Glucocorticoid metabolism related SNP site detection primer group and kit

Abstract

The invention discloses a glucocorticoid metabolism related SNP site detection primer group and a kit, the primer group is designed for main mutation sites related to glucocorticoid metabolism, specific detection of site mutation related to glucocorticoid metabolism can be realized, the accuracy is high, the detection result covers two mutation sites of PAI-1 and ABCB1 genes, and the primer group can be applied to the detection of glucocorticoid metabolism related SNP sites. The detection period can be greatly shortened, and the detection cost is reduced; moreover, a nucleic acid mass spectrum platform can be used for realizing detection of multiple gene loci on a single platform, and reference is provided for individualized glucocorticoid treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of glucocorticoid-related SNP site detection, in particular to a glucocorticoid metabolism-related SNP site detection primer set and a kit. Background Art

[0002] Glucocorticoids are a class of steroid hormones secreted by the zona fasciculata in the adrenal cortex, mainly cortisol, which regulates the biosynthesis and metabolism of sugar, fat, and protein, and also inhibits immune response, anti-inflammatory, anti-toxic, and anti-shock effects. Glucocorticoids have a powerful effect, but the systemic toxic side effects they cause to the body cannot be ignored. Among them, avascular necrosis of the femoral head is one of its common serious adverse reactions. Glucocorticoid administration is the most common cause of secondary osteoporosis and the main cause of non-traumatic osteonecrosis. The incidence of osteonecrosis in patients receiving long-term treatment is 9%-40%. In multiple studies on patients with femoral head necrosis, it was found that PAI-1 (4G / 5G) and ABCB1 (3435C>T) site gene polymorphisms are associated with the occurrence of glucocorticoid-induced femoral head necrosis (SONFH). PAI-1 is the main inhibitor of the plasma fibrinolytic system, which can lead to reduced fibrinolytic activity, cause thrombosis, cause intraosseous hypertension, reduced bone blood flow, osteocyte ischemia and hypoxia, and ultimately lead to osteocyte necrosis. The P-type glycoprotein encoded by ABCB1 is an energy-dependent efflux pump located on the cell membrane that can pump most therapeutic drugs, including glucocorticoids, out of the cell. It also plays an important role in the pharmacokinetic processes of drug absorption, distribution, and metabolism. Genetic testing can be performed before long-term or large-scale use of glucocorticoids to predict the risk of femoral head necrosis in patients, help better understand the characteristics of the disease, develop individualized treatment plans, and reduce the risk of adverse reactions and treatment failure. [1,2,3,4,5] .

[0003] At present, there are many detection methods with their own characteristics. PCR-based detection methods, such as ARMS-PCR, fluorescent quantitative PCR, digital PCR, etc., are easy to operate, fast, and simple to analyze data. However, due to the limitation of throughput, they cannot meet the clinical needs for multiple genes and multiple sites detection. High-throughput gene detection technology, such as high-throughput sequencing technology, solves the limitation of throughput, but it is costly, has a long detection cycle, and has high technical requirements for experimental operators and data analysts. Therefore, it has not yet been widely used in clinical detection.

[0004] In summary, there is an urgent need for an economical, rapid, and easy-to-operate detection method to predict the dosage of glucocorticoids for different individuals in a personalized manner, in order to meet the needs of precise medication and treatment.

[0005] References:

[0006] [1] Liu Meng, Wu Simiao. Standardized application of glucocorticoids in nervous system diseases [J / OL]. West China Medical Journal: 1-8 [2023-07-11]. http: / / kns.cnki.net / kcms / detail / 51.1356.R.20221222.1144.013.html.

[0007] [2]D'haeseleer M. Systemic use of corticosteroids in neurological disorders. Journal of Translational Internal Medicine. 2015; 2(2):70-72.

[0008] [3] Parissis D, et al. Corticosteroids in neurological disorders: Thedark side. JClin Neurosci. 2017Oct; 44:1-5.

[0009] [4] “Technical Guidelines for Drug Metabolizing Enzymes and Drug Target Gene Detection (Trial)” 2015 edition. National Health and Family Planning Commission.

