Gene detection kit for medication guidance of anti-epileptic drugs and use method of gene detection kit

Abstract

The invention belongs to the technical field of anti-epileptic drug medication guidance gene detection, and particularly relates to an anti-epileptic drug medication guidance gene detection kit and a use method, the anti-epileptic drug medication guidance gene detection kit comprises primers and probes for detecting 15 specific sites of 9 genes, and the gene sites comprise the rs144012689 site, the rs181596323 site and the rs3094589 site of an HLA-B gene; the rs17179220 site of the HLA-A gene is selected from the rs17179220 site; the rs3812718 site of the SCN1A gene is a rs3812718 site; the rs1799853 site and the rs1057910 site of the CYP2C9 gene are shown in the specification; the rs4244285 site and the rs4986893 site of the CYP2C19 gene are respectively used for detecting the rs4244285 site and the The rs2304016 site of the SCN2A gene is a rs2304016 site; the rs4240803 of the SLC7A5 gene; the rs2606345 site of the CYP1A1 gene is a rs2606345 site; the rs1800497 site of the Ankk1 gene is a rs1800497 site; the kit comprises rs3087374 sites and rs2307441 sites of the POLG gene, has the characteristics of high sensitivity, low cost, simplicity and convenience in operation, short detection period, intuitive interpretation and the like, and can be used for quickly and accurately detecting the polymorphism of the individualized medication gene of the antiepileptic drug.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection for guiding the use of anti-epileptic drugs, and in particular relates to a gene detection kit for guiding the use of anti-epileptic drugs and a method for using the kit. Background Art

[0002] Epilepsy is a group of chronic brain diseases with recurrent, paroxysmal and transient central nervous system dysfunction caused by abnormal excessive discharge of brain neurons. Drug therapy is the main means of controlling epileptic seizures. Clinical studies in various countries have shown that 70% to 80% of newly diagnosed epilepsy patients can be controlled, and 60% to 70% of patients can stop taking drugs after 2 to 5 years of treatment. After the drug enters the human body, the absorption, distribution, metabolism and excretion processes in the human body are related to the human body's mechanism of action, which is regulated by a series of genes encoding drug metabolizing enzymes, transporters, receptors and other proteins in the human body. Gene mutations may lead to enhanced, weakened or missing protein functions, causing changes in the drug's action process in the body, or causing changes or disappearance of the drug's ability to bind to the target site. Ultimately, it affects the drug effect or produces toxicity, resulting in individual differences. Common side effects of anti-epileptic drugs include nausea, stomach pain, loss of appetite, poor coordination, hair growth and gingival enlargement. Serious side effects include drowsiness, liver problems, hypotension, Stevens-Johnson syndrome and toxic epidermal necrolysis. Medication-guided genetic testing is to determine the patient's responsiveness to drugs by testing the drug's metabolism, transport, and target gene sites, and then select the most appropriate treatment plan.

[0003] At present, there are no genetic testing kits specifically for guiding the use of anti-epileptic drugs on the market. Most of the kits in the published patents are used for full detection of anti-epileptic drug sites, with redundant detection sites, and most of them cover more than 30 sites. There are a large number of sites for which clinical evidence is not clear, and the multi-site detection methods are limited to expensive time-of-flight mass spectrometry and NGS sequencing, which brings unnecessary expenses to clinical patients. As a result, the current application of genetic testing for guiding the use of anti-epileptic drugs in clinical practice is poor and has been unable to meet clinical testing needs. Summary of the invention

[0004] In order to solve the problems in the prior art, the present invention provides a gene detection kit for guiding the use of anti-epileptic drugs and a method of use, so that the gene detection kit for guiding the use of anti-epileptic drugs has the characteristics of high sensitivity, low cost, simple operation, short detection cycle, intuitive interpretation, etc., and can quickly and accurately detect the gene polymorphism of individualized anti-epileptic drug use.

