Individualized medication gene detection kit and application thereof

Abstract

The invention discloses an individualized medication gene detection kit and application thereof.The individualized medication gene detection kit comprises PCR reaction liquid, Taq DNA enzyme, positive control and negative control, the PCR reaction liquid comprises two primer pairs and two fluorescent probes designed for CYP3A5 and TPMT genes, and the two fluorescent probes comprise CYPF2 with the nucleotide sequence as shown in SEQ ID NO.1, CYP3A5 with the nucleotide sequence as shown in SEQ ID NO.2, TPMT with the nucleotide sequence as shown in SEQ ID NO.3, TPMT with the nucleotide sequence as shown in SEQ ID NO.4 and TPMT with the nucleotide sequence as shown in SEQ ID NO.4; the nucleotide sequence of the CYPR2 is as shown in SEQ ID NO. 2; the nucleotide sequence of the CYPP1 is as shown in SEQ ID NO.3; the nucleotide sequence of the TPMTF is as shown in SEQ ID NO. 4; the nucleotide sequence of the TPMTR2 is as shown in SEQ ID NO. 5; the nucleotide sequence of the TPMTP4 is as shown in SEQ ID NO. 6. According to the individualized medication gene detection kit and the application thereof, primer and fluorescent probe sequences are designed aiming at polymorphic sites of CYP3A5 and TPMT genes, and rapid and accurate detection of the polymorphic sites of the genes in a patient sample is achieved through a real-time fluorescent quantitative PCR technology.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a gene detection kit for individualized medication and application thereof. Background Art

[0002] About one-third of deaths worldwide are caused by irrational use of drugs, so drug effectiveness and adverse reactions are the key to determining the success or failure of treatment.

[0003] Drug treatment is not a "one-size-fits-all" approach. It requires "tailoring the medicine" according to the specific situation of each individual, that is, individualized treatment. In addition to traditional considerations such as pathology, physiological state, gender, age, height, weight, and patient compliance, genetic factors have also been proven to be a major factor leading to individual differences in drug response. Personalized medication in precision medicine is based on a comprehensive consideration of these factors, aiming to develop a treatment plan that is both safe and reasonable, effective and economical, in order to achieve the best treatment effect. Summary of the invention

[0004] The purpose of the present invention is to provide a personalized drug gene detection kit and its application, aiming to provide medication tips based on the possible impact of individual gene differences on drug absorption, metabolism, transport and emission, and provide a reference for clinical medication.

[0005] To achieve the above-mentioned object, the present invention provides a personalized drug gene detection kit, comprising a PCR reaction solution, Taq DNA enzyme, a positive control and a negative control, the PCR reaction solution comprising two sets of primer pairs and two sets of fluorescent probes designed for CYP3A5 and TPMT genes.

[0006] Preferably, the primer pair designed for the CYP3A5 gene is:

[0007] CYPF2, the nucleotide sequence is shown in SEQ ID NO.1;

[0008] CYPR2, the nucleotide sequence is shown in SEQ ID NO.2;

[0009] The fluorescent probes designed for the CYP3A5 gene are:

[0010] CYPP1, the nucleotide sequence is shown in SEQ ID NO.3.

[0011] Preferably, the primer pair designed for the TPMT gene is:

[0012] TPMTF, the nucleotide sequence is shown in SEQ ID NO.4;

[0013] TPMTR2, the nucleotide sequence is shown in SEQ ID NO.5;

[0014] The fluorescent probes designed for the TPMT gene are:

[0015] TPMTP4, the nucleotide sequence is shown in SEQ ID NO.6.

[0016] Preferably, the positive control is a DNA recombinant plasmid containing all target gene target sequence polymorphic sites, and the negative control is purified water.

[0017] In another aspect, the present invention provides an application of the above-mentioned personalized drug gene detection kit, which uses PCR-melting curve method to qualitatively detect SNPs in DNA samples, including the following steps:

[0018] S1. Extract sample DNA;

[0019] S2. Calculate the total amount of PCR reaction solution and Taq DNA enzyme according to the number of reaction reagents required and each test reaction system, mix them well, and dispense them into eight PCR thin-walled tubes according to 1 per test reaction system;

[0020] S3, add 2 μL of negative control or 2 μL of positive control or 2 μL of sample DNA to the thin-walled eight-tube strip in step S2 to make the total reaction volume 20 μL;

[0021] S4, placing each reaction tube in turn into a fluorescent quantitative PCR instrument for PCR amplification;

[0022] S5. Compare the melting curve Tm values ​​generated by the specific fluorescent probe and the target sequence to perform SNP typing.

