Human UGT1A4 gene polymorphism detection kit

Abstract

The invention relates to a human UGT1A4 gene polymorphism detection kit as well as a preparation method and application thereof. The invention provides a primer and probe composition for detecting human UGT1A4 * 3 gene polymorphism. The primer and probe composition comprises a forward primer F, a reverse primer R, a wild type detection probe, a mutant type detection probe, an internal standard upstream primer, an internal standard downstream primer and an internal standard probe. The invention also provides a PCR reaction solution containing the primer and the probe and a kit. The primer and probe composition, the PCR reaction liquid and the kit can be used for preparing a detection reagent for detecting gene polymorphism or a reagent for guiding medication of lamotrigine. And gene detection is combined with clinical and diagnostic information of a patient before medication, so that a doctor can quickly determine a dosing scheme and a drug dosage, and the processes of earlier-stage drug trial, dose adjustment and the like of the patient are reduced.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a human UGT1A4 gene polymorphism detection kit and a preparation method and application thereof. Background Art

[0002] Lamotrigine (LTG) is an anticonvulsant used to treat epilepsy and bipolar disorder. Common side effects include drowsiness, headache, vomiting, coordination difficulties and rash. Serious side effects include lack of red blood cells, leading to Stevens-Johnson syndrome and allergic reactions. The Chinese Guidelines for the Diagnosis and Treatment of Epilepsy and the Clinical Pharmacist Guidelines for Individualized Dosing of Lamotrigine have listed the reference concentration range and warning values ​​of lamotrigine for the treatment of epilepsy, as well as the warning values ​​for the treatment of bipolar disorder, as a reference for dose adjustment and prediction of adverse reactions.

[0003] Lamotrigine LTG is mainly metabolized in liver microsomes by uridine diphosphate glucuronosyltransferase (UGT), of which the main metabolizing enzyme is UGT1A4, which is involved in the metabolism of a variety of endogenous and exogenous substances. Several studies on the correlation between UGT1A4*3 gene polymorphism and lamotrigine pharmacokinetics have shown that UGT1A4*3 (142T>G) mutation can reduce the blood concentration of lamotrigine.

[0004] There are many methods for detecting UGT1A4*3 (142T>G) gene polymorphism, mainly including DNA direct sequencing, nucleic acid mass spectrometry, and multiplex PCR combined with fragment length analysis. Sanger sequencing is complex and expensive. CN110184345A and CN113249460A both use nucleic acid mass spectrometry to invent a gene group detection kit Pannel for medication guidance for patients with mental illness or anxiety. CN109825574A uses multiplex PCR combined with fragment length analysis to disclose a multiplex gene detection kit for medication guidance for anti-epileptic drugs; both contain design information of UGT1A4*3 polymorphism sites, but such designs are complex, contain many detection drugs, and have relatively high detection costs. They are more suitable for preliminary diagnosis of patients to conduct a major examination before drug selection and trial. However, when several drugs are basically locked based on clinical information, choosing the above design and inspection scheme may result in high economic waste.

[0005] TaqMan probe is a dual-labeled, self-quenched hydrolysis probe. Two specific probes with different fluorescent labels at both ends are added to the PCR reaction to identify different alleles. The 5' and 3' ends are labeled with a fluorescent group and a quenching group, respectively. When the probe structure is complete, the two are close to each other, and the signal of the fluorescent group can be quenched. During the PCR amplification process, if the TaqMan probe and the target sequence are completely matched, the probe can be bound to the DNA template. At this time, when the Taq enzyme extends to the probe position, its exonuclease activity will cut the hydrolysis probe, release the fluorescent group, and enhance the fluorescence signal. Moreover, as the number of amplified products increases, the fluorescent signal becomes stronger and stronger, so that the change process of the fluorescent signal can be monitored in real time by the instrument. At present, there is no TaqMan kit for single-site UGT1A4*3 gene polymorphism on the market. Therefore, it is very necessary to develop a product for detecting UGT1A4*3 gene polymorphism to achieve high sensitivity, high specificity, rapid and low-cost, more convenient and fast UGT1A4*3 gene polymorphism detection. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention provides a kit for detecting human UGT1A4*3 gene polymorphism using a PCR fluorescent probe method (improved version), a preparation method and an application thereof. The kit can accurately distinguish the three genotypes of the UGT1A4*3 locus, significantly improve the accuracy of the detection, and reduce the minimum detection limit. This improvement makes the application of the reagent in genotyping detection more reliable and efficient. Before medication, genetic testing combined with the patient's clinical and diagnostic information can help doctors quickly determine the dosing regimen and drug dosage, reducing the patient's early drug testing and dosage adjustment processes.