[0005] The present invention solves the technical problem by adopting the following technical solutions:

[0006] The present invention aims to provide a gene detection kit for guiding the use of anti-epileptic drugs, comprising primers and probes for detecting 15 specific sites of 9 genes, wherein the gene sites include rs144012689, rs181596323 and rs3094589 of the HLA-B gene; rs17179220 of the HLA-A gene; rs3812718 of the SCN1A gene; rs1799 853 and rs1057910 of the CYP2C19 gene; rs4244285 and rs4986893 of the SCN2A gene; rs4240803 of the SLC7A5 gene; rs2606345 of the CYP1A1 gene; rs1800497 of the Ankk1 gene; rs3087374 and rs2307441 of the POLG gene.

[0007] Further, the primer and probe sequences used to detect 15 sites are shown in the following table:

[0008]

[0009]

[0010] Furthermore, the kit also includes primers and probes for monitoring an internal control gene, and the internal control gene is GAPDH.

[0011] Furthermore, the primer and probe sequences used to detect the internal control gene GAPDH are:

[0012] GF: SEQ IQ NO.113;

[0013] GR: SEQ IQ NO.114;

[0014] G-WP probe: SEQ IQ NO.115.

[0015] Furthermore, the fluorescent group at the 5' end of the G-WP probe is one of FAM, (HEX or VIC or JOE), (ROX or Texas Red), CY5, and the fluorescent group at the 5' end of the site to be tested is one of the remaining three, and the three signals are evenly distributed, that is, each signal corresponds to 8 probes. The quenching group at the 3' end is TAMRA, BHQ1, BHQ2, MGB or Dabcy1 fluorescent quenching group suitable for fluorescence quantitative PCR.

[0016] Furthermore, the fluorescent group at the 5' end of the probe is one of FAM, VIC, ROX, and CY5, and the quenching group at the 3' end is MGB.

[0017] Furthermore, the kit also includes a PCR reaction solution, a positive quality control product and a negative quality control product.

[0018] Furthermore, the PCR reaction solution contains hot-start Taq enzyme, UNG enzyme, buffer, magnesium ions, and dNTP substances required for the PCR reaction.

[0019] Furthermore, the method for obtaining positive quality control products is as follows: according to the SNP site information published in the NCBI database, the wild-type and mutant sequence information are used to construct plasmids to synthesize sequence gene fragments, and then the fragments are inserted into the T vector, and the Escherichia coli DH5α strain is used to transform and extract the plasmids, and the plasmids are mixed in equal proportions to obtain the positive quality control product.

[0020] Furthermore, the negative quality control product is purified water for IVD reagents.

[0021] A method for using a gene detection kit for guiding the use of anti-epileptic drugs comprises the following steps:

[0022] 1) Extract nucleic acid from the sample to be tested using common methods;

[0023] 2) premixing and subpackaging gene detection reagents to obtain PCR reaction solution 1, PCR reaction solution 2, PCR reaction solution 3, PCR reaction solution 4, PCR reaction solution 5, PCR reaction solution 6, PCR reaction solution 7, and PCR reaction solution 8 to form a PCR reaction system;

[0024] 3) adding the DNA obtained in step 1) to the PCR reaction system in step 2), mixing and then performing PCR amplification;

[0025] 4) After the reaction is completed, genotyping is performed.

[0026] Furthermore, the conditions for PCR amplification in step 3) are: the conditions for UNG enzyme reaction are: 25°C, 10 minutes; the conditions for UNG enzyme inactivation are: 95°C, 2 minutes; the conditions for system amplification are a two-step method: denaturation: 95°C, 5 seconds; annealing extension: 58°C, 30 seconds, setting fluorescence signal collection; the number of cycles is set to 40.

[0027] Genotyping interpretation: Under the above PCR reaction system and cycling program conditions, the FAM, VIC, ROX and Cy5 fluorescence detection signals of positive quality control products No. 1-8 should form a logarithmic amplification "S" curve; negative quality control products No. 1-8 should have no amplification curve or the Ct value is 0; observe whether the FAM, VIC, ROX and Cy5 fluorescence detection signals of No. 1-8 form a logarithmic amplification "S" curve, among which the presence or absence of an "S" curve of the signal carried by G-WP represents whether the sample and system are normal.

