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CN113755588A - Genetic polymorphism detection kit for fluorouracil adverse reaction and curative effect prediction, detection method and application thereof - Google Patents

Genetic polymorphism detection kit for fluorouracil adverse reaction and curative effect prediction, detection method and application thereof Download PDF

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CN113755588A
CN113755588A CN202111048288.6A CN202111048288A CN113755588A CN 113755588 A CN113755588 A CN 113755588A CN 202111048288 A CN202111048288 A CN 202111048288A CN 113755588 A CN113755588 A CN 113755588A
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fluorouracil
nqo1
tyms
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刘丹
孙悦
陈立波
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Fist Shanghai Biotechnology Co ltd
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Abstract

The invention discloses a gene polymorphism detection kit for fluorouracil adverse reaction and curative effect prediction, a detection method and application thereof, wherein the detection kit is used for detecting gene polymorphisms of fluorouracil metabolism-related genes TYMS, GSTP1, NQO1 and MTHFR, the kit is designed with specific amplification primers and sequencing primers aiming at TYMS, GSTP1, NQO1 and MTHFR, and the kit comprises the following components: sample processing liquid, magnetic beads, amplification reagent 1, amplification reagent 2, TYMS, GSTP1, NQO1, MTHFR sequencing primer and positive control. The invention uses rapid DNA preparation, constant temperature PCR amplification and pyrosequencing technology as a combination to detect the fluorouracil adverse reaction and the gene polymorphism of curative effect prediction, and provides a gene angle suggestion for clinical personalized medication.

Description

Genetic polymorphism detection kit for fluorouracil adverse reaction and curative effect prediction, detection method and application thereof
Technical Field
The invention relates to a gene polymorphism detection kit for fluorouracil adverse reaction and curative effect prediction, and a detection method and application thereof, and belongs to the field of gene detection.
Background
Fluorouracil drugs are one of tumor chemotherapy drugs widely used in clinic, including 5-fluorouracil, capecitabine, tegafur, carmofur, floxuridine, doxifluridine and the like, which are all converted into 5-FU in vivo, and the main approaches are as follows: converting into 5-fluoro-2-deoxyuridine, inhibiting thymidylate synthase, and blocking the conversion of deoxyuridine into deoxythymidine, thereby inhibiting DNA biosynthesis. In addition, fluorouracil triphosphate (pseudo metabolite) can permeate into human RNA, inhibit RNA synthesis by preventing uracil and orotic acid from permeating into human RNA, influence protein biosynthesis, inhibit granulation tissue proliferation, and prevent scar formation. In recent years, researches show that active metabolites of the thymidine and the 5-fluorouracil deoxynucleoside and the methyltetrahydrofolic acid can form a triple complex with thymidine synthetase, prevent the activity of the thymidine synthetase from being exerted, and inhibit the synthesis of DNA. Because 5-FU has several action routes and different individuals have differences in drug metabolism, part of patients cannot reach effective blood concentration when being administrated in a conventional body surface area conversion mode, and other parts of patients can have cumulative poisoning or adverse reaction.
Thymidylate Synthase (TS) encoded by the TYMS gene is a rate-limiting enzyme in pyrimidine nucleotide synthesis and plays an important role in tumor growth. TS can methylate deoxyuridine monophosphate into deoxythymidine triphosphate as an analogue of pyrimidine together with a methyl prosthetic group, 5-FU metabolite fluorouracil deoxynucleotide forms a trimer together with TS and the methyl prosthetic group to block the formation of the deoxythymidine triphosphate, and finally, DNA synthesis is prevented, so that the growth or proliferation of cells is inhibited. TYMS is an important target of 5-FU as a basic tumor chemotherapeutic drug effect. The TYMSTTAAAG/del (rs151264360) mutation was associated with sensitivity to 5-FU, and studies showed: individuals of the TTAAAG/TTAAAG, TTAAAG/del, del/del genotypes have successively reduced sensitivity to fluorouracil.
GSTs are the most important II-phase metabolic enzymes in human bodies, are a group of proteins with various physiological functions which participate in a supergene family of the detoxification of organisms, and can catalyze the combination of harmful polar compounds in the organisms and glutathione to achieve the purpose of detoxification; meanwhile, the compound has peroxidase and foreign body enzyme activities, and participates in the processes of antioxidation in organisms and the synthesis, storage and transportation of substances in protected cells. Whether a carcinogen can cause carcinogenesis in a target cell depends largely on the activities of these two classes of enzymes and the equilibrium relationship between them. Since phase II metabolic enzymes GSTs are involved in the detoxification of carcinogens in vivo, and the genes encoding many members of the enzyme system (e.g., GSTP1) are polymorphic, genetic variation can alter the ability of the corresponding enzyme to activate or inactivate heterologous substrates. When a chemotherapy scheme of oxaliplatin and fluorouracil medicines is applied to a patient with late intestinal cancer, the medicines with different genotypes of GSTP1 have different metabolic capacities, and the genotype with poor metabolic capacity can cause toxic and side effects to be enhanced, so that serious systemic adverse reactions are caused.
