponding to multiple target sites in the same reaction system; (4) "Single base extension" refers to the method of using primers to perform a single base extension reaction in the region adjacent to the target site to achieve allele recognition; (5) “Generating rate” refers to the proportion of loci from which a clear genotyping result can be obtained out of the total number of detected loci; (6) "Concordance rate" refers to the proportion of the detection results of this invention that are consistent with the control method (first generation sequencing) at comparable sites.

[0022] Example 1 This embodiment proposes a primer set for gene detection of general anesthesia medications, the primer set comprising: Multiplex PCR amplification primer pairs were used to amplify DNA fragments corresponding to genetic polymorphic sites related to general anesthesia medication, and their sequences are shown in SEQ ID NO.1-206; Single-base extension primers, used to perform single-base extension reactions on the genetic polymorphic sites, have sequences shown in SEQ ID NO.207-309.

[0023] The 5' end of the multiplex PCR amplification primer pair contains the tag sequence ACGTTGGATG.

[0024] Example 2 This embodiment presents a kit for gene detection of drugs used in general anesthesia. The kit contains the primer set described in Example 1, and reagents for matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) SNP genotyping. The reagents include: multiplex PCR amplification reaction solution, SAP reaction solution, SAP enzyme, extension buffer, single-base extension termination mixture, extension reaction enzyme, desalting purification resin, standards, calibrators, and deionized water.

[0025] The multiplex PCR amplification reaction solution contains Tris-HCl, MgCl2, dNTPs, and thermostable DNA polymerase; the SAP reaction solution contains Tris-HCl and MgCl2; the single-base extension reaction solution contains a mixture of extension buffer and single-base extension termination solution. The genetic polymorphism sites include 103 sites, and the sites are single nucleotide polymorphism (SNP) sites.

[0026] Multiplex PCR amplification primer pairs and / or the single-base extension primers are divided into multiple reaction systems according to the mass window and peak shape balance requirements of mass spectrometry typing, preferably into Plex1-Plex5.

[0027] The reference material is a human genomic DNA reference material or a mixture of oligonucleotides containing the target site sequence.

[0028] Example 3 This embodiment presents a method for in vitro detection of genetic polymorphisms related to general anesthesia medications using the aforementioned kit, comprising the following steps: (a) Extracting DNA from the sample to be tested; (b) Perform multiplex PCR amplification on the DNA to obtain the amplification product of the target site; (c) Treat the amplification products with shrimp alkaline phosphatase (SAP); (d) Perform a single-base extension reaction on the SAP-treated product; (e) Desalting and purifying the extended reaction products; (f) The purified product was analyzed by MALDI-TOF time-of-flight mass spectrometry and the typing results were obtained.

[0029] The typing results can be further analyzed to generate explanatory information, which includes at least one or more of the following: metabolic phenotype prediction, drug sensitivity / efficacy-related information, and adverse reaction risk information.

[0030] The sample to be tested is human peripheral blood.

[0031] The desalting and purification reagent is a cation exchange resin, used to remove salt ions from the extended reaction products for mass spectrometry detection.

[0032] In vitro detection statement: The method described in this invention is a method for in vitro detection and data analysis of nucleic acids extracted from collected human samples, and does not include diagnostic or treatment steps performed in humans or animals; the interpretation information described in this invention is used to assist clinical decision-making, and the final medication plan shall be determined by a qualified clinician in combination with the patient's specific condition.

[0033] Example 4: Drug and Site Determination and Primer Design The selection of anesthetics, analgesics, and muscle relaxants can be determined by referring to publicly available guidelines / consensus statements and clinical expert opinions. After determining the drug range, genetic polymorphic sites related to drug metabolism, transport, or target sites can be screened based on publicly available databases and literature evidence, and multiplex PCR amplification primers and single-base extension primers can be designed accordingly.

[0034] (1) Data source and site screening reliability The selection of drugs and genetic loci can be based on cross-validation of publicly available evidence from multiple sources. Preferably, the range of drugs can be determined by referring to publicly available guidelines / consensus and clinical medication practices, such as the "Guidelines for Analgesia and Sedation in Chinese Adult ICUs," the "Expert Consensus on Prevention and Treatment of Malignant Hyperthermia in China," and the "Expert Consensus on Goal-Oriented Perioperative Analgesia Management in China (2021)." Locus sources may include, but are not limited to: (a) publicly available regulatory / guideline resources (e.g., publicly available lists from the FDA Pharmacogenetic Associations, CPIC guidelines); (b) drug and pharmacogenomics databases (e.g., DrugBank, PharmGKB); (c) gene allelic and variant annotation databases (e.g., PharmVar, dbSNP); and (d) publicly available SCI literature (prioritizing studies that target Asian populations, have large sample sizes, and are statistically significant).