[0010] [5]Liu Y, Fernandez CA, Smith C, et al. Genome-wide study links PNPLA3 variant with elevated hepatic transaminase after acute lymphoblastic leukemiatherapy: PNPLA3 and asparaginase transaminasemia[J]. Clinical Pharmacology&Therapeutics, 2017, 102(1). Summary of the invention

[0011] The present invention provides a primer set and a kit for detecting SNP sites related to glucocorticoid metabolism, so as to detect two mutation sites of PAI-1 and ABCB1 genes related to glucocorticoid metabolism in a single well, greatly shorten the detection cycle, reduce the detection cost, and realize the guidance of glucocorticoid medication.

[0012] In view of this, the scheme of the present invention is:

[0013] The first aspect of the present invention provides a primer set for detecting SNP sites related to glucocorticoid metabolism, comprising a first primer pair for detecting the rs1799762 site of the PAI-1 gene, and a second primer pair for detecting the rs1045642 site of the ABCB1 gene; the nucleotide sequence of the first primer pair is shown in SEQ ID NO: 1-2, and the nucleotide sequence of the second primer pair is shown in SEQ ID NO: 3-4.

[0014] Furthermore, the first primer set further includes a single base extension primer having a nucleotide sequence as shown in SEQ ID NO:5, and the second primer set further includes a single base extension primer having a nucleotide sequence as shown in SEQ ID NO:6.

[0015] The second aspect of the present invention provides the use of the primer set described in the first aspect above in the preparation of a glucocorticoid metabolism-related SNP site detection product or a glucocorticoid metabolism level prediction product.

[0016] The third aspect of the present invention is a glucocorticoid metabolism-related SNP site detection kit based on a nucleic acid mass spectrometry platform, comprising a first primer set and a second primer set; the first primer set comprises a PCR amplification primer pair with a nucleotide sequence as shown in SEQ ID NO: 1-2, and a single base extension primer with a nucleotide sequence as shown in SEQ ID NO: 5; the second primer set comprises a PCR amplification primer pair with a nucleotide sequence as shown in SEQ ID NO: 3-4, and a single base extension primer with a nucleotide sequence as shown in SEQ ID NO: 6.

[0017] Furthermore, the kit also includes one or more of a PCR reaction reagent, a SAP reaction reagent and a single base extension reaction reagent.

[0018] Preferably, the PCR reaction reagents include PCR reaction buffer, Mg 2+ , dNTPs, DNA polymerase, one or more in water; the SAP reaction is a shrimp alkaline phosphatase digestion reaction, and the reagents include SAP reaction buffer, SAP enzyme, one or more in water; the single base extension reaction reagents include single base extension reaction buffer, single base extension reaction termination solution, single base extension reaction enzyme, one or more in water.

[0019] The fourth aspect of the present invention proposes a glucocorticoid metabolism-related SNP site detection reaction system based on a nucleic acid mass spectrometry platform, comprising a PCR amplification reaction system and a single base extension reaction system; the PCR amplification reaction system contains a first primer set and a second primer set; the first primer set includes a PCR amplification primer pair with a nucleotide sequence as shown in SEQ ID NO: 1-2, and the second primer set includes a PCR amplification primer pair with a nucleotide sequence as shown in SEQ ID NO: 3-4; the single base extension reaction system contains a single base extension primer, which is used to detect the same site as the amplification primer pair in the PCR amplification reaction system.

[0020] Furthermore, the single-base extension primer includes a single-base extension primer set having a nucleotide sequence as shown in SEQ ID NO: 5-6.

[0021] Furthermore, the concentration of each amplification primer used in the PCR amplification reaction system is 0.25 μM.

[0022] Furthermore, the concentrations of the primers in the single-base extension primer set with the nucleotide sequences as shown in SEQ ID NOs: 5-6 are 3.75 μM and 10 μM, respectively.

[0023] Furthermore, the reaction system also includes one or more of a PCR reaction reagent, a SAP reaction reagent and a single base extension reaction reagent.

[0024] Preferably, the PCR reaction reagents include PCR reaction buffer, Mg 2+ , dNTPs, DNA polymerase, one or more in water; the SAP reaction is a shrimp alkaline phosphatase digestion reaction, and the reagents include SAP reaction buffer, SAP enzyme, one or more in water; the single base extension reaction reagents include single base extension reaction buffer, single base extension reaction termination solution, single base extension reaction enzyme, one or more in water.