The NQO1 gene encodes reduced coenzyme/quinone oxidoreductase, also known as DT-diaphorase (DT-diaphorase), a flavoenzyme, and NQO1 is widely distributed in organs, but has the highest levels in liver, kidney, and gastrointestinal tract. The NQO1 enzyme can be induced by various chemicals, such as polycyclic aromatic hydrocarbons, hydroquinones, acrylates, and the like. NQO1 enzyme belongs to II-phase metabolic enzyme, and forms a metabolic network for exogenous toxic substances in vivo together with other I and II-phase metabolic enzymes, and plays an important role in detoxification and metabolism of organisms. The polymorphism of NQO1 reduces the detoxification ability of NQO1 enzyme to chemical substances, increasing the risk of blood diseases. The protein coded by NQO1 gene is a key enzyme for metabolizing benzene in human bone marrow, the enzyme is a protective reductase in cells, and can protect cells from external quinone substances and oxidative damage, such as transformation of benzoquinone into low-toxicity hydroquinone and hydroxyl compounds, thereby avoiding damage of affinity quinones to cell DNA and ensuring normal physiological functions of organisms. The 609C/T polymorphism of the NQO1 gene is related to the activity of the NQO1 enzyme, wherein the 609CC genotype of the NQO1 gene has high activity, the 609CT genotype has medium activity, and the 609TT genotype has no activity.
5-fluorouracil is a common chemotherapeutic drug for tumors of the digestive tract. 5-fluorouracil entering human body is absorbed by cells and converted into a plurality of active metabolites, wherein fluorouracil monophosphate deoxynucleoside forms a ternary complex with TS (thymidylate synthase) through a covalent substrate (reduced folic acid) of the fluorouracil monophosphate deoxynucleoside, the uracil deoxynucleoside synthase is blocked from being converted into thymidylate, MTHFR influencing the biosynthesis of DNA is a key enzyme in the folic acid metabolism process, the reduced folic acid can be converted into 5-methyltetrahydrofolic acid, so that the ternary complex formed by the fluorouracil monophosphate deoxynucleoside, frightened and reduced folic acid is reduced, and the anti-tumor effect of the 5-fluorouracil is weakened. It catalyzes an irreversible reaction to convert 5, 10-formyltetrahydrofolate (5, 10MTHF) to 5-methyltetrahydrofolate (5-MTHF), and studies have shown that increasing tumor 5, 10-MTHF concentration prior to 5-FU use enhances thymidylate synthase inhibition, but also results in increased cytotoxicity associated with 5-FU. Variation of the polymorphic site of the MTHFR portion is the primary mechanism that results in a reduction or loss of enzymatic activity.
At present, the methods for detecting gene polymorphism are various, mainly adopt fluorescence PCR, and also comprise a high-resolution melting curve method, a taqman fluorescence probe method and an allele specific amplification method. The high-resolution melting curve method has simple steps, but has low specificity and higher requirements on instruments and equipment; the allele specific amplification method adopts ARMS primers to carry out specific amplification, and has simple operation method, but strict detection condition requirements, and easy occurrence of primer mismatching in actual operation to generate false positive. the taqman fluorescence probe method has higher test cost.
At present, the blood DNA is obtained mainly by the traditional column extraction and the magnetic bead extraction, and the two methods both take longer time and are relatively complicated to operate. CN201610022581.8 proposes a real-time fluorescent quantitative PCR method for extracting nucleic acid and amplifying by magnetic beads in one tube, adding lysis solution mixed with magnetic beads and a sample to be detected into a PCR amplification tube, mixing uniformly, standing, performing magnetic attraction, sucking out mixed solution, and washing the obtained magnetic beads once; and adding the prepared PCR reaction solution into the PCR amplification tube to perform real-time fluorescent quantitative PCR reaction on the target nucleic acid. The invention also requires washing of the magnetic beads. Therefore, there is an urgent need for a simple, rapid, efficient, inexpensive, and highly specific pyrosequencing method for detecting gene polymorphisms.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to obtain a gene polymorphism detection kit for fluorouracil adverse reaction and curative effect prediction, and a detection method and application thereof.