[0035] In a preferred embodiment, the following screening rules can be used to improve the reliability of site selection: 1) Sites recommended or clearly associated in publicly available resources such as FDA / DrugBank; 2) Sites with an evidence level higher than 2B (or equivalent) in CPIC; 3) Sites with an evidence level higher than or equal to 3 in PharmGKB; 4) Combining the definition of asterisk alleles with PharmVar and the definition of rs number and alleles with dbSNP; and manually reviewing or conservatively including sites with inconsistent conclusions from different sources.

[0036] The database / document search mentioned above should use the most recent published version before the application date (or the version published on the search date); the determination of the level of evidence should follow the publicly available definitions in the relevant databases. By aggregating evidence from multiple sources, the bias of a single source can be reduced and the traceability and stability of site selection can be improved.

[0037] After selecting the drug, gene, and corresponding site, the design logic and optimization process of the 103-key primer set in the kit described in this invention are as follows: 1. Peak staggering design for mass spectrometry: This invention performed full-spectrum simulation on 103 extension primers. A tag sequence (ACGTTGGATG) was added to the 5' end of the amplification primers to increase the primer molecular weight and reduce the interference of primer peaks on the mass spectrometry elution position in the multiplexing system; primer specificity can be verified using conventional methods.

[0038] Technical effect: This modification does not participate in genome hybridization, but only increases the molecular weight. It successfully achieved a uniform distribution of all target peaks within the detection window of 4000-9000 Da. The clear panoramic peak diagram shown in Figure 1 verifies the effectiveness of this design.

[0039] 2' and 3' End Locking and Dimer Suppression: Given the presence of nearly 200 amplification primers and 103 extension primers in the reaction system, non-specific binding between primers is the main cause of background noise. This invention utilizes a thermodynamic algorithm to perform an "all-vs-all" interaction scan of the primer pool.

[0040] Optimization scheme: The complementarity of the 3' ends of the primers was specifically screened. Primer pairs with complementary pairing within the last three bases of the 3' end (훥G < -5 kcal / mol) were marked and their sequences were redesigned or their binding positions adjusted. Experimental verification: After this optimization, different genotypes at the same locus could be significantly distinguished by mass spectrometry (see Figure 2-3), demonstrating the accuracy of the primer library.

[0041] 3. Primer grouping algorithm logic: To maximize detection throughput, this invention does not employ random grouping, but instead uses a clustering grouping strategy based on a genetic algorithm. The 103 sites are divided into 5 reaction groups (Plex) according to their amplicon read length, GC content, and extension product mass. Design highlights: Ensuring uniform distribution of amplicon long reads within each Plex avoids competitive inhibition of long amplicon fragments by short-end amplicon fragments, thereby ensuring a detection rate of >99% for 103 sites in the kit.

[0042] Table 1 Examples of commonly used drugs in general anesthesia Example 5: Kit composition and reaction system In one embodiment, the kit includes multiplex PCR amplification primers, single-base extension primers, multiplex PCR amplification reaction solution, SAP reaction solution, SAP enzyme, extension buffer, single-base extension termination mixture, extension reaction enzyme, desalting purification resin, standards, calibrators, and deionized water.

[0043] The optimal final concentration of different primers in a multiplex system can be optimized based on amplification efficiency, non-specific amplification inhibition, and peak shape balance. To avoid limiting the protection range to a specific single primer concentration, it is preferable to describe it using the "feasible range + preferred range + allowable fine-tuning principle": (a) The final concentration of multiplex PCR amplification primers (single primers) can be 0.05–0.30 μmol / L, preferably 0.08–0.15 μmol / L; (b) The final concentration of single-base extension primers (single primers) can be 0.30–1.50 μmol / L, preferably 0.60–1.00 μmol / L; Since the mass spectrometry response capabilities of different extension primers vary, the single-base primers can be fine-tuned within the above range (±30%) if necessary to obtain a more balanced mass spectrometry peak shape and a higher overall gradation rate for the same Plex.

[0044] As a non-limiting example, in a specific optimization scheme, the final concentration of a few amplification primers can be adjusted to 0.12, 0.13, or 0.14 μmol / L, while the remaining amplification primers are at approximately 0.10 μmol / L; and the final concentration of a few extension primers can be adjusted to 0.76, 0.94, or 1.15 μmol / L, while the remaining extension primers are at approximately 0.80 μmol / L.

