CN113528629A - Detection kit for methotrexate metabolic marker, detection method and application thereof - Google Patents

Abstract

The invention discloses a detection kit for methotrexate metabolic markers, and a detection method and application thereof, wherein the kit is used for detecting gene polymorphisms of methotrexate metabolic markers ABCB1(C3435T), MTHFR (C677T) and SLCO1B1(c.1865+4846T > C). The invention uses multiple RPA amplification and optimized pyrosequencing technology as a combination to detect the methotrexate drug dosage and the gene polymorphism related to adverse reaction prediction, and the kit can simultaneously detect ABCB1(C3435T), MTHFR (C677T) and SLCO1B1(c.1865+4846T > C) gene polymorphism methotrexate drugs and provides a suggestion of gene angle for clinical personalized medication.

Description

Detection kit for methotrexate metabolic marker, detection method and application thereof

Technical Field

The invention relates to a detection kit for a methotrexate metabolic marker, and a detection method and application thereof, and belongs to the field of gene detection.

Background

Methotrexate (MTX) is a folate antagonist that interferes with DNA synthesis by binding dihydrofolate reductase, making tetrahydrofolate dysregulated, thereby inhibiting tumor cell proliferation. HDMTX chemotherapy can cause various toxic and side effects, mainly has myelosuppression, is manifested by leucopenia, and can have whole blood suppression in severe cases, and gastrointestinal tract reactions, such as nausea, vomiting and the like, and mucosal ulcers of oral cavity, perianal and the like. The liver and kidney function can be damaged by large dose or long-term application. Adverse reactions caused by chemotherapy drugs cause chemotherapy suspension or termination, which affects disease outcome. Pharmacogenomics studies show that Single Nucleotide Polymorphism (SNP) of genes related to drug transport, metabolism and action targets is one of the key factors determining the curative effect and adverse reaction of drugs.

Methylene tetrahydrofolate reductase (MTHFR) is one of rate-limiting enzymes in a folate metabolic pathway, can catalyze and reduce 5, 10-dimethyl tetrahydrofolate into 5-methyl tetrahydrofolate, and plays a key role in regulation and control on synthesis, activation and repair of DNA; the 677C-T genetic polymorphism of the enzyme seriously affects the enzyme activity, thereby causing a series of reactions, and the most important adverse reaction is the toxic reaction of the methotrexate drug. In addition, there are two main classes of transporters of methotrexate: adenosine triphosphate binding cassette transporter (ABC transporter) and solute protein transporter family (SLC family).

ABCB1 is a phosphorylated and glycosylated transmembrane efflux transporter, which is the most important genetic material in the ATP binding cassette protein (ATP binding cassette transporter) family, and ABCB1 gene is located in the long arm 21 region (7q21) of human chromosome 7 and can encode an ATP-dependent efflux pump P-glycoprotein (P-gp), which can transport intracellular drugs to the outside of cells, thereby causing the generation of drug resistance. ABCB 1C 3435T is located on exon 26, C3435T of ABCBL gene can change the specificity of P-gp to its substrate by affecting the secondary structure of the encoded protein without changing the encoded amino acid sequence, and the function or expression level of P-gp can change the pharmacokinetics of MTX in vivo, thus changing the effective concentration in target cells, and ultimately affecting the therapeutic effect.

The organic anion transport polypeptide 1B1(OATP1B1) encoded by the SLCO1B1 gene is one of the major uptake transporters in the body, mediating the transmembrane transport of a variety of exogenous and endogenous substances from the blood to hepatocytes. SLCO1B1 has high genetic polymorphism, and the gene polymorphism can influence the transport capacity of OATP1B1, at present, there are many methods for detecting the gene polymorphism, such as a direct sequencing method, a chip method, a high-resolution melting curve method, an allele specific amplification method, a taqman fluorescence probe method and the like. The sequencing method and the chip method have the disadvantages of complicated operation steps, long detection period and easy pollution of amplification products; the high-resolution melting curve method has simple steps, low specificity and higher requirements on instruments and equipment; the allele specific amplification method adopts ARMS primers for specific amplification, the design of the primers is difficult to optimize, and the detection condition is strict. The Taqman fluorescent probe method has high test cost and low amplification flux for a plurality of genes. Therefore, it is necessary to establish a simple, rapid, efficient, inexpensive, and highly specific method for detecting gene polymorphisms.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to obtain a detection kit for a methotrexate metabolic marker based on multiple RPA amplification and pyrosequencing technology, and a detection method and application thereof.