[0028] When the above conditions are met, observe the amplification curve of the reaction tube of the sample to be tested. If a logarithmic amplification "S"-shaped curve is formed, the sample to be tested contains the corresponding base mutation; if there is no logarithmic amplification "S"-shaped curve or the Ct value is 0, there is no corresponding base mutation.

[0029] Compared with the prior art, the beneficial technical effects of the present invention are:

[0030] (1) The present invention packages 8 kinds of PCR reaction solutions of gene detection reagents related to medication guidance of anti-epileptic drugs into an eight-link PCR reaction strip, each reaction strip includes one person's detection reagent, pre-mixed packaging, convenient detection, and realizes multi-gene variation analysis in one reaction system, reducing costs while improving detection efficiency.

[0031] (2) The existing detection technologies are either single-point detection or single-drug detection technologies, which have a small detection coverage and cannot provide good guidance on medication. Or they are time-of-flight mass spectrometry or NGS sequencing technologies, which have high requirements on the detection platform and increase the detection cost. Compared with the existing detection technologies, the present invention is based on ARMS technology and TaqMan fluorescent probe technology, which can accurately guide the use of anti-epileptic drugs, has the characteristics of high sensitivity, low cost, simple operation, short detection cycle, intuitive interpretation, etc., and can quickly and accurately detect the gene polymorphism of individualized medication of anti-epileptic drugs.

[0032] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented according to the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is an amplification curve diagram of the positive quality control product of a single well position in the kit of the present invention.

[0034] Figure 2 It is an amplification curve diagram of a negative quality control product at a single well position in the kit of the present invention. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0036] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0037] The inventors analyzed the individual differences in the efficacy of anti-epileptic drugs in clinical practice, screened out polymorphic sites of genes closely related to anti-epileptic drugs, prepared a composition for detecting the polymorphic sites and a detection kit using the composition, and established an accurate detection method to provide effective medication guidance for epilepsy drugs.

[0038] Example 1: Establishment and optimization of detection system

[0039] 1. Arrange the wild-type and mutant sequence information of rs144012689, rs181596323, rs3094589, rs17179220, rs3087374, rs2307441, rs3812718, rs1799853, rs1057910, rs4244285, rs4986893, rs2304016, rs4240803, rs2606345 and rs1800497 on NCBI, and dilute all plasmids to 10 -4 ng / ul simulated sample, 10 -4 ng / ul simulated wild-type and mutant plasmids were mixed in equal volumes to obtain heterozygous simulated samples.

[0040]

[0041]

[0042]

[0043]

[0044] 2 Based on the sequence characteristics of the genetic polymorphism sites, design the primers and probes used for sample testing and amplify the analysis results.

[0045] (1) According to the sequence characteristics of the genetic polymorphism site, the primers and probes used for sample detection are designed to obtain the mismatched MGB probe used in the following PCR detection system, the forward primer and the reverse primer SEQ IQ NO.1 to SEQ IQ NO.115. Among them, the probe modification method is shown in the following Table 1:

[0046] Table 1.

[0047]

[0048] (2) Prepare the single-plex PCR detection system respectively. The detection system is shown in Table 2.

[0049] Table 2: Wild-type single-plex detection system

[0050] Reagent name Volume (μL) TaKaRa Taq HS (5U / μL) 0.15 10×PCR Buffer (Mg2+plus) 2.5 dU plus dNTP Mixture (12.5X) 4ul UNG (2U / μL) 0.1 Forward primer 0.5 Reverse primer 0.5 Wild-type MGB probe 0.3 / - Mutant MGB probe 0.3 DNA (template) 2 DEPC water Make up to 25ul

[0051] The DNA templates are the wild-type and mutant simulation samples synthesized and diluted in step 1, and the primers and probes are the primers and probes in step 2 (1). For specific sequence information, please refer to the Nucleotide Sequence Information Table.

[0052] In each point PCR mutant detection system, a pair of amplification primers is added to the wild-type and mutant probes each time until all primer and probe combinations in step 2 obtain detection results, thereby screening amplification primers. In each point PCR detection system, the DNA samples added are the corresponding wild-type and mutant plasmids, with a final concentration of 10 -4 ng / ul.