In order to realize one of the above purposes, the technical scheme of the gene polymorphism detection kit for fluorouracil adverse reaction and curative effect prediction adopted by the invention is as follows:
the kit for detecting the genetic polymorphism of adverse reactions and curative effect prediction of fluorouracil is designed with specific amplification primers and sequencing primers aiming at polymorphisms of TYMS (1494delTTAAAG), GSTP1(A313G), NQO1(C609T) and MTHFR (C677T), and comprises the following components: sample treatment solution, magnetic beads, amplification reagent 1, amplification reagent 2, TYMS (1494delTTAAAG), GSTP1(A313G), NQO1(C609T), MTHFR (C677T) sequencing primer, and positive control.
Specifically, the specific primer sequences are shown in the following table:
Figure BDA0003251821220000031
Figure BDA0003251821220000041
preferably, the sequence of the specific primer group of the TYMS (1494delTTAAAG) is shown as the sequence tables SEQ ID NO 1-SEQ ID NO 2; the sequence of the specific primer group of GSTP1(A313G) is shown as sequence tables SEQ ID NO. 3-SEQ ID NO. 4; the specific primer group sequence of the NQO1(C609T) is shown as SEQ ID NO: 5-SEQ ID NO:6 of the sequence table; the sequence of the specific primer group of the MTHFR (C677T) is shown in a sequence table SEQ ID NO. 7-SEQ ID NO. 8.
Preferably, the sequencing primers of TYMS (1494delTTAAAG), GSTP1(A313G), NQO1(C609T) and MTHFR (C677T) are respectively shown as SEQ ID NO: 9-SEQ ID NO:12 of the sequence table.
More preferably, the sequencing primer is a nucleic acid analogue, the skeleton of which is a peptide bond rather than a phosphodiester bond, and the peptide bond skeleton is connected with a corresponding base. The structure has stable biological properties, and is not easy to degrade by protease or nuclease. Binding to DNA is more stable than DNA/DNA binding.
Preferably, the sequencing region corresponding to the TYMS (1494delTTAAAG) sequencing primer is a to-be-detected sequence of TYMS (1494delTTAAAG), and is shown as the sequence table SEQ ID NO: 13. The sequencing region corresponding to the GSTP1(A313G) sequencing primer is a to-be-detected sequence of GSTP1(A313G), and is shown as a sequence table SEQ ID NO: 14. The sequencing region corresponding to the NQO1(C609T) sequencing primer is the sequence to be detected of NQO1(C609T), and is shown as the sequence table SEQ ID NO: 15. The sequencing region corresponding to the MTHFR (C677T) sequencing primer is an MTHFR (C677T) to-be-detected sequence, and is shown as a sequence table SEQ ID NO: 16.
Preferably, TYMS (1494delTTAAAG), GSTP1(A313G), NQO1(C609T) and MTHFR (C677T) share one allocation command as shown in SEQ ID NO:17 of the sequence Listing. "ddA" in SEQ ID NO:17 indicates that ddATP, the last base added to the sequencing reaction of GSTP1(A313G), is added to terminate the sequencing reaction. "ddT" in SEQ ID NO:17 indicates that NQO1(C609T) was added to the last base ddTTP of the sequencing reaction, which was added to terminate the sequencing reaction. "ddC" in SEQ ID NO:17 indicates that NQO1(C609T) was added to the last base ddCTP of the sequencing reaction, which addition of the base terminated the sequencing reaction. Finally sequencing is carried out according to an allocation instruction TYMS (1494 delTTAAAG). 17 "-" in SEQ ID NO means that the addition of the reagent was suspended for 3 min. Subsequent sequencing primers will be added during this pause.
Preferably, the sample treatment solution, which does not contain a guanidine salt, is used to lyse the sample under alkaline conditions and with a surfactant. Comprises 0.1-0.6% of lithium dodecyl sulfate, 0.1-0.5% of triton X-100, 2-50mg/mL of sodium hydroxide, 5-15% of trehalose, 3-7 mmol/L of BSA, 20-80mM of Tris-HCl and 100mM of NaCl, wherein the pH value is 8.5-9.5.
More preferably, the sample treatment solution comprises 0.3-0.5% of lithium dodecyl sulfate, 0.2-0.4% of Triton X-100, 10-20mg/mL of sodium hydroxide, 8-12mM of betaine, 8-12% of trehalose, 4-6 mM of BSA, 50mM of Tris-HCl, 100mM of NaCl, and pH 9.