In order to realize one of the purposes of the invention, the technical scheme of the detection kit for the methotrexate metabolic marker adopted by the invention is as follows:

the kit provided by the invention is used for designing specific amplification primers and sequencing primers aiming at polymorphism of three genes, namely ABCB1(C3435T), MTHFR (C677T) and SLCO1B1(c.1865+4846T > C), and comprises the following components: reagent 1, reagent 2, ABCB1(C3435T) sequencing primer, MTHFR (C677T) sequencing primer, SLCO1B1(c.1865+4846T > C) sequencing primer, positive control.

Preferably, the specific primers are designed as shown in the following table:

preferably, the sequence of the specific primer group of the ABCB1(C3435T) is shown as the sequence tables SEQ ID NO 1-SEQ ID NO 2; the sequence of the specific primer group of the MTHFR (C677T) is shown as SEQ ID NO. 3-SEQ ID NO. 4 of the sequence table; the sequence of the specific primer group of the SLCO1B1(c.1865+4846T > C) is shown as the sequence tables SEQ ID NO: 5-SEQ ID NO: 6.

Preferably, the ABCB1(C3435T) sequencing primer, the MTHFR (C677T) sequencing primer and the SLCO1B1(c.1865+4846T > C) sequencing primer are respectively shown as SEQ ID NO: 7-SEQ ID NO:9 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 sequencing primer of ABCB1(C3435T) is a to-be-detected sequence of ABCB1(C3435T), and is shown as SEQ ID NO:10 of the sequence table; 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. 11. The sequencing region corresponding to the sequencing primer of SLCO1B1(c.1865+4846T > C) is a sequence to be detected of SLCO1B1(c.1865+4846T > C), and is shown as a sequence table SEQ ID NO: 12.

Preferably, ABCB1(C3435T), MTHFR (C677T), SLCO1B1(c.1865+4846T > C) share a common assignment instruction as set forth in SEQ ID NO:13 of the sequence Listing. "ddG" in SEQ ID NO:13 indicates that the last base ddGTP was added to the sequencing reaction of ABCB1(C3435T), and this addition of base can terminate the sequencing reaction. "ddC" in SEQ ID NO:13 indicates the last addition of base ddCTP to MTHFR (C677T) sequencing reaction, and "-" in SEQ ID NO:13 indicates the pause of reagent addition for 3 min. During this pause, the next gene sequencing primer will be added.

Preferably, the reagent 1 comprises: amplification buffer, 18mM magnesium acetate;

preferably, the reagent 2 comprises: ABCB1(C3435T) rear primer (0.32uM), ABCB1(C3435T) rear primer (0.32uM), MTHFR (C677T) front primer (0.32uM), MTHFR (C677T) rear primer (0.32uM), SLCO1B1(c.1865+4846T > C) front primer (0.32uM), SLCO1B1(c.1865+4846T > C) rear 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), recombinase binding single-stranded nucleic acid (4.8 ng/. mu.L), trehalose (0.2%); the simultaneous amplification of ABCB1(C3435T), MTHFR (C677T), SLCO1B1(c.1865+4846T > C) can be performed under isothermal conditions.

Preferably, the reaction volume is 25ul, and the reaction conditions are as follows: 25min at 39 ℃.

Preferably, the positive control comprises ABCB1(C3435T), MTHFR (C677T) and SLCO1B1(c.1865+4846T > C) hybrid 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 method for detecting the gene polymorphism related to methotrexate drug dosage and adverse reaction prediction by adopting the kit, which comprises the following steps:

a. amplifying the amplification reaction solution and 5ul of genome DNA to be detected by adopting multiple RPA amplification;

b. binding the binding solution (containing the microbeads) with the amplification product;

c. treating the denatured liquid to obtain a single-chain product;

d. adding a washing buffer solution for rinsing;

e. adding a sequencing enzyme and a sequencing substrate to each sequencing tube;

f. taking an 8-calandria, and sequentially adding dATP, dTTP, dGTP, dCTP, ABCB1(C3435T) sequencing primer, MTHFR (C677T) sequencing primer, SLCO1B1(c.1865+4846T > C) sequencing primer, ddGTP and ddCTP from one round smooth end to the 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;

g. pyrosequencing;

h. determining the genotypes of the ABCB1(C3435T) site, MTHFR (C677T) site, SLCO1B1(c.1865+4846T > C) site methotrexate drug dose and adverse reaction site.