[0045] (2) Key reaction liquid components and concentration range To ensure that this invention can be implemented by those skilled in the art and to avoid limiting the scope of protection to a single formulation, the key reaction solution may adopt the following composition and dosage / concentration range (preferred values ​​are given in parentheses), and conventional adjustments may be made within the range described above: ① Multiplex PCR amplification reaction solution (final concentration): Tris-HCl 5–20 mM (15 mM), MgCl2 2.0–6.0 mM (4.0 mM), dNTP 100–600 μM (500 μM), thermostable DNA polymerase approximately 0.5–2.0 U / 5 μL or 1–4 U / 10 μL (approximately 1 U / 5 μL or 2 U / 10 μL); the final concentration of each amplification primer is usually approximately 0.05–0.20 μmol / L (preferably approximately 0.10 μmol / L). Without affecting the balance of multiplex amplification, a few primers can be fine-tuned to approximately 0.12, 0.13, or 0.14 μmol / L; template DNA 5–50 ng / reaction (preferably approximately 10 ng).

[0046] ②SAP reaction solution: Commercially available SAP Buffer (10× stock solution) can be used. After addition, the effective concentration in the reaction system can be about 0.2–0.3×. The amount of SAP enzyme can be about 0.2–0.5 U / well (preferably about 0.3 U / well; 0.30 μL of 1.0 U / μL SAP enzyme is added). The final concentration in the SAP reaction system is 0.005-0.05 U / μL, preferably 0.015-0.03 U / μL.

[0047] ③ Extension buffer: Commercial iPLEX Buffer Plus (10× stock solution) can be used, and its effective concentration in the extension reaction system after addition can be about 0.2–0.3×; In a non-limiting example of a self-prepared equivalent buffer, the 10× stock solution may contain Tris-HCl 500–2000 mM (1M) and MgCl2 100–400 mM (250 mM).

[0048] ④ Single-base terminator mixture: Contains mass-modified ddNTP terminators (mass-modified ddATP, ddCTP, ddGTP, ddTTP), with each ddNTP component having a high concentration of 2-5 mM in the storage solution. This is designed to minimize the introduction of impurity ions from exogenous sources and optimize crystallization during matrix-assisted laser desorption / ionization. In a non-limiting example of a self-prepared equivalent system, a small amount of dNTPs (e.g., 0–0.2 mM each) can be optionally added to optimize the peak shape of the extended reaction.

[0049] ⑤ Single-base extension primer mixture: The 103 single-base extension primers shown in Table 15 are classified according to their respective Plex groups. Each primer is diluted to 7 μM working solution using ultrapure water or TE buffer. Then, they are mixed according to their final concentrations to obtain the single-base extension primer premix for each Plex. For sites with weak mass spectrometry signals, the concentration of the primer for that site can be appropriately increased to 1.5 μM to balance the peak shape.

[0050] In addition to the aforementioned key reaction solutions, deionized water, reference standards, desalination resins, etc., can be conventional commercial products or equivalent materials in this field.

[0051] Table 2 Multiplex PCR amplification reaction system Table 3 Multiplex PCR amplification reaction procedure Table 4 SAP enzyme treatment reaction system Table 5. SAP Enzyme Treatment Reaction Procedures (Examples) Table 6 Single-base extension reaction system Table 7 Single-base extension reaction procedure Table 8. Components of the reagent kit (96 doses) In one embodiment, the kit can be transported and stored at -18°C or lower; preferably, the shelf life is 12 months. Once opened, the enzymes and mixtures are recommended to be used within 3 months, and the number of freeze-thaw cycles should preferably not exceed 5.

[0052] Example 6: Validation of system accuracy (10 sites, compared with first-generation sequencing) This example was used to verify the genotyping accuracy of this kit on the MALDI-TOF mass spectrometry platform for representative SNP loci. Human genomic reference NA12878 (3 technical replicates) and clinical peripheral blood DNA samples (3 technical replicates per sample) were analyzed. Ten representative loci were selected from the 103 SNP loci covered by this kit for accuracy verification. These loci covered different gene classes (drug-metabolizing enzymes, transporters, receptors) and were distributed across different Plex groups to represent the genotyping performance of the multiplex system under different amplification / extension conditions. 4 μL of DNA template was added to each well; the sample DNA concentration was preferably not less than 5.0 ng / μL (corresponding to an input volume ≥20 ng / reaction).

[0053] The detection process may include: multiplex PCR amplification, SAP enzyme treatment, single-base extension, desalting and purification, MALDI-TOF mass spectrometry detection, and data analysis. The genotyping parameters are configured as follows: mass offset: 500 ppm, primer SNR threshold: 3, product SNR threshold: 3, and minimum signal-to-noise ratio (SNR) of two products: 0.3. Using first-generation sequencing genotyping results as a control, the MALDI-TOF genotyping results are compared site-by-site, and the overall genotyping rate and concordance rate are statistically analyzed. In this embodiment, the overall genotyping rate of the reference sample is 100%, and the concordance rate with the first-generation sequencing control result is 100%.