[0008] (II) Technical solution

[0009] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0010] In one aspect, the present invention provides a primer and probe composition for detecting human UGT1A4*3 gene polymorphism, which is used to detect the UGT1A4142T>G site. It comprises a forward primer F, a reverse primer R, a wild-type detection probe and a mutant detection probe. It also comprises an internal standard upstream primer, an internal standard downstream primer and an internal standard probe.

[0011] In one aspect, the present invention provides specific sequences of a forward primer F, a reverse primer R, a wild-type detection probe and a mutant detection probe: the forward primer F is selected from the nucleotide sequence shown in SEQ ID NO: 1-3; the reverse primer R is selected from the nucleotide sequence shown in SEQ ID NO: 4-6; the wild-type detection probe is selected from the nucleotide sequence shown in SEQ ID NO: 7-8; the mutant detection probe is selected from the nucleotide sequence shown in SEQ ID NO: 9-10.

[0012] In one aspect, the present invention provides specific sequences of an internal standard upstream primer, an internal standard downstream primer and an internal standard probe: the internal standard upstream primer is the nucleotide sequence shown in SEQ ID NO:11; the internal standard downstream primer is the nucleotide sequence shown in SEQ ID NO:12; and the internal standard probe is the nucleotide sequence shown in SEQ ID NO:13.

[0013] In one aspect, the 5' ends of all detection probes are connected to a luminescent reporter gene selected from ROX, FAM or VIC; the 3' ends of all detection probes are connected to a phosphate group and a quencher group capable of absorbing the fluorescent signal emitted by the 5' end, and the quencher group is selected from BHQ2 or NFQ.

[0014] In one aspect, the present invention provides an amplification PCR reaction solution, which comprises primers, probes, dNTPs, Tris-HCl, antibody-modified TaqDNA polymerase, MgCl2, dUTP, 37°C non-thermosensitive UDG enzyme, KCl, Glycerol, DMSO, Formamide, Triton100 and other components.

[0015] In one aspect, the present invention provides a kit for human UGT1A4*3 gene polymorphism, the kit comprising the following components: sample lysate, forward primer, reverse primer, wild-type detection probe, mutant detection probe, internal standard upstream primer, internal standard downstream primer, internal standard probe, 2*PCR reaction solution, positive control and negative control.

[0016] In one aspect, the present invention provides a method for preparing a kit for human UGT1A4*3 gene polymorphism, comprising the following steps: packaging a sample lysate, a forward primer, a reverse primer, a wild-type detection probe, a mutant detection probe, an internal standard upstream primer, an internal standard downstream primer, an internal standard probe, a 2*PCR reaction solution, a positive control, and a negative control into suitable containers.

[0017] In one aspect, the present invention provides an application of a primer and probe composition, a PCR reaction solution and a kit for detecting human UGT1A4*3 gene polymorphism, wherein the application is an application in preparing a detection reagent for detecting gene polymorphism or in preparing a reagent for guiding lamotrigine medication.

[0018] In one embodiment, the forward primer F is preferably the nucleotide sequence shown in SEQ ID NO:2, the reverse primer R is preferably the nucleotide sequence shown in SEQ ID NO:6, the wild-type detection probe is preferably the nucleotide sequence shown in SEQ ID NO:7, and the mutant detection probe is preferably the nucleotide sequence shown in SEQ ID NO:9.