The invention also discloses a kit for predicting methotrexate drug dosage and adverse reaction and application of the method, wherein the detection kit is used for detecting ABCB1(C3435T), MTHFR (C677T) and SLCO1B1(c.1865+4846T > C) so as to guide methotrexate drug dosage and adverse reaction prediction from a gene level.

The target single-stranded DNA containing the biotin label is captured by streptavidin through Recombinase Polymerase Amplification (RPA) one-tube amplification of ABCB1(C3435T), MTHFR (C677T) and SLCO1B1(c.1865+4846T > C). After rinsing the product, firstly adding an ABCB1(C3435T) sequencing primer and a sequencing raw material to perform pyrosequencing, and adding ddNTP to the last base to terminate the sequencing reaction; then adding MTHFR (C677T) sequencing primer and corresponding dNTP for sequencing, and adding ddNTP to the last base to terminate the sequencing reaction; and finally, adding an SLCO1B1(c.1865+4846T > C) sequencing primer and corresponding dNTP for sequencing, carrying out sequencing on three sites in sequence by one-time sequencing treatment, and reducing the operation time and improving the sequencing flux.

Compared with the prior art, the kit provided by the invention detects the methotrexate drug dosage and adverse reaction prediction related gene polymorphism by taking multiple RPA amplification and optimized pyrosequencing technology as a combination, can simultaneously detect ABCB1(C3435T), MTHFR (C677T) and SLCO1B1(c.1865+4846T > C) gene polymorphisms of the methotrexate drug, and provides a gene angle suggestion for clinical personalized medication.

Drawings

FIG. 1 is a diagram showing an example of the sequencing results of ABCB 13435 CC, MTHFR 677CC, SLCO1B1 c.1865+4846TT type provided by the invention;

FIG. 2 is a diagram showing an example of the sequencing results of ABCB 13435 CT, MTHFR 677CT and SLCO1B1 c.1865+4846TC provided by the present invention;

FIG. 3 is an exemplary diagram of ABCB 13435 TT, MTHFR 677TT, SLCO1B1 c.1865+4846CC type sequencing results provided by the invention.

Detailed Description

The following examples are provided to further detail and fully illustrate the detection kit for methotrexate metabolic markers and the detection method and application thereof. 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.

Example 1 preparation of kit

The kit provided by the invention designs specific amplification primers and sequencing primers aiming at ABCB1(C3435T), MTHFR (C677T) and SLCO1B1(c.1865+4846T > C) and is used for isothermal amplification and pyrosequencing detection. The design of the primer based on the recombinase polymerase amplification technology is one of the keys of the invention, and the primer design of the technology cannot be carried out by auxiliary software and only depends on manual design. In order to ensure the amplification speed and the detection sensitivity, the length of the primer should be controlled to be 30-35 bp, the non-specific amplification is increased easily to cause false positive if the primer is designed to be too short, and the amplification cannot be performed easily if the primer is designed to be too long. Gene polymorphism sequences are subject to published sequences in Genebank.

The primer sequences of this example are as follows:

(II) the detection kit of the embodiment comprises the following components:

(III) the detection kit reagent 1 of the embodiment is prepared by the following single-person preparation system:

composition (I)	Volume (ul)
		Amplification buffer	18.8
300mM magnesium acetate	1.2

(IV) the detection kit reagent 2 of the embodiment is configured by the following single-person system:

the concentration of each component of the reagent 2 is as follows: ABCB1(C3435T) front primer (0.32uM), ABCB1(C3435T) rear primer (0.32uM), MTHFR (C677T) front primer (0.32uM), MTHFR (C677T) rear primer (0.32uM), SLCO1B1(c.1865+4846T > C) front primer (0.32uM), SLCO1B1(c.1865+4846T > C) (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%);

after the preparation, 115.5 ul/tube is subpackaged and freeze-dried.

Example 2 detection of Pyrophosphoric acid

The apparatus used in the present invention is as follows: a thermostat;

pyrophosphoric acid sequencer: wuhan Firstet Biotech, Inc.