Preferably, the magnetic beads are carboxyl magnetic beads, the particle size is 600mm, the suspension property is good, the magnetic property is strong, and the adsorption capacity is large. The concentration of magnetic beads in the reaction solution is 0.2mg/25 mu L, and the DNA adsorption amount in the blood sample is 30 ng-260 ng, which completely meets the DNA amount required by amplification.
Preferably, the amplification reagent 1 comprises: amplification buffer, 15mM magnesium acetate;
preferably, the amplification reagent 2 comprises: TYMS (1494delTTAAAG) pre-primer (0.32uM), TYMS (1494delTTAAAG) post-primer (0.32uM), GSTP1(A313G) pre-primer (0.32uM), GSTP1(A313G) post-primer (0.32uM), NQO1(C609T) pre-primer (0.32uM), NQO1(C609T) post-primer (0.32uM), MTHFR (C677T) pre-primer (0.32uM), MTHFR (C67 677T) post-primer (0.32uM), dNTPS (0.3mM), strand displacement DNA polymerase (1.2 ng/. mu.L), single-stranded DNA binding protein (3.2 ng/. mu.L), single-stranded nucleic acid binding recombinase (4.8 ng/. mu.L).
More preferably, the amplification reagent 2 comprises: trehalose (0.2%), 10mM manganese acetate, 0.1M sorbitol, 5ug/mL BSA. Trehalose has nonspecific protection effect on bioactive substances, can improve the thermal stability of DNA polymerase, reduce the melting temperature of a DNA template, and reduce the secondary structure formed by self-complementary pairing of a G-C rich region, thereby improving the specificity of PCR reaction. Sorbitol and manganese acetate have the stabilizing effect of the PCR premix, and have the stabilizing effect in the freeze drying process. The bovine serum albumin can improve the amplification efficiency of the PCR reaction and reduce the influence of PCR inhibitors in the system on the reaction.
Preferably, the reaction volume is 25ul, and the reaction conditions are as follows: 30min at 42 ℃.
Preferably, the positive control comprises TYMS (1494delTTAAAG), GSTP1(A313G), NQO1(C609T) and MTHFR (C677T) heterozygous genomic DNA at a concentration of 20 ng/ul. The positive control corresponds to the heterozygosis of the detected gene locus, provides reference for the type determination of an unknown sample, and simultaneously performs quality control on the effectiveness of the reaction solution.
The invention also discloses a gene polymorphism detection method for fluorouracil adverse reaction and curative effect prediction by adopting the kit, which comprises the following steps:
a. mixing 100ul of sample treatment solution, 4ul of magnetic beads and 30ul of EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood sample, and standing at room temperature for 5 min;
b. placing the PCR amplification tube on a magnetic frame, and sucking out the mixed solution from the opposite side of the magnetic beads after the magnetic beads are completely adsorbed to one side;
c. adding the prepared PCR reaction solution into the PCR amplification tube obtained in the step b), fully and uniformly mixing magnetic beads and the PCR reaction solution, centrifuging, and carrying out constant-temperature reaction;
d. binding the binding solution (containing the microbeads) with the amplification product;
e. treating the denatured liquid to obtain a single-chain product;
f. adding a washing buffer solution for rinsing;
g. adding a sequencing enzyme and a sequencing substrate to each sequencing tube;
h. taking an 8-row pipe, and adding dATP, dTTP, dGTP, dCTP, TYMS (1494delTTAAAG), GSTP1(A313G), NQO1(C609T), MTHFR (C677T) sequencing primer, ddATP, ddTTP and ddCTP from one round smooth end to the flat end in sequence; lightly knocking the bottom of the calandria against the tabletop to enable the bases to be flatly paved at the bottom of the calandria;
i. and (4) pyrosequencing.
The invention also discloses application of the gene polymorphism detection kit for adverse reaction and curative effect prediction of fluorouracil, and the detection kit is used for detecting TYMS (1494delTTAAAG), GSTP1(A313G), NQO1(C609T) and MTHFR (C677T) so as to reflect adverse reaction risk of fluorouracil from a gene level and further provide a suggestion of the gene level for guiding dosage of drugs related to fluorouracil.