[0019] In one embodiment, the 5' end of the internal standard probe is connected to the fluorescent group ROX, the 5' end of the wild-type detection probe is connected to the fluorescent group FAM, and the 5' end of the mutant detection probe is connected to the fluorescent group VIC. The 3' end of the internal standard probe is connected to the quencher group BHQ2; the 3' ends of the wild-type detection probe and the mutant detection probe are connected to the quencher group NFQ, and a minor groove binder (MGB) that can insert into the minor groove of DNA is added to the 3' end of the probe.

[0020] In one embodiment, the ratio of forward primer F, reverse primer R, wild-type detection probe and mutant probe in the kit is preferably F:R:FAM:VIC=(0.8):(0.8):(0.125):(0.25). The anti-pollution system in the kit is 37°C non-thermosensitive UDG enzyme and dUTP. The concentration of dNTP in the PCR reaction solution is 0.3mM, and the concentration of MgCl2 is 3mM. The PCR reaction solution also contains 5% Glycerol, 4% DMSO, 2% Formamide and 0.3% Triton 100.

[0021] In one embodiment, the positive control includes a positive control W (UGT1A4*1 wild-type plasmid) and a positive control M (UGT1A4*3 mutant plasmid). The UGT1A4*1 wild-type plasmid has a nucleotide sequence as shown in SEQ ID NO: 14, and the UGT1A4*3 mutant plasmid has a nucleotide sequence as shown in SEQ ID NO: 15. The negative control is a sample lysate without nucleic acid.

[0022] In one embodiment, the method for using the sample lysis solution in the kit is as follows: add the sample lysis solution to the sample, mix well and centrifuge instantly, heat at 95°C for 5 minutes, wait for cooling to room temperature and centrifuge at 12000rpm for 2 minutes, and the supernatant can be used as the loading solution.

[0023] (III) Beneficial effects

[0024] The present invention provides a human UGT1A4 gene polymorphism detection kit and a preparation method and application thereof. Compared with the prior art, it has the following beneficial effects:

[0025] 1. The UGT1A family genes are highly similar in homology. Highly specific probes are designed for the wild type and mutant types of the UGT1A4*3 polymorphic site. Special primers with differences between the second to last or third base at the 3' end and the continuous bases at the end and the homologous sequence can clearly distinguish the three genotypes of the UGT1A4*3 site, greatly improving the accuracy and specificity of the detection reagents to 100%.

[0026] 2. Sample pretreatment is simple and quick. It only needs to be heated at 95℃ for 5 minutes and centrifuged at 4000rpm or 12000rpm for 2 minutes before it can be used for subsequent amplification gene detection and typing. It is not necessary to extract nucleic acids from samples by column or magnetic bead method, which can simplify the complex operation process of sample pretreatment, reduce the probability of cross contamination caused by long-term tube opening, shorten the detection time and save the economic cost of sample processing.

[0027] 3. Due to the high specificity and sensitivity of the detection reagents and the release of intracellular nucleic acid substances by the sample processing reagents, the minimum detection limit LOD value is extremely low. Whether the normal blood sample white blood cells 10^9 are diluted 1000 times before sample processing, or the blood sample is processed and the nucleic acid-containing loading solution is diluted 500 times, the genotyping test can still accurately detect, and the sensitivity can reach 10pg / μL. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is the gel electrophoresis diagram in Example 1.