[0025] The fifth aspect of the present invention provides a glucocorticoid metabolism-related SNP site detection system based on a nucleic acid mass spectrometry platform, comprising the reaction system described in the fourth aspect, a desalting resin and a spotting chip.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects:

[0027] The primer set of the present invention is designed for the main mutation sites related to glucocorticoid metabolism, and can realize specific detection of site mutations related to glucocorticoid metabolism with high accuracy. The detection results cover a total of 2 mutation sites of PAI-1 and ABCB1 genes, which can greatly shorten the detection cycle and reduce the detection cost. In addition, the detection of multiple gene sites on a single platform is realized, providing a reference for individualized glucocorticoid treatment.

[0028] The primer set provided by the present invention is obtained through modification optimization and large sample test screening. All primer sequences can accurately type the samples and can also meet the requirements of mass spectrometry detection technology, thereby realizing the rapid and effective detection of glucocorticoid metabolism using the MassARRAY platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a MassArray result diagram of the rs1799762 site provided in an embodiment of the present invention.

[0030] Figure 2 This is a MassArray result diagram of the rs1045642 site provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In one embodiment, a primer set for detecting SNP sites related to glucocorticoid metabolism is proposed, and the main mutation sites related to glucocorticoid metabolism are designed, and specific detection can be achieved for site mutations related to glucocorticoid metabolism, with high accuracy, and the test results cover a total of 2 mutation sites of PAI-1 and ABCB1 genes, which can greatly shorten the detection cycle and reduce the detection cost; and, the detection of multiple gene sites on a single platform is achieved, providing a reference for individualized glucocorticoid treatment. In addition, the primer set provided by the present invention is obtained by changing optimization and large sample test screening, and all primer sequences can accurately type samples, and can also meet the requirements of mass spectrometry detection technology, and realize the application of MassARRAY platform to quickly and effectively detect glucocorticoid metabolism.

[0033] In an embodiment of the present invention, the SNP sites corresponding to glucocorticoid metabolism include: rs1799762 and rs1045642.

[0034] In a preferred embodiment, the primer pair is a PCR primer pair. When an extension primer is required in actual application, the primer set also includes two extension primers. When designing the above two target SNP sites, other SNP sites upstream and downstream of each site that account for more than 1% of the total are confirmed through professional software, and coverage is avoided when designing amplification primers and extension primers to reduce their impact on detection.

[0035] It can be understood that the primer pairs and extension primers correspond one to one, and the corresponding primer pairs and extension primers are used to detect the nucleotide at the same site.

[0036] Further, the genotypes of each site are listed, as shown in the following table:

[0037] Gene rs number Mutation information PAI-1 rs1799762 NM_000602.4(SERPINE1):c.-820_-817G(4_5) ABCB1 rs1045642 NC_000007.14:g.87509329A>G

[0038] In an embodiment of the present invention, the glucocorticoid metabolism detection is performed by detecting the human PAI-1 gene and ABCB1 gene through gene detection technology to find out the glucocorticoid metabolism levels of different individuals.

[0039] It should be noted that in order to save samples and costs as much as possible in the detection of glucocorticoid metabolism, all two SNP sites were detected in the same detection well;

[0040] It is understandable that there will be multiple pairs of PCR amplification primers in the detection well. To ensure the accuracy and specificity of the detection, primers are designed for multiplex PCR when designing amplification primers, and the design results are compared with the human genome using a professional website (https: / / www.ncbi.nlm.nih.gov / ) to ensure that there is no non-specific match;

[0041] It is understandable that in order to avoid interference of amplification primers on the detection results, a specific prefix is ​​added during primer design to increase the primer quality and eliminate interference.

[0042] It is understandable that there will be multiple extension primers in the detection well. In order to ensure that the detection results of each site do not interfere with each other, professional software is used to design the extension primers to different masses. At the same time, the mass of each primer before and after extension is calculated through manual verification, and unqualified primers are designed separately.

[0043] Specifically, the correspondence between the detection sites and the types of diseases is shown in the following table.

[0044]

[0045] According to a second aspect of the present invention, there is provided use of the above-mentioned primer set for SNP sites related to glucocorticoid metabolism detection in the preparation of a glucocorticoid metabolism detection product.