[0023] Preferably, each test reaction system includes 17 μL of PCR reaction solution and 1 μL of Taq DNA enzyme.

[0024] Preferably, in step S3, the PCR amplification procedure includes:

[0025] Pre-denaturation: 95°C, 10 min, 1 cycle; amplification: 95°C, 15 sec, 58°C, 40 sec, 40 cycles, fluorescence signal reading at 58°C; melting curve: 95°C, 2 min, 45°C, 0.03°C / s heating, 2 min, 85°C, 5 sec, one cycle, fluorescence signal reading at 85°C.

[0026] Preferably, in step S3, the fluorescence quantitative PCR instrument selects FAM channel and HEX channel for fluorescence detection, selects none for the quenching group, and selects none for the reference fluorescence.

[0027] Preferably, in step S4, the SNP typing determination criteria are:

[0028] For the FAM channel, the high melting peak Tm value was 59 ± 1.5 °C, and the genotype was the wild type of CYP3A5;

[0029] or the low melting peak Tm value is 55±1.5℃, the high melting peak Tm value is 59±1.5℃, and the genotype is a heterozygous mutation of CYP3A5;

[0030] or the low melting peak Tm value is 55±1.5℃, and the genotype is a homozygous mutation of CYP3A5;

[0031] For the HEX channel, the high melting peak Tm value was 56 ± 2°C, and the genotype was the wild type of TPMT;

[0032] or the low melting peak Tm value is 51±2℃, the high melting peak Tm value is 56±2℃, and the genotype is a heterozygous mutation of TPMT;

[0033] Or the low melting peak Tm value is 51±2°C, and the genotype is a homozygous mutation of TPMT.

[0034] Therefore, the personalized drug gene detection kit and its application of the present invention have the following beneficial effects:

[0035] (1) The kit of the present invention uses real-time fluorescence quantitative PCR technology, combined with asymmetric PCR, fluorescent probe and melting curve analysis technology, to qualitatively detect SNPs in DNA samples;

[0036] (2) Design primers and fluorescent probe sequences for the polymorphic sites of CYP3A5 and TPMT genes, and use real-time fluorescence quantitative PCR technology to achieve rapid and accurate detection of these gene polymorphic sites in patient samples;

[0037] (3) The design of the kit of the present invention fully considers the characteristics of different gene polymorphic sites, ensuring the specificity and sensitivity of primers and fluorescent probes;

[0038] (4) During the detection process, specific primers and probes are used to perform high-precision PCR amplification of the target sequence. By analyzing and comparing the changes in the Tm value of the melting curve generated by the specific fluorescent probe and the target sequence, it is possible to accurately determine whether specific genetic polymorphism sites exist in the patient sample, thereby providing a scientific basis for personalized medication.

[0039] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0041] Figure 1 The detection spectrum of the fluorescence quantitative PCR instrument in the test example 1 of the present invention;

[0042] Figure 2 For the second test example of the present invention DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0044] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0045] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels.

[0046] Embodiment 1

[0047] A personalized drug gene detection kit comprises a PCR reaction solution, Taq DNA enzyme, a positive control (a DNA recombinant plasmid containing all target gene target sequence polymorphic sites) and a negative control (purified water).

[0048] The PCR reaction solution consisted of pH 8.3 Tris-HCl, 3 mM MgCl 2 , deoxynucleotide triphosphates (dNTPs, final concentration 0.2 mM), primers and fluorescent probes, upstream primer: downstream primer: probe = 1:5:1. The sequences of primers and fluorescent probes are shown in Table 1 below:

[0049] Table 1 Primer and fluorescent probe sequences

[0050]

[0051]

[0052] The sequences of primers and fluorescent probes were designed to target the polymorphic sites of CYP3A5 and TPMT genes, aiming to ensure accurate detection of target gene mutations.

[0053] When using the kit, the target sequence is amplified by PCR with high precision through specific primers and probes. The SNP typing is determined by analyzing and comparing the changes in the Tm value of the melting curve generated by the specific fluorescent probe and the target sequence.

[0054] Embodiment 2

[0055] An application of a personalized drug gene detection kit comprises the following steps:

[0056] S1. Sample collection: The sample can be human peripheral blood specimen or collected whole blood.

[0057] Whole blood collection: Use a disposable sterile syringe to draw 2-3 mL of the subject's venous blood and inject it into a blood collection tube containing EDTA anticoagulant. Immediately and gently invert the blood collection tube 5-10 times to mix the anticoagulant and venous blood thoroughly.