[0030] Figure 2 is a graph showing the primer and probe amplification curves in Example 2. Figure 2A It is an amplification curve diagram of forward primer F2+reverse primer R5+wild-type detection probe 7+mutant detection probe 9, forward primer F2+reverse primer R6+wild-type detection probe 7+mutant detection probe 9, forward primer F3+reverse primer R5+wild-type detection probe 7+mutant detection probe 9, forward primer F3+reverse primer R6+wild-type detection probe 7+mutant detection probe 9; Figure 2BIt is an amplification curve diagram of forward primer F2+reverse primer R5+wild-type detection probe 7+mutant detection probe 10, forward primer F2+reverse primer R6+wild-type detection probe 7+mutant detection probe 10, forward primer F3+reverse primer R5+wild-type detection probe 7+mutant detection probe 10, forward primer F3+reverse primer R6+wild-type detection probe 7+mutant detection probe 10; Figure 2C It is an amplification curve diagram of forward primer F2+reverse primer R5+wild-type detection probe 8+mutant detection probe 9, forward primer F2+reverse primer R6+wild-type detection probe 8+mutant detection probe 9, forward primer F3+reverse primer R5+wild-type detection probe 8+mutant detection probe 9, forward primer F3+reverse primer R6+wild-type detection probe 8+mutant detection probe 9; Figure 2D These are the amplification curves of forward primer F2 + reverse primer R5 + wild-type detection probe 8 + mutant detection probe 10, forward primer F2 + reverse primer R6 + wild-type detection probe 8 + mutant detection probe 9, forward primer F3 + reverse primer R5 + wild-type detection probe 8 + mutant detection probe 10, and forward primer F3 + reverse primer R6 + wild-type detection probe 8 + mutant detection probe 10.

[0031] FIG3 is a graph showing a clinical sample amplification curve in Example 4. Figure 3A Amplification curves of sample 153, sample 166, sample 175, sample 183, sample 184, and sample 197;

[0032] Figure 3B These are the amplification curves of sample 204, sample 248, sample 251, sample 252, wild-type quality control, and homozygous quality control.

[0033] Figure 4 is a diagram of the sequencing results in Example 4. Figure 4A The sequencing result graphs of sample 153, sample 166, sample 175, sample 204, and sample 248; Figure 4B These are the sequencing result graphs for sample 251, sample 183, sample 184, sample 197, and sample 252.

[0034] Figure 5 This is the amplification curve of the blood and diluted samples in Example 4. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are 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.

[0036] In order to better understand the technical solution, the technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0037] Example 1: Primer and probe design

[0038] Amplification primers and probes were designed with reference to the gene sequences of wild type and mutant type of UGT1A4*3 (142T>G) in the prior art, and amplification primers and probes were designed for the internal standard gene (RNase P). All amplification primers and probes were optimized based on the premise of being suitable for the PCR fluorescent probe method, combined with parameters such as primer and probe length, Tm value, GC content, amplification product length, probe binding position, specificity and base distribution to form the following sequence.

[0039] Forward primer F1: 5'-AGGTTCCCCTGCCGCG-3' (SEQ ID NO: 1);

[0040] Forward primer F2: 5′-GCCACAGGACTGCTGCTCC-3′ (SEQ ID NO: 2);

[0041] Forward primer F3: 5'-CCACTGATGGCAGCCCC-3' (SEQ ID NO: 3); Reverse primer R4: 5'-GAGTGTAGCCCAGCGTAACG-3' (SEQ ID NO: 4);

[0042] Reverse primer R5: 5′-TCGAAGAACCCTTGAGTGTAGC-3′ (SEQ ID NO: 5);

[0043] Reverse primer R6: 5'-CCTTGAGTGTAGCCCAGCGTA-3' (SEQ ID NO: 6);

[0044] Wild-type detection probe 7: 5′-CCCGCAAGGCCTCC-3′ (SEQ ID NO: 7);

[0045] Wild-type detection probe 8: 5′-AGCTCCCGCAAGGCC-3′ (SEQ ID NO: 8);

[0046] Mutation detection probe 9: 5'-CCGCACGGCCTCC-3' (SEQ ID NO: 9);

[0047] Mutation detection probe 10: 5'-TCCCGCACGGCCTC-3' (SEQ ID NO: 10);

[0048] Internal standard upstream primer 11: 5'-AGATTTGGACCTGCGAGCG-3' (SEQ ID NO: 11);

[0049] Internal standard downstream primer 12: 5′-GAGCGGCTGTCTCCACAAGT-3′ (SEQ ID NO: 12);

[0050] Internal standard probe 13: 5′-TTCTGACCTGAAGGCTCTGCGCG-3′ (SEQ ID NO: 13).