The rapid amplification method is optimized mainly from three aspects, and on one hand, the method adopts a mode of extracting and amplifying the same tube, so that the risks of extracting multiple tube moving and losing nucleic acid are avoided; on the other hand, the rapid constant temperature amplification is carried out by adding an anti-inhibitor; in the third aspect, multiplex PCR is adopted to amplify four sites of GSTP1(A313G), NQO1(C609T), MTHFR (C677T) and TYMS (1494delTTAAAG), and pyrosequencing is carried out on the four sites in one reaction. The sequencing reaction is terminated by adding GSTP1(A313G) sequencing primer and sequencing material to perform pyrosequencing, and adding ddATP to the last base. NQO1(C609T) was added to the sequencing primer and sequencing was performed, and the last base was added ddTTP to terminate the sequencing reaction. Then adding MTHFR (C677T) sequencing primer and sequencing, adding ddCTP to the last base to terminate the sequencing reaction, finally adding TYMS (1494delTTAAAG) sequencing primer to perform dNTP sequencing, and sequencing four sites in sequence by one treatment, thereby reducing the operation time and improving the sequencing flux. The invention aims to obtain a gene polymorphism detection kit for fluorouracil adverse reaction and curative effect prediction based on constant temperature PCR and pyrophosphoric acid detection, and a detection method and application thereof.
Compared with the prior art, the invention adopts the sample processing liquid to rapidly release the DNA from the sample, the sample amount is 30ul of whole blood, and compared with a one-step cracking method and direct amplification (about 2-10ul), enough genome DNA can be obtained and adsorbed on the magnetic beads for subsequent analysis. Most of the inhibitor can be removed by removing the sample and lysis mix. The DNA template was amplified by isothermal PCR at the gene sites TYMS (1494delTTAAAG), GSTP1(A313G), NQO1(C609T), MTHFR (C677T) to produce a large amount of biotin-labeled single-stranded DNA. The biotin-labeled single-stranded DNA is combined with streptavidin, and a sequencing primer and a sequencing raw material are added after washing to perform pyrosequencing, so that the sequencing process and time are simplified. The invention uses rapid DNA preparation, constant temperature PCR amplification and pyrosequencing technology as a combination to detect the fluorouracil adverse reaction and the gene polymorphism of curative effect prediction, and provides a gene angle suggestion for clinical personalized medication.
Drawings
FIG. 1 is an exemplary diagram of the detection results of GSTP1(1375CT), NQO1(609CC), MTHFR (677CC), TYMS (1494delTTAAAG) pyrophosphate provided by the present invention;
FIG. 2 is a diagram showing an example of the detection results of GSTP1(1375TT), NQO1(609CC), MTHFR (677CT), TYMS (1494ins/delTTAAAG) pyrophosphate;
FIG. 3 is an exemplary diagram of the detection results of GSTP1(1375TT), NQO1(609CT), MTHFR (677TT), TYMS (1494insTTAAAG) pyrophosphate provided by the present invention;
FIG. 4 is an exemplary diagram of the detection results of GSTP1(1375CC), NQO1(609CC), MTHFR (677CT), TYMS (1494delTTAAAG) pyrophosphate provided by the present invention;
FIG. 5 is an exemplary diagram of the detection results of GSTP1(1375TT), NQO1(609TT), MTHFR (677CC), TYMS (1494insTTAAAG) pyrophosphate provided by the present invention.
Detailed Description
The following examples are provided to further describe the kit for detecting genetic polymorphism, the detection method and the application of the kit for detecting fluorouracil adverse reaction and therapeutic effect prediction in detail and in a complete manner. The following examples are illustrative only and are not to be construed as limiting the invention.
The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all commercially available unless otherwise specified.
(I) design of specific primers
The kit of the invention designs specific amplification primers and sequencing primers aiming at TYMS (1494delTTAAAG), GSTP1(A313G), NQO1(C609T) and MTHFR (C677T) gene polymorphism, and is used for pyrophosphate PCR detection. Gene polymorphism sequences are subject to published sequences in Genebank, and primer sequences are shown in the following table 1:
TABLE 1
Figure BDA0003251821220000071
Figure BDA0003251821220000081
(II) kit composition
The detection kit comprises the components shown in the following table 2:
TABLE 2 kit component table
Serial number Composition of Indicating the amount of filling
1 Sample treatment liquid 2000 uL X1 tube
2 Magnetic bead 80 μ L X1 tube
3 Amplification reagent 1 500 μ L X1 tube
4 Amplification reagent 2 Dry powder
5 TYMS (1494delTTAAAG) sequencing primer 70ul X1 tube
6 GSTP1(A313G) sequencing primer 70ul X1 tube
7 NQO1(C609T) sequencing primer 70ul X1 tube
8 MTHFR (C677T) sequencing primer 70ul X1 tube
9 Positive control 50 μ L X1 tube
(III) the sample treatment solution preparation system comprises the following steps:
sample treatment solution containing 0.4% lithium lauryl sulfate, 0.3% Triton X-100, 15mg/mL sodium hydroxide, 10mM betaine, 10% trehalose, 5mM BSA, 50mM Tris-HCl, 100mM NaCl, pH 9.