[0053] (3) The prepared detection system is subjected to PCR amplification. The PCR reaction procedure is as follows:

[0054] Stage 1: 25°C, 10 minutes;

[0055] Second stage: 95℃, 2 minutes;

[0056] The third stage: 95°C, 5 seconds; 58°C, 30 seconds, the number of cycles is set to 40; fluorescence is collected at 58°C.

[0057] (4) Experimental results: PCR was performed on the above primers and probes respectively, and the Ct value of the detection system was directly read. According to the Ct value results, firstly, a probe with a wild-type detection system wild-type plasmid between 22-30, a mutant plasmid with no Ct value or greater than 37 was selected, and a probe with a mutant detection system mutant plasmid between 22-30, a wild-type plasmid with no Ct value or greater than 37 was selected, and then a probe with a wild-type probe and a mutant probe Ct value difference within 2 was selected. The final selected primer probe combination is shown in the following table:

[0058] Table 3

[0059]

[0060]

[0061] 3 Based on the specific primers screened by the PCR system, add the internal control to prepare the PCR reaction solution for each site, as shown in Table 4:

[0062] Table 4

[0063] Reagent name Volume (μL) TaKaRa Taq HS (5U / μL) 0.15 10×PCR Buffer (Mg2+plus) 2.5 dU plus dNTP Mixture (12.5X) 4ul UNG (2U / μL) 0.1 Forward primer 0.5 Reverse primer 0.5 Wild-type probe 0.3 Mutation Probe 0.3 GAPDH-F 0.2 GAPDH-R 0.2 GAPDH-P 0.2 DNA (template) 2 DEPC water Make up to 25ul

[0064] The DNA templates are the diluted heterozygous simulated samples synthesized in step 1, and the primers and probes are the primers and probes screened in step 2.

[0065] Each reaction system added a forward primer, a reverse primer, a wild-type probe, and a mutant probe of one site. The DNA samples added to each site reaction system were the corresponding heterozygous plasmids, with a final concentration of 10 -4 ng / ul.

[0066] The prepared PCR reaction solution for each site is subjected to PCR amplification. The PCR reaction procedure is as follows:

[0067] Stage 1: 25°C, 10 minutes;

[0068] Second stage: 95℃, 2 minutes;

[0069] The third stage: 95°C, 5 seconds; 58°C, 30 seconds, the number of cycles is set to 40; fluorescence is collected at 58°C.

[0070] Experimental results: Read the CT values ​​of FAM, VIC and ROX signals at each point and record the experimental results of each group with the highest signal value at each point

[0071] 4 Based on the reaction results of step 3, adjust the amount of primers and probes at each site to make the CT value and signal value of each site closer.

[0072] 5. Design of the kit: The kit of the present invention adopts an eight-tube premix system designed for different genotypes.

[0073] Example 2: Use of Gene Detection Kit

[0074] 1. Sample collection and processing

[0075] Peripheral blood was collected from patients using a vacuum tube containing EDTA anticoagulant to collect 5 ml of blood specimens. Nucleic acid was extracted using a conventional nucleic acid extraction kit. The experimental steps were carried out according to the instructions.

[0076] 2. Preparation of reaction system

[0077] Preparation of the reaction system The reaction system was prepared according to the PCR reaction solutions 1-8 in Example 1.

[0078] 3. PCR reaction

[0079] The DNA extracted from the sample is added to the prepared reaction system, and the amount of template added is 1-100ng. When performing the PCR reaction, the sample to be tested, the positive quality control product and the negative quality control product need to be tested in parallel on the machine, and each sample needs to be added to eight reaction solutions at the same time for reaction.

[0080] 4. Instrument channel and reaction volume selection

[0081] ① Select FAM channel (Reporter: FAM, Quencher: MGB), VIC channel (Reporter: VIC, Quencher: MGB), ROX channel (Reporter: ROX, Quencher: MGB) and CY5 channel (Reporter: CY5, Quencher: BHQ2) to detect amplification;

[0082] ②The reaction volume (Sample Volume) is 25μL.

[0083] ③ Reference Dye: For specific detection channel settings, please refer to the instructions for use of each instrument.