[0058] Whole blood should be stored at 2-8℃ for no more than 1 month; at -20℃±5℃ for no more than 3 months; avoid repeated freezing and thawing.

[0059] S2. Sample processing: Use commercial extraction reagents to extract sample nucleic acid.

[0060] S3. Reagent preparation:

[0061] Take out the PCR reaction solution and Taq DNA enzyme from the gene detection kit, melt the reaction solution at room temperature, shake it gently to mix, and centrifuge it at 2000rpm for 10 seconds. Calculate the number of reaction reagents required for testing n, and the configuration of each test reaction system is shown in Table 2 below:

[0062] Table 2 Reaction system configuration

[0063] Components Reaction tube PCR reaction solution 17μL TaqDNA enzyme 1μL Total volume 18μL

[0064] The calculation formula for the number of reaction reagent tests n required is:

[0065] n = number of samples + number of negative controls + number of positive controls.

[0066] Calculate the amount of each reagent in the reaction system according to n tests, add it to a centrifuge tube of appropriate volume and mix well. Dispense 18 μL into 8-tube PCR thin-wall tubes, and then transfer to the sample processing area for subsequent sample addition.

[0067] S4, add sample:

[0068] The total volume of a single reaction is 20 μL, so add 2 μL of negative control, 2 μL of positive control, and 2 μL of sample to the PCR thin-walled tube containing 18 μL of reaction solution in step S3 in order, cover the eight-tube strip tightly, then gently mix the eight-tube strip, centrifuge it instantly, and finally transfer it to the PCR detection area.

[0069] S5. PCR amplification and fluorescence detection:

[0070] Each reaction tube was sequentially placed in a fluorescent quantitative PCR instrument for PCR amplification, and the sequence was shown in Table 3 below:

[0071] Table 3 PCR reaction program

[0072]

[0073]

[0074] The reaction volume was 20 μL, and the fluorescence detection options were: FAM channel, HEX channel, quencher group selected none, and reference fluorescence selected none (no quencher group and reference fluorescence).

[0075] Quality control requirements:

[0076] (1) The threshold setting principle is that the threshold line just exceeds the highest point of the normal negative control. Adjust the analysis mode according to the situation and switch between automatic mode and manual mode.

[0077] (2) The negative control should not have an obvious melting peak corresponding to the Tm. If a melting peak appears after analysis, it may be due to contamination of the reagents or contamination during the operation. Please eliminate the source of contamination and retest.

[0078] (3) If all the above requirements are met, the test is successful and the sample can be interpreted. Otherwise, the test is considered invalid and errors in instruments, reagents, amplification conditions, etc. should be checked.

[0079] S6. Results Analysis

[0080] The target gene and melting peak Tm value reference table corresponding to each reaction tube is shown in Table 4 below. According to the melting peak Tm value range shown in the table below, the corresponding genotype is interpreted:

[0081] Table 4 Melting peak Tm value range of target gene and positive control

[0082]

[0083]

[0084] Note: “ / ” indicates no obvious melting peak. If the detected peak is at the critical value between the high peak and the low peak, re-test is required.

[0085] Test Example 1

[0086] An application of a personalized drug gene detection kit, the application method is the same as that of Example 2, using the detection spectrum of the Hongshi SLAN-96P real-time fluorescence quantitative PCR instrument as shown in Figure 1 As shown, the blue curve is the FAM channel and the green curve is the HEX channel.

[0087] FAM channel: The melting peak temperature of CYP3A5 (rs776746, T>C) is 55.45℃ and 59.43℃. According to the interpretation rules, the result should be TC type (heterozygous mutation).

[0088] HEX channel: The melting peak temperatures of TPMT (rs1142345, T>C) are 51.06°C and 56.23°C. According to the interpretation rules, the result should be TC type (heterozygous mutation).

[0089] Test Example 2

[0090] An application of a personalized drug gene detection kit, the application method is the same as that of Example 2, using the detection spectrum of the Hongshi SLAN-96P real-time fluorescence quantitative PCR instrument as shown in Figure 2 As shown, the blue curve is the FAM channel and the green curve is the HEX channel.

[0091] FAM channel: The melting peak temperatures of CYP3A5 (rs776746, T>C) are 55.46°C and 59.36°C. According to the interpretation rules, the result should be TC type (heterozygous mutation).

[0092] HEX channel: The melting peak temperatures of TPMT (rs1142345, T>C) are 51.27°C and 56.46°C. According to the interpretation rules, the result should be TC type (heterozygous mutation).