(IV) the single-person configuration system of the detection kit amplification reagent 1 of the embodiment is as follows:
composition (I) Volume (ul)
Amplification buffer 24
200mM magnesium acetate 1
(V) the detection kit amplification reagent 2 of the present embodiment is configured as follows in a single-person configuration system:
TYMS (1494delTTAAAG) pre-primer (0.32uM), TYMS (1494delTTAAAG) post-primer (0.32uM), GSTP1(A313G) pre-primer (0.32uM), GSTP1(A313G) post-primer (0.32uM), NQO1(C609T) pre-primer (0.32uM), NQO1(C609T) post-primer (0.32uM), MTHFR (C677T) pre-primer (0.32uM), MTHFR (C677T) post-primer (0.32uM), dNTPS (0.3mM), strand displacement DNA polymerase (1.2 ng/. mu.L), single-stranded DNA binding protein (3.2 ng/. mu.L), single-stranded nucleic acid binding recombinase (4.8 ng/. mu.L), trehalose (0.2 mM), 10mM manganese sorbate, 0.1M, 5 ng BSA/. mu.5 mL. The PCR system is shown in Table 3:
TABLE 3 PCR systems
Figure BDA0003251821220000091
Figure BDA0003251821220000101
After the preparation, 149.6 ul/tube is subpackaged and freeze-dried.
Example 2 kit detection procedure
The apparatus used in the present invention is as follows: thermostats, pyrosequencing instruments (Wuhan Firster Biotech, Inc.).
1) Taking 30 mu L of EDTA anticoagulated whole blood sample in a PCR amplification tube;
2) adding 100 μ L sample treatment solution and 4ul magnetic beads, and standing for 5 min;
3) placing the PCR amplification tube on a magnetic frame, and sucking out the mixed solution from the opposite side of the magnetic beads after the magnetic beads are completely adsorbed to one side;
4) adding the amplification reagent 1 into the amplification reagent 2 dry powder, and fully dissolving and uniformly mixing;
5) adding 25ul of prepared PCR reaction liquid into the PCR amplification tube obtained in the step 4), fully and uniformly mixing magnetic beads and the PCR reaction liquid, centrifuging, and carrying out constant-temperature reaction;
6) and (3) amplifying by adopting a PCR instrument, wherein the reaction system is 25 mu L, and the amplification conditions are as follows:
temperature of amplification Time Number of cycles
42℃ 30min 1
7) Adding 40 mu L of binding solution and 3ul of agarose gel particles into a PCR reaction tube, adding 20 mu L of PCR product into the PCR reaction tube, placing the PCR reaction tube on a table type oscillator, and oscillating at 1100rpm for 10min to ensure that the microbeads and the PCR product are fully bound;
8) centrifuging at 7,000 Xg for 1min, and discarding the supernatant;
9) adding 22uL of diluted working solution of the denatured liquid, standing for 5min, centrifuging for 1min at 7,000 Xg, and collecting by an EP tube to obtain a single-chain product;
10) to the EP tube, 150uL of washing buffer was added, and centrifuged at 7,000 Xg for 1 min. (repeat 3 times);
11) transferring the single-stranded product in the EP tube to a sequencing tube, and adding 3uL sequencing enzyme and 3uL sequencing substrate to each sequencing tube;
12) respectively adding 3uL sequencing enzyme and 3uL sequencing substrate into a sequencing tube;
13) taking an 8-calandria, and sequentially adding dATP, dTTP, dGTP, dCTP, TYMS (1494delTTAAAG) sequencing primer, GSTP1(A313G) sequencing primer, NQO1(C609T) sequencing primer, MTHFR (C677T) sequencing primer, ddATP, ddTTP and ddCTP from one round smooth end to a flat end; lightly knocking the bottom of the calandria against the tabletop to enable the bases to be flatly paved at the bottom of the calandria;
14) pyrosequencing; the sequencing results are shown in FIGS. 1 to 5.
15) And (5) judging the result.
i. And (3) judging the effectiveness:
the blank control of the kit does not pass, and the detection result of the positive control is TYMS (1494delTTAAAG), GSTP1(A313G), NQO1(C609T) and MTHFR (C677T) hybrid mutant.