[0051] The 5' end of all detection probes is connected to a luminescent reporter group, the 5' end of the internal standard probe is connected to a fluorescent group ROX; the 5' end of the wild-type detection probe is connected to a fluorescent group FAM, and the 5' end of the mutant detection probe is connected to a fluorescent group VIC.

[0052] The 3' ends of all detection probes are connected to a phosphate group and a quenching group that can absorb the fluorescent signal emitted by the 5' end. The 3' end of the internal standard probe is connected to the quenching group BHQ2; the 3' ends of the wild-type detection probe and the mutant detection probe are connected to the quenching group NFQ. A minor groove binder (MGB) that can insert into the minor groove of DNA is added to the 3' end of the probe, which can greatly improve the Tm value of the probe and increase the specificity of the hybridization reaction. In addition, MGB fits with the minor groove of the DNA helix and improves the accuracy and stability of hybridization by stabilizing the DNA double helix structure.

[0053] The penultimate base at the 3' end of the forward primer is the same difference point of the highly homologous sequences UGT1A3, UGT1A5, and UGT1A8; the two last bases at the 3' end of the reverse primer are also the same difference point of the highly homologous sequences UGT1A3, UGT1A5, and UGT1A8; the penultimate or third single-site base at the 3' end of the primer and the terminal continuous bases are "mismatched" to improve the specificity of primer extension and prevent mismatches. TaqDNA polymerase lacks 3'→5' exonuclease activity, so for primers with 3' end mismatches, they are extended at a speed lower than that of normal end-paired primers. When the number of mismatched bases reaches a certain level or the conditions reach a certain level of rigor, the 3' terminal base cannot be extended due to the difficulty in forming a phosphodiester bond, and no amplified band of specific length can be obtained.

[0054] Example 2: Primer and probe screening

[0055] The designed primers, probes and artificial plasmids were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd. The wild-type plasmid sequence (SEQ ID NO: 14) and mutant plasmid sequence (SEQ ID NO: 15) were used as templates for the screening of primers and wild-type / mutant detection probes, respectively.

[0056] There were 9 primer combinations, including 3 sets of forward primers and 3 sets of reverse primers. The primer amplification efficiency was detected by PCR amplification and gel electrophoresis.

[0057] Gel electrophoresis results (see Figure 1 ) showed that the forward primer F1 had a lower amplification efficiency than other primers, and the reverse primers R4 and R5 had similar amplification efficiencies. Forward primers F2 and F3 and reverse primers R5 and R6 were preferred for probe sequence optimization.

[0058] Fluorescence quantitative PCR test was performed on 16 types of primer and probe combinations, including 2 sets of forward primers, 2 sets of reverse primers, 2 sets of wild-type detection probes, and 2 sets of mutant detection probes, to screen for appropriate primer and probe combinations.

[0059] The test data of the screening primer-probe combination test (see Figure 2) show that the amplification curve is a standard "S" curve, and the background signal has no non-specific amplification. The combination of forward primer F2, reverse primer R6, wild-type detection probe 7 and mutant detection probe 9 is preferred. Its amplification "S" type, signal peak, amplification efficiency and specificity are better than other primer-probe combinations. This primer-probe combination is used as the basis for the optimization of the kit formula process.

[0060] Example 3: Preparation of a human UGT1A4 gene polymorphism detection kit

[0061] A human UGT1A4 gene polymorphism detection kit was prepared according to the following components.

[0062] Table 1 Kit composition

[0063]

[0064] The concentration of each primer is 0.4μM, the concentration of the wild-type detection probe is 0.0625μM, the concentration of the mutant detection probe is 0.125μM, the concentration of the internal standard probe is 0.0625μM, and the 2*PCR reaction solution contains: Tris-HCl buffer 10mM, MgCl23mM, dUTP 0.3mM, UNG enzyme 0.01U / μL, Taq DNA polymerase 0.1U / μL, 5% Glycerol, 4% DMSO, 2% Formamide, and 0.3% Triton100.