[0054] Table 9. Examples of NA12878 locus genotyping results Table 10 Summary of accuracy results for 10 loci Example 7: System repeatability validation (intra-batch / inter-batch) This embodiment is used to verify the repeatability of the detection sites of this kit, including intra-batch repeatability and inter-batch repeatability. (1) Intra-batch repeatability: Two standards and 10 clinical samples were selected and independently tested three times within the same batch of kit, by the same operator, and on the same day. The genotyping consistency rate and overall genotyping rate between each replicate were calculated. (2) Inter-batch repeatability: Ten samples were tested repeatedly under different dates, different batches of kit, and different operator conditions. The genotyping consistency rate was calculated. In this embodiment, the intra-batch and inter-batch repeatability consistency rates were both 100%.

[0055] Table 11 Intra-batch consistency test results Table 12 Results of inter-batch consistency test Example 8: System sensitivity verification (minimum DNA input amount) The reference DNA was diluted to multiple concentration gradients using reagent kit references and deionized water (template-free control, ddH2O) and three technical replicates were set up. Detection was performed according to the method in Example 2. 4 μL of DNA template was added to each well, corresponding to different input volumes. The overall genotyping rate was statistically analyzed for different input volumes. The results showed that when the input volume was ≥20 ng / reaction, the overall genotyping rate reached 100%; no effective genotyping peak was detected in the negative control (Table 13).

[0056] Table 13 Overall genotyping rate under different DNA concentrations / input volumes Table 14: Primer sequences for multiplex PCR amplification (SEQ ID NO.1-206) Note: Table 14 lists 103 sets of multiplex PCR amplification primer pairs, covering 103 SNP sites.

[0057] Table 15: Single-base extension primer sequences (SEQ ID NO.207-309) Table 15 lists single-base extension primers that match the amplification primer pairs described in Table 14 for genotyping of the 103 SNP sites on the MALDI-TOF mass spectrometry platform.

[0058] The embodiments of the present invention have been described above. However, the embodiments are not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make more equivalent embodiments under the guidance of the present embodiments, and all of them are within the protection scope of the present embodiments.

Claims

1. A primer set for gene detection of drugs used in general anesthesia, characterized in that, The primer set includes: Multiplex PCR amplification primer pairs were used to amplify DNA fragments corresponding to genetic polymorphic sites related to general anesthesia medication, and their sequences are shown in SEQ ID NO.1-206; Single-base extension primers, used to perform single-base extension reactions on the genetic polymorphic sites, have sequences shown in SEQ ID NO. 207-309.

2. The primer set according to claim 1, characterized in that, The 5' end of the multiplex PCR amplification primer pair contains the tag sequence ACGTTGGATG.

3. A kit for gene detection of drugs used in general anesthesia, characterized in that, The kit contains the primer set as described in claim 1 or 2, and reagents for matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) SNP typing, the reagents including: multiplex PCR amplification reaction solution, SAP reaction solution, SAP enzyme, extension buffer, single-base extension termination mixture, extension reaction enzyme, desalting purification resin, standards, calibrators, and deionized water.

4. The reagent kit according to claim 3, characterized in that, The multiplex PCR amplification reaction solution contains Tris-HCl, MgCl2, dNTPs, and thermostable DNA polymerase; the SAP reaction solution contains Tris-HCl and MgCl2; and the single-base extension reaction solution contains a mixture of extension buffer and single-base extension termination solution.

5. The kit according to claim 3 or 4, characterized in that, The reference material is a human genomic DNA reference material or a mixture of oligonucleotides containing the target site sequence.

6. A method for in vitro detection of genetic polymorphisms related to general anesthesia medications using the kit described in any one of claims 3-5, characterized in that, Includes the following steps: (a) Extracting DNA from the sample to be tested; (b) Perform multiplex PCR amplification on the DNA to obtain the amplification product of the target site; (c) Treat the amplification products with shrimp alkaline phosphatase (SAP); (d) Perform a single-base extension reaction on the SAP-treated product; (e) Desalting and purifying the extended reaction products; (f) The purified product was analyzed by MALDI-TOF time-of-flight mass spectrometry and the typing results were obtained.

7. The method according to claim 6, characterized in that, The sample to be tested is human peripheral blood.

8. Use of the kit according to any one of claims 3-5 in the preparation of a genetic testing product for personalized medication guidance in general anesthesia.

9. The primer set according to claim 1, characterized in that, The genetic polymorphism sites include 103 sites, and the sites are single nucleotide polymorphism (SNP) sites.

10. The primer set according to claim 1 or 9, characterized in that, The multiplex PCR amplification primer pairs and / or the single-base extension primers are divided into Plex1-Plex5 reaction systems according to the mass window and peak shape balance requirements of mass spectrometry typing.

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