Criteria for determination of results
In the peak image of DNA sequencing of TYMS (1494delTTAAAG),
the frequency of T is larger than or equal to 90 percent, the frequency of A is smaller than or equal to 10 percent, and the type is deletion type;
the frequency of 40% to T is less than or equal to 60%, the frequency of 40% to A is less than or equal to 60%, and the hybrid is obtained;
the frequency of A is not less than 90%, the frequency of T is not less than 10%, and the insertion type is obtained;
in the DNA sequencing peak map of GSTP1(A313G),
the frequency of A is not less than 90 percent, the frequency of G is not less than 10 percent, and the product is AA type;
the frequency of 40% to A is 60% and the frequency of 40% to G is 60%, which is AG type;
the frequency of G is not less than 90 percent, the frequency of A is not less than 10 percent, and the product is GG type;
in the DNA sequencing peak map of NQO1(C609T),
the frequency of C is not less than 90 percent, the frequency of T is not less than 10 percent, and the model is CC;
the frequency of 40% to C is less than or equal to 60%, and the frequency of 40% to T is less than or equal to 60%, which is CT type;
the frequency of T is not less than 90 percent, the frequency of C is not less than 10 percent, and the model is TT;
in the DNA sequencing peak map of MTHFR (C677T),
the frequency of C is not less than 90 percent, the frequency of T is not less than 10 percent, and the model is CC;
the frequency of 40% to C is less than or equal to 60%, and the frequency of 40% to T is less than or equal to 60%, which is CT type;
the frequency of T is not less than 90 percent, the frequency of C is not less than 10 percent, and the model is TT;
correlation between gene detection results and adverse reaction and curative effect prediction of fluorouracil
Figure BDA0003251821220000111
Figure BDA0003251821220000121
Fifth, the performance test result of the kit
5.1. Specificity of
The specific sample (including non-human DNA template and dilution of amplification product of different sites or homologous sites of the same human gene) is detected, and the result is negative.
5.2. Accuracy of
The detection of reference substances of different genotypes (including TYMS (1494delTTAAAG), GSTP1(A313G), NQO1(C609T) and MTHFR (C677T) in the kit range can detect the corresponding genotypes.
5.3. Minimum detection limit
The minimum detection limit should not be higher than 2 ng/ul.
5.4. Repeatability of
In the detection kit, each reference substance is subjected to 10 times of detection, the results are corresponding mutation types, and the Coefficient of Variation (CV) of the Ct value of a corresponding detection channel is less than or equal to 5.0%.
Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.
Figure BDA0003251821220000131
Figure BDA0003251821220000141
Figure BDA0003251821220000151
Figure BDA0003251821220000161
Figure BDA0003251821220000171
Figure BDA0003251821220000181
Figure BDA0003251821220000191
Figure BDA0003251821220000201
Sequence listing
<110> Firstat (Shanghai) Biotech Co., Ltd
<120> gene polymorphism detection kit for fluorouracil adverse reaction and curative effect prediction, and detection method and application thereof
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(30)
<400> 1
acatgtatgt gcatttcaat cccacgtact 30
<210> 2
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(31)
<400> 2
ctattttagg aaggaactga gcagataagt g 31
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(30)
<400> 3
caggaggcag ccctggtgga catggtgaat 30
<210> 4
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(30)
<400> 4
tgcactcgcc cccatgaccc gttacttggc 30
<210> 5
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(30)
<400> 5
ctttctgtat cctcagagtg gcattctgca 30
<210> 6
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(30)
<400> 6
ccttccagga tttgaattcg ggcgtctgct 30
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(30)
<400> 7
gaggactctc tctgcccagt ccctgtggtc 30
<210> 8
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(30)
<400> 8
tgcccatgtc ggtgcatgcc ttcacaaagc 30
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(20)
<400> 9
gtagagtgtg gttatgaact 20
<210> 10
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(17)
<400> 10
ttggtgtaga tgaggga 17
<210> 11
<211> 19
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<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(19)
<400> 11
ccaatgctat atgtcagtt 19
<210> 12
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
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<400> 12
ctgcgtgatg atgaa 15
<210> 13
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
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<222> (1)..(9)
<400> 13
tttawagtt 9
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(10)
<400> 14
gaygtatttg 10
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<211> 10
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(10)
<400> 15
gagrttctaa 10
<210> 16
<211> 10
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(10)
<400> 16
tcgrctcccg 10
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<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(39)
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tgcactgtdd atgcagatcd dtgtacgact ddcgtatag 39

Claims (10)

1. The kit is characterized by being used for detecting the gene polymorphism of fluorouracil metabolism-related genes TYMS, GSTP1, NQO1 and MTHFR, and designing specific amplification primers and sequencing primers aiming at TYMS, GSTP1, NQO1 and MTHFR, wherein the kit comprises the following components: sample processing liquid, magnetic beads, amplification reagent 1, amplification reagent 2, TYMS, GSTP1, NQO1, MTHFR sequencing primer and positive control.