[0065] Example 4: Performance verification of gene polymorphism detection kit

[0066] Part I: Sample Processing

[0067] Randomly collect clinical samples including 50 EDTA anticoagulated peripheral blood samples, 20 clinical samples of patients taking lamotrigine, 10 of which had known first-generation sequencing results, 1 positive control (W), 1 positive control (M), 1 positive plasmid (W:M=1:1), and 1 negative control. Take 75 μL of each of the above samples, add 75 μL of sample lysis buffer to each sample, mix well and centrifuge instantly, heat at 95 °C for 5 min, wait to cool to room temperature and centrifuge at 12000 rpm for 2 min, and the supernatant can be used as the loading solution.

[0068] Part II: Accuracy Verification

[0069] Take 14 portions of 15 μL of the detection system reagent into eight-tube strips, and then detect 10 clinical samples with known first-generation sequencing results and positive control (W), positive control (M), positive plasmid (W:M=1:1), and negative control, and add an equal amount of 5 μL of template solution to each reaction.

[0070] Set the PCR reaction conditions according to the table below, select the FAM channel to detect the wild type, select the VIC channel to detect the mutant type, select the ROX channel to detect the internal standard gene, and complete the fluorescence quantitative PCR reaction conditions.

[0071] Table 2 PCR reaction conditions

[0072]

[0073] The PCR reaction results showed that the negative controls FAM, VIC, and ROX, which used sample lysate without nucleic acid as template, did not produce any lines and had no Ct values.

[0074] The FAM signal amplification curve using the positive control W as the template was S-shaped and the Ct value was ≤36, the VIC signal amplification curve using the positive control M as the template was S-shaped and the Ct value was ≤36, and the FAM and VIC signal amplification curves using the positive control (W:M=1:1) as the template were both S-shaped and the Ct value was ≤36; this indicated that the above kit could be used to effectively and sensitively detect the UGT1A4*3 gene polymorphism.

[0075] Table 3 Judgment criteria for UGT1A4*3 polymorphism results

[0076]

[0077]

[0078] Ten clinical samples of patients taking lamotrigine and with known first-generation sequencing results were tested. The amplification curves of the ten samples (see Figure 3) were all consistent with the known first-generation sequencing results (see Figure 4). The above results indicate that the detection capability, specificity and sensitivity of the kit are very high, reaching 100%.

[0079] Part III: Establishment and verification of the minimum detection limit LOD

[0080] Three samples were selected from the samples with known first-generation sequencing results, namely wild-type AA, heterozygous AC, and homozygous CC. The three blood sample stock solutions were diluted 2 times with PBS, 10 times with PBS, 50 times with PBS, and 100 times with PBS, and the samples were processed according to the first part; after the three blood sample stock solutions were heated and centrifuged, the supernatant solutions (about 5ng / μL) were diluted 10 times with sterile water, 50 times with sterile water, 100 times with sterile water, and 500 times with sterile water (about 10pg / μL); the above 27 samples and 1 negative control sample were tested according to the PCR reaction conditions set in the second part.

[0081] The negative controls FAM, VIC, and ROX in the PCR reaction using the sample lysate without nucleic acid as the template did not produce any line and had no Ct value. The 27 blood and diluted samples were all accurately detected according to the effective gradient (see Figure 5 ), indicating that the minimum detection limit LOD value of the invented kit is very low, and it is possible to expand its application to other different types of samples.