[0093] Therefore, the present invention provides a personalized drug gene detection kit, which designs primers and fluorescent probe sequences for the polymorphic sites of CYP3A5 and TPMT genes, and realizes rapid and accurate detection of these gene polymorphic sites in patient samples through real-time fluorescence quantitative PCR technology; the design of the kit fully considers the characteristics of different gene polymorphic sites, and ensures the specificity and sensitivity of primers and fluorescent probes; in the detection process, the target sequence is amplified by high-precision PCR using specific primers and probes, and by analyzing and comparing the changes in the Tm value of the melting curve generated by the specific fluorescent probe and the target sequence, it can be accurately determined whether there is a specific gene polymorphic site in the patient sample, thereby providing a scientific basis for personalized drug use.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A personalized drug gene detection kit, comprising a PCR reaction solution, Taq DNA enzyme, a positive control and a negative control, characterized in that: The PCR reaction solution includes two sets of primer pairs designed for CYP3A5 and TPMT genes and two sets of fluorescent probes.

2. The personalized drug gene detection kit according to claim 1, characterized in that: The primer pairs designed for the CYP3A5 gene are: CYPF2, the nucleotide sequence is shown in SEQ ID NO.1; CYPR2, the nucleotide sequence is shown in SEQ ID NO.2; The fluorescent probes designed for the CYP3A5 gene are: CYPP1, the nucleotide sequence is shown in SEQ ID NO.

3.

3. The personalized drug gene detection kit according to claim 1, characterized in that: The primer pairs designed for the TPMT gene are: TPMTF, the nucleotide sequence is shown in SEQ ID NO.4; TPMTR2, the nucleotide sequence is shown in SEQ ID NO.5; The fluorescent probes designed for the TPMT gene are: TPMTP4, the nucleotide sequence is shown in SEQ ID NO.

6.

4. The personalized drug gene detection kit according to claim 1, characterized in that: The positive control was a DNA recombinant plasmid containing all the target gene target sequence polymorphic sites, and the negative control was purified water.

5. A use of the personalized drug gene detection kit according to any one of claims 1 to 4, characterized in that: The PCR-melting curve method is used to qualitatively detect SNPs in DNA samples, including the following steps: S1. Extract sample DNA; S2. Calculate the total amount of PCR reaction solution and Taq DNA enzyme according to the number of reaction reagents required and each test reaction system, mix them well, and dispense them into eight PCR thin-walled tubes according to 1 per test reaction system; S3, add 2 μL of negative control or 2 μL of positive control or 2 μL of sample DNA to the thin-walled eight-tube strip in step S2 to make the total reaction volume 20 μL; S4, placing each reaction tube in turn into a fluorescent quantitative PCR instrument for PCR amplification; S5. Compare the melting curve Tm values ​​generated by the specific fluorescent probe and the target sequence to perform SNP typing.

6. The use of a personalized drug gene detection kit according to claim 5, characterized in that: Each test reaction system includes 17 μL of PCR reaction solution and 1 μL of Taq DNA enzyme.

7. The use of a personalized drug gene detection kit according to claim 5, characterized in that: In step S3, the PCR amplification procedure includes: Pre-denaturation: 95°C, 10 min, 1 cycle; amplification: 95°C, 15 sec, 58°C, 40 sec, 40 cycles, fluorescence signal reading at 58°C; melting curve: 95°C, 2 min, 45°C, 0.03°C / s heating, 2 min, 85°C, 5 sec, one cycle, fluorescence signal reading at 85°C.

8. The use of a personalized drug gene detection kit according to claim 5, characterized in that: In step S3, the fluorescence quantitative PCR instrument selects FAM channel and HEX channel for fluorescence detection, selects none for quenching group, and selects none for reference fluorescence.

9. The use of a personalized drug gene detection kit according to claim 5, characterized in that: In step S4, the SNP typing criteria are: For the FAM channel, the high melting peak Tm value was 59 ± 1.5 °C, and the genotype was the wild type of CYP3A5; or the low melting peak Tm value is 55±1.5℃, the high melting peak Tm value is 59±1.5℃, and the genotype is a heterozygous mutation of CYP3A5; or the low melting peak Tm value is 55±1.5℃, and the genotype is a homozygous mutation of CYP3A5; For the HEX channel, the high melting peak Tm value was 56 ± 2°C, and the genotype was the wild type of TPMT; or the low melting peak Tm value is 51±2℃, the high melting peak Tm value is 56±2℃, and the genotype is a heterozygous mutation of TPMT; Or the low melting peak Tm value is 51±2°C, and the genotype is a homozygous mutation of TPMT.

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