[0046] According to a third aspect of the present invention, a product for detecting glucocorticoid metabolism is provided, the product comprising the above-mentioned primer set.

[0047] This product can specifically detect site mutations related to glucocorticoid metabolism, with low detection cost, short cycle, simple operation and high accuracy. It has great clinical application value and very broad market promotion prospects.

[0048] In an embodiment of the present invention, the product for detecting glucocorticoid metabolism further comprises a reagent and / or device for detecting SNP sites.

[0049] It is to be understood that the reagents and / or devices for detecting SNP sites may be conventional general reagents and / or devices commercially available in the art for detecting SNP site nucleotides, for example, may be reagents or kits for detecting SNP site nucleotides, or may be kits comprising reagents for detecting SNP site nucleotides.

[0050] In an embodiment of the present invention, the reagents include 10×PCR Buffer, dNTP Mix, MgCl2, PrimerMix, PCR Enzyme and ultrapure water; the device includes a device for detecting SNP site nucleotides using MassARRAY, and the device includes MassARRAY CPM.

[0051] In another embodiment, a method for detecting SNP sites related to glucocorticoid metabolism is provided, the method comprising using the above primer set to detect nucleotides at the SNP site in the genome of the sample to be tested.

[0052] The method can detect two SNP sites of two genes related to glucocorticoid metabolism by using the primer set provided by the present invention, and has the characteristics of high accuracy, high sensitivity, good repeatability, low cost, short detection cycle, intuitive results, etc.

[0053] It is understandable that the present invention can detect the entire genome of the sample to be tested, and can also screen specific genes for detection (for example, detecting specific genes such as rs1799762 and rs1045642). When detecting specific genes, there is less interference and the test results are more accurate.

[0054] In some preferred embodiments, the above primer set is used to perform PCR amplification and base extension reaction on the genome of the sample to be tested, and then the MassARRAY system is used to detect the reaction product to determine the nucleotide of the SNP site in the genome of the sample to be tested.

[0055] MassARRAY gene analysis technology is based on MALDT-TOF time-of-flight mass spectrometry technology. It first amplifies the target sequence through PCR, and then adds SNP sequence-specific extension primers as needed to extend a single base at the SNP site. This technology uses the high sensitivity of mass spectrometry analysis to effectively distinguish two gene sequences with only one different base, and then infers the SNP typing.

[0056] In the preferred embodiment, the MassARRAY system is used for detection, which realizes the detection method of multiple gene loci using a single platform, greatly improving the detection efficiency, and is particularly suitable for batch detection, providing a reference for personalized glucocorticoid treatment. In addition, the required samples are highly adaptable, and both peripheral blood and oral exfoliated cells can be well detected.

[0057] Preferably, the method further comprises the step of dephosphorylating the PCR product before the base extension reaction. Specifically, the PCR product is dephosphorylated using shrimp alkaline phosphatase.

[0058] Preferably, the method further comprises the step of purifying the reaction product after the base extension reaction, and then applying MassARRAY to detect the reaction product. Specifically, the reaction product can be purified by resin salt removal.

[0059] In some preferred embodiments, the primer set is divided into the following group 1: a primer set comprising the detection of rs1799762 and rs1045642.

[0060] The primer grouping takes into account the size of the extension primers to ensure that there is no interference between primers in the group.

[0061] In a preferred embodiment, the method further includes a step of purifying the reaction product after the base extension reaction, and then applying MassARRAY to detect the reaction product. Specifically, the reaction product can be purified by resin salt removal, and then spotted onto a chip with a matrix through an instrument to form a co-crystal with the matrix, and then the composite crystal is irradiated with a laser, and the irradiated matrix molecules absorb and accumulate energy and then rapidly heat up, so that the crystal sublimates into a gas phase, and the nucleic acid molecules are also desorbed and transformed into metastable ions (mostly single-charged ions); these single positively charged ions can obtain kinetic energy in the accelerating electric field, and fly toward the detector after flying through the free drift zone, and the smaller the ion mass, the faster the flight speed, and the earlier it arrives at the detector, and nucleic acid molecules of different molecular weights are detected due to different arrival times at the detector.