[0082] Part IV: Statistical analysis of 70 clinical samples

[0083] The 70 clinical samples were processed according to the first part and the PCR amplification test and result interpretation were performed in the second part. The interpretation method is shown in Table 3. The statistical results are as follows:

[0084] Table 4 PCR amplification detection and interpretation results

[0085]

[0086]

[0087] Table 5 Database statistics

[0088] Classification Wild-type TT Heterozygous TG Homozygous GG CHB(Beijing, China) 71.8％ 25.2％ 2.9％ CHD(China South) 61％ 29.5％ 9.5％ average 66.4％ 27.35％ 6.2％

[0089] Table 6 National Research Institute of Hanyin Testing Data

[0090] Classification Wild-type TT Heterozygous TG Homozygous GG Number of cases 50 19 5 Proportion (%) 67.56％ 25.67％ 6.76％

[0091] The experimental results showed that the wild-type TT accounted for 67.56%, which was very close to the population distribution frequency of 66.4% in the database; the homozygous GG accounted for 6.76%, which was slightly different from the population distribution frequency of 6.2% in the database, indicating that the detection consistency rate of the independently developed gene polymorphism detection kit was very consistent with the population distribution frequency in the database.

[0092] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer and probe combination for detecting human UGT1A4*3 gene polymorphism, characterized in that: The composition comprises a forward primer F, a reverse primer R, a wild-type detection probe and a mutant detection probe, wherein the forward primer F is selected from the nucleotide sequences shown in SEQ ID NOs: 1-3; the reverse primer R is selected from the nucleotide sequences shown in SEQ ID NOs: 4-6; the wild-type detection probe is selected from the nucleotide sequences shown in SEQ ID NOs: 7-8; and the mutant detection probe is selected from the nucleotide sequences shown in SEQ ID NOs: 9-10.

2. The primer and probe composition according to claim 1, characterized in that The forward primer F is preferably the nucleotide sequence shown in SEQ ID NO: 2, the reverse primer R is preferably the nucleotide sequence shown in SEQ ID NO: 6, the wild-type detection probe is preferably the nucleotide sequence shown in SEQ ID NO: 7, and the mutant detection probe is preferably the nucleotide sequence shown in SEQ ID NO:

9.

3. The primer and probe combination according to claim 1 or 2, characterized in that: The composition also comprises an internal standard upstream primer, an internal standard downstream primer and an internal standard probe, wherein the internal standard upstream primer is the nucleotide sequence shown in SEQ ID NO: 11; the internal standard downstream primer is the nucleotide sequence shown in SEQ ID NO: 12; and the internal standard probe is the nucleotide sequence shown in SEQ ID NO:

13.

4. The primer and probe composition according to claim 3, characterized in that All detection probes are connected to a luminescent reporter group at their 5' ends, and the luminescent reporter gene is selected from ROX, FAM or VIC; all detection probes are connected to a phosphate group and a quencher group capable of absorbing the fluorescent signal emitted by the 5' end at their 3' ends, and the quencher group is selected from BHQ2 or NFQ.

5. A PCR reaction solution, characterized in that: The PCR reaction solution comprises the primer and probe combination shown in any one of claims 1 to 4.

6. The PCR reaction solution according to claim 5, characterized in that: The PCR reaction solution also contains dNTP, Tris-HCl, antibody-modified TaqDNA polymerase, MgCl2, dUTP, 37°C non-thermosensitive UDG enzyme, KCl, Glycerol, DMSO, Formamide, and Triton100.

7. A kit for human UGT1A4*3 gene polymorphism, characterized in that: The kit comprises the primer and probe combination as shown in any one of claims 1 to 4 or the PCR reaction solution as described in claim 5.

8. The kit according to claim 7, characterized in that The kit also includes a sample lysate, a positive control and a negative control.

9. The method for preparing the kit according to claim 7 or 8, characterized in that: Dispense sample lysate, forward primer, reverse primer, wild-type detection probe, mutant detection probe, internal standard upstream primer, internal standard downstream primer, internal standard probe, 2*PCR reaction solution, positive control and negative control into appropriate containers.

10. Use of the primer and probe combination according to any one of claims 1 to 4, the PCR reaction solution according to claim 5 or 6, or the kit according to any one of claims 7 to 9, characterized in that: The application is in the preparation of a detection reagent for detecting gene polymorphism or in the preparation of a reagent for guiding the use of lamotrigine.

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