2. The kit for detecting the genetic polymorphism of fluorouracil adverse reaction and curative effect prediction according to claim 1, wherein the sequence of the specific primer set of TYMS is shown in sequence tables SEQ ID NO 1-SEQ ID NO 2; the sequence of the specific primer group of GSTP1 is shown in a sequence table SEQ ID NO. 3-SEQ ID NO. 4; the specific primer group sequence of NQO1 is shown in sequence tables SEQ ID NO. 5-SEQ ID NO. 6; the sequence of the specific primer group of the MTHFR is shown in a sequence table SEQ ID NO. 7-SEQ ID NO. 8.
3. The kit for detecting the genetic polymorphism of fluorouracil adverse reaction and curative effect prediction according to claim 1, wherein the sequencing primers TYMS, GSTP1, NQO1 and MTHFR are respectively shown as SEQ ID NO 9-SEQ ID NO 12 of the sequence table.
4. The kit for detecting the genetic polymorphism of fluorouracil adverse reaction and efficacy prediction according to claim 1, wherein TYMS, GSTP1, NQO1 and MTHFR share one assignment instruction as shown in sequence table SEQ ID NO. 17.
5. The kit for detecting the genetic polymorphism of adverse reaction and curative effect prediction of fluorouracil according to claim 1, wherein the sample treatment solution comprises 0.1-0.6% of lithium dodecyl sulfate, 0.1-0.5% of triton X-100, 2-50mg/mL sodium hydroxide, 5-15% of trehalose, 3-7 mmol/L BSA, 20-80mM Tris-HCl and 100mM NaCl, and the pH is 8.5-9.5.
6. The kit for detecting genetic polymorphism capable of predicting adverse reaction and curative effect of fluorouracil according to claim 1, wherein the magnetic beads are carboxyl magnetic beads, and the particle size is 600 mm.
7. The kit for detecting the genetic polymorphism of fluorouracil adverse reaction and therapeutic effect prediction according to claim 1, wherein the amplification reagent 1 comprises: amplification buffer, 15mM magnesium acetate.
8. The kit for detecting the genetic polymorphism of fluorouracil adverse reaction and therapeutic effect prediction according to claim 1, wherein the amplification reagent 2 comprises: TYMS pre-primer 0.32uM, TYMS post-primer 0.32uM, GSTP1 pre-primer 0.32uM, GSTP1 post-primer 0.32uM, NQO1 pre-primer 0.32uM, NQO1 post-primer 0.32uM, MTHFR pre-primer 0.32uM, MTHFR post-primer 0.32uM, dNTPS0.3 mM, strand displacement DNA polymerase 1.2 ng/. mu.L, single-stranded DNA binding protein 3.2 ng/. mu.L, recombinase binding to single-stranded nucleic acid 4.8 ng/. mu.L.
9. A method for detecting fluorouracil adverse reaction and gene polymorphism with predicted curative effect according to any one of claims 1 to 8, which comprises the following steps:
a) mixing 100ul of sample processing liquid, 4ul of magnetic beads and 30ul of EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood sample, and standing at room temperature for 5 min;
b) placing the PCR amplification tube on a magnetic frame, and sucking out the mixed solution from the opposite side of the magnetic beads after the magnetic beads are completely adsorbed to one side;
c) adding the prepared PCR reaction solution into the PCR amplification tube obtained in the step b), fully and uniformly mixing magnetic beads and the PCR reaction solution, centrifuging, and carrying out constant-temperature reaction;
d) combining the binding solution containing the microbeads with the amplification product;
e) treating the denatured liquid to obtain a single-chain product;
f) adding a washing buffer solution for rinsing;
g) adding a sequencing enzyme and a sequencing substrate to each sequencing tube;
h) taking an 8-row pipe, and sequentially adding dATP, dTTP, dGTP, dCTP, TYMS, GSTP1, NQO1, MTHFR sequencing primer, ddATP, ddTTP and ddCTP from one round smooth end to the flat end;
i) and (4) pyrosequencing.
10. The kit for detecting the genetic polymorphism for the adverse reaction and the curative effect prediction of fluorouracil according to any one of claims 1 to 9, and the application of the detection method thereof, are characterized in that the kit detects TYMS, GSTP1, NQO1 and MTHFR to reflect the risk of adverse reaction of fluorouracil from a gene level.
CN202111048288.6A 2021-09-08 2021-09-08 Genetic polymorphism detection kit for fluorouracil adverse reaction and curative effect prediction, detection method and application thereof Pending CN113755588A (en)

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