[0062] The following are preferred embodiments for verifying the technical solution and technical effects of the present invention. Unless otherwise specified, the operations involved are basic means mastered by those skilled in the art.

[0063] The main reagent information used in the following examples is shown in Table 1.

[0064] Table 1: Main reagents

[0065]

[0066] The main instrument information used in the following examples is shown in Table 2.

[0067] Table 2: Main instruments

[0068]

[0069] Example 1 Primer design and reaction system establishment

[0070] Through the primer design software (Assay Design Suite) on the MassARRAY website, adjust the relevant parameters, complete the primer design of PCR and UEP at the two sites, export the designed primers and parameter files, and synthesize the primers. For fragments of different molecular weights, the detection efficiency of the nucleic acid mass spectrometer is different. In order to make the detection efficiency of each site reach a more consistent level, it is necessary to adjust the extension primer UEP in the system to an appropriate concentration. The specific method is as follows:

[0071] Dilute 1μL UEP mix to 25μL with ultrapure water, perform nucleic acid mass spectrometry detection according to the general process, and compare the peak height of each UEP primer. According to the amount of each UEP primer added and the peak height, add the UEP primer of the corresponding site in direct proportion, and finally make the lowest peak height of each site UEP half of the highest peak height. Figure 1 , Figure 2The specific peak graphs before and after the adjustment of UEP primers are listed respectively. (Table 3 and Table 4 are the preparation tables of PCR primer mix and UEP primer mix after adjustment respectively), and then the primers are tested and optimized.

[0072] Table 3: PCR primer mix preparation

[0073] Well SNP_ID add(μl) W1 rs1799762 0.5 / 0.5 W1 rs1045642 0.5 / 0.5 W1 water 199

[0074] Table 4: UEP primer mix preparation

[0075] Well SNP_ID add(μl) W1 rs1799762 1.5 W1 rs1045642 4.0 W1 water 194.5

[0076] The specific steps are as follows:

[0077] (1) Dilute the genomic DNA sample to 10 ng / μL and prepare the PCR reaction MIX according to the table below (the following is the amount of a single sample):

[0078] Table 5: PCR reaction system

[0079]

[0080] Seal the film, vortex mix for 30 seconds, centrifuge at 4000 rpm for 1 minute, and place the plate on the PCR instrument for the following thermal cycles:

[0081]

[0082]

[0083] (2) Shrimp alkaline phosphatase digestion (SAP)

[0084] Take out the PCR plate, centrifuge at 4000 rpm for 1 minute, and prepare the SAP reaction system according to the following table (the following is the amount of a single sample):

[0085] Table 6: SAP reaction system

[0086]

[0087] Add 2 μL of SAP mixture to each reaction well, seal the film, vortex mix for 30 seconds, centrifuge at 4000 rpm for 1 minute, and place the plate on the PCR instrument for the following thermal cycle:

[0088] Temperature(℃) time 37 40min 85 5min 10 Insulation

[0089] (3) Single base extension (EXT)

[0090] Take out the PCR plate, centrifuge at 4000 rpm for 1 minute, and prepare the EXT reaction system according to the following table, where Extend PrimerMix is ​​a mixture of four sets of extension primers with different sites (the following is the amount of a single sample):

[0091] Table 7: EXT reaction system

[0092]

[0093] Add 2 μL of extension mixture to each reaction well, seal the membrane, vortex mix for 30 seconds, centrifuge at 4000 rpm for 1 minute, and place the plate on a PCR instrument for the following thermal cycles:

[0094]

[0095]

[0096] (4) Resin desalting and dispensing

[0097] The samples were desalted by resin using MassARRAY CPM and spotted onto the corresponding SpectroCHIP (chip).

[0098] (5) MALDI-TOF

[0099] The data were obtained using a MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometer. Taking the EDT12267925B1D1 sample test as an example, the MassARRAY test results of each point are shown in Figure 1-2 .

[0100] For specific primer sequences, see the primer sequences in Tables 8 and 9.