[0084] 5. PCR amplification reaction procedure

[0085] Stage 1: 25°C, 10 minutes;

[0086] Second stage: 95℃, 2 minutes;

[0087] The third stage: 95°C, 5 seconds; 58°C, 30 seconds, the number of cycles is set to 40; fluorescence is collected at 58°C.

[0088] 6. Experimental results

[0089] After the reaction program is completed, the results are saved and interpreted. If the amplification Ct value is ≤35, it is detected; otherwise, it is judged as not detected.

[0090] The fluorescence detection signals of FAM, VIC, ROX and CY5 in reaction tubes 1-8 of the positive control product form a logarithmic amplification "S" curve (see attached Figure 1 ); negative control tubes 1-8 have no amplification curve (see attached Figure 2 );

[0091] 7. Determination of the genotype determination criteria of the kit

[0092] Directly read the Ct value of the PCR detection system, and judge the genotype according to the CT value of the FAM, VIC and ROX signals of the PCR detection system. Based on the premise that the Ct value of this kit detected 90 human peripheral blood gDNA samples is 35, when the Ct value corresponding to each signal is ≥35, the genotype needs to be judged one by one in combination with the relevant points and signal combinations in Tables 1 and 3.

[0093] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0094] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A gene detection kit for guiding the use of anti-epileptic drugs, characterized in that: It includes primers and probes for detecting 15 specific sites of 9 genes, including rs144012689, rs181596323 and rs3094589 of HLA-B gene; rs17179220 of HLA-A gene; rs3812718 of SCN1A gene; rs1799853 and rs1057910 of CYP2C9 gene; rs4244285 and rs4986893 of CYP2C19 gene; rs2304016 of SCN2A gene; rs4240803 of SLC7A5 gene; rs2606345 of CYP1A1 gene; rs1800497 of Ankk1 gene; rs3087374 and rs2307441 of POLG gene.

2. A gene detection kit for guiding the use of anti-epileptic drugs as claimed in claim 1, characterized in that: The primer and probe sequences for detecting 15 loci are as follows:

3. A gene detection kit for guiding the use of anti-epileptic drugs as claimed in claim 2, characterized in that: The kit also includes primers and probes for an internal control gene to be monitored, and the internal control gene is GAPDH.

4. A gene detection kit for guiding the use of anti-epileptic drugs as claimed in claim 3, characterized in that: The primer and probe sequences for detecting the internal control gene GAPDH are: GF: SEQ IQ NO.113; GR: SEQ IQ NO.114; G-WP probe: SEQ IQ NO.

115.

5. A gene detection kit for guiding the use of anti-epileptic drugs as claimed in claim 4, characterized in that: The fluorescent group at the 5' end of the probe is one of FAM, VIC, ROX, and CY5, and the quenching group at the 3' end is MGB.

6. A gene detection kit for guiding the use of anti-epileptic drugs as claimed in claim 1, characterized in that: The kit also includes a PCR reaction solution, which contains a hot-start Taq enzyme, UNG enzyme, buffer, magnesium ions, and dNTP substances required for the PCR reaction.

7. A method for using a gene detection kit for guiding the use of anti-epileptic drugs according to any one of claims 1 to 6, characterized in that: The following steps are involved: 1) premixing and subpackaging gene detection reagents to obtain PCR reaction solution 1, PCR reaction solution 2, PCR reaction solution 3, PCR reaction solution 4, PCR reaction solution 5, PCR reaction solution 6, PCR reaction solution 7, and PCR reaction solution 8 to form a PCR reaction system; 2) adding the sample to be tested to the PCR reaction system in step 1), mixing and then performing PCR amplification; 3) After the reaction is completed, genotyping is performed.

8. The method for using the gene detection kit for guiding the use of anti-epileptic drugs as claimed in claim 7, characterized in that: The conditions for PCR amplification in step 2) are: the conditions for UNG enzyme reaction are: 25°C, 10 minutes; the conditions for UNG enzyme inactivation are: 95°C, 2 minutes; the conditions for system amplification are a two-step method: denaturation: 95°C, 5 seconds; annealing extension: 58°C, 30 seconds, set fluorescence signal collection; the number of cycles is set to 40.

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