[0101] Table 8: PCR primer sequences

[0102]

[0103] Table 9: UEP primer sequences

[0104] Location UEP Primers SEQ ID NO rs1799762 CCAGCACATCAATGA 5 rs1045642 TTGTGGCTATGATTG 6

[0105] Example 2 Verification of the reaction system

[0106] During the system optimization process, each detection site has 51 samples with known results (8 Sanger and 42 MassArray) for verification. The comparison results are consistent, confirming that the results of the detection of the present invention are accurate. After confirming the optimal reaction system, the present invention conducted an accuracy verification experiment. The specific verification scheme is as follows:

[0107] (1) Accuracy test verification plan: 51 samples were used to complete the accuracy test (see Table 10), and the test results were compared with the original results. If the consistency was greater than 95%, the verification was passed.

[0108] The specific verification process is as follows: first, the amplification primer MIX and the extension primer MIX are prepared according to the system addition table provided in Example 1 of the present invention, and then according to the operating steps in Example 1, PCR amplification, shrimp alkaline phosphatase consumption, single base extension, resin desalting and MassARRAY CPM spot analysis are performed respectively, and the results are analyzed. The accuracy and precision results are shown in Tables 10 and 11 below.

[0109] Table 10: Raw validation results for accuracy

[0110]

[0111]

[0112] Table 11: MassARRAY validation results for accuracy

[0113]

[0114]

[0115] Comparison of the MassARRAY results of 51 samples with the original results shows that the accuracy of the system verification experiment of the present invention is 100%.

[0116] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A primer set for detecting SNP sites related to glucocorticoid metabolism, characterized in that: It comprises a first primer pair for detecting the rs1799762 site of the PAI-1 gene and a second primer pair for detecting the rs1045642 site of the ABCB1 gene; the nucleotide sequence of the first primer pair is shown in SEQ ID NO: 1-2, and the nucleotide sequence of the second primer pair is shown in SEQ ID NO: 3-4.

2. The primer set according to claim 1, characterized in that The first primer set further includes a single base extension primer having a nucleotide sequence as shown in SEQ ID NO:5, and the second primer set further includes a single base extension primer having a nucleotide sequence as shown in SEQ ID NO:

6.

3. Use of the primer set described in claim 1 or 2 in preparing a glucocorticoid metabolism-related SNP site detection product or a glucocorticoid metabolism level prediction product.

4. A glucocorticoid metabolism-related SNP site detection kit based on a nucleic acid mass spectrometry platform, characterized in that: It includes a first primer set and a second primer set; the first primer set includes a PCR amplification primer pair with a nucleotide sequence as shown in SEQ ID NO: 1-2, and a single base extension primer with a nucleotide sequence as shown in SEQ ID NO: 5; the second primer set includes a PCR amplification primer pair with a nucleotide sequence as shown in SEQ ID NO: 3-4, and a single base extension primer with a nucleotide sequence as shown in SEQ ID NO:

6.

5. The kit according to claim 4, characterized in that The kit further comprises one or more of a PCR reaction reagent, a SAP reaction reagent and a single base extension reaction reagent.

6. A glucocorticoid metabolism-related SNP site detection reaction system based on a nucleic acid mass spectrometry platform, characterized in that: It comprises a PCR amplification reaction system and a single base extension reaction system; the PCR amplification reaction system comprises a first primer group and a second primer group; the first primer group comprises a PCR amplification primer pair having a nucleotide sequence as shown in SEQ ID NO: 1-2, and the second primer group comprises a PCR amplification primer pair having a nucleotide sequence as shown in SEQ ID NO: 3-4; the single base extension reaction system comprises a single base extension primer for detecting the same site as the amplification primer pair in the PCR amplification reaction system.

7. The reaction system according to claim 6, characterized in that The single-base extension primers include a single-base extension primer set having nucleotide sequences as shown in SEQ ID NOs: 5-6.

8. The reaction system according to claim 7, characterized in that: The concentration of each amplification primer used in the PCR amplification reaction system is 0.25 μM; And / or, the concentration of each primer in the single base extension primer set with the nucleotide sequence as shown in SEQ ID NO: 5-6 is 3.75 μM and 10 μM respectively.

9. The reaction system according to claim 6, characterized in that: The reaction system also includes one or more of a PCR reaction reagent, a SAP reaction reagent and a single base extension reaction reagent.

10. A glucocorticoid metabolism-related SNP site detection system based on a nucleic acid mass spectrometry platform, characterized in that: The method comprises the reaction system according to any one of claims 6 to 9, and a desalting resin and a spotting chip.

Images