(1) Reagent preparation (reagent preparation Chamber)

The reagent was removed in advance and reagent 1 was vortexed for 15 seconds and centrifuged at low speed until use. 440ul of reagent 1 was added directly to reagent 2 (lyophilized) and mixed well by vortexing for 15 seconds. And determining the reaction number N, wherein N is the number of samples to be detected (N), the number of quality control products (1) and a blank control. It is recommended that positive control and blank control analyses be performed simultaneously for each PCR experiment. Then, the reaction solution was dispensed into a PCR reaction tube at a volume of 20. mu.L/tube.

(2) Application of sample detection (sample preparation room)

Adding the sample DNA, the positive control and the blank control into a PCR reaction tube according to the sample adding amount of 5 mu L, covering the tube cover tightly, centrifuging at low speed for 15 seconds to completely throw liquid on the tube wall to the tube bottom, and then immediately carrying out PCR amplification reaction.

(3) PCR amplification (between amplifications)

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
			39℃	15min	1

(4) Pyrophosphoric acid sequencing

1) Adding 40 mu L of binding solution and 3ul of agarose gel particles into a PCR reaction tube, adding 10 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;

2) centrifuging at 7,000 Xg for 1min, and discarding the supernatant;

3) 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.

4) To the EP tube, 150uL of washing buffer was added, and centrifuged at 7,000 Xg for 1 min. (repeat 3 times)

5) The single stranded product from the EP tube was transferred to sequencing tubes and 3uL of sequencing enzyme and 3uL of sequencing substrate was added to each sequencing tube.

6) Respectively adding 3uL sequencing enzyme and 3uL sequencing substrate into a sequencing tube;

7) taking an 8-calandria, and adding dATP, dTTP, dGTP, dCTP, ABCB1(C3435T) sequencing primer, MTHFR (C677T) sequencing primer, SLCO1B1(c.1865+4846T > C) sequencing primer, ddGTP and ddCTP from one round 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.

8) Pyrosequencing; the detection results are shown in FIGS. 1 to 3.

(5) Interpretation of results

1) And (3) judging the effectiveness:

the blank control of the kit does not pass, and the detection results of the positive control are ABCB1(C3435T), MTHFR (C677T) and SLCO1B1(c.1865+4846T > C).

2) Criteria for determination of results

In the DNA sequencing peak map of ABCB1(C3435T),

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, namely TT type

SLCO1B1(c.1865+4846T > C) in a DNA sequencing peak map,

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;

the frequency of 40% to T is less than or equal to 60%, and the frequency of 40% to C is less than or equal to 60%, which is CT type;

the frequency of C is not less than 90 percent, the frequency of T is not less than 10 percent, namely the type CC

Example 3 correlation between Gene detection results and methotrexate drug dose and adverse reactions

Correspondence between gene locus and drug

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.

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Claims (10)

1. The kit is used for detecting the genetic polymorphisms of methotrexate metabolic markers ABCB1(C3435T), MTHFR (C677T) and SLCO1B1(c.1865+4846T > C), and comprises the following components: ABCB1(C3435T) amplification primer, ABCB1(C3435T) sequencing primer, MTHFR (C677T) amplification primer, MTHFR (C677T) sequencing primer, SLCO1B1(c.1865+4846T > C) amplification primer, SLCO1B1(c.1865+4846T > C) sequencing primer and positive control.

2. The detection kit for the methotrexate metabolic marker according to claim 1, wherein the ABCB1(C3435T) amplification primer is shown as a sequence table SEQ ID NO 1-2.

3. The detection kit for the methotrexate metabolic marker according to claim 1, wherein the MTHFR (C677T) sequencing primer is shown in a sequence table SEQ ID NO. 3-4.

4. The detection kit for the methotrexate metabolic marker according to claim 1, wherein the SLCO1B1(c.1865+4846T > C) amplification primer is shown as a sequence table SEQ ID NO. 5-6.

5. The detection kit for the methotrexate metabolic marker according to claim 1, wherein the ABCB1(C3435T) sequencing primer is shown as a sequence table SEQ ID NO:7, the MTHFR (C677T) sequencing primer is shown as a sequence table SEQ ID NO:8, and the SLCO1B1(c.1865+4846T > C) sequencing primer is shown as a sequence table SEQ ID NO: 9.

6. The detection kit for the methotrexate metabolic marker according to claim 1, wherein the sequencing primer is a conjugate of agarose gel particles and amino-labeled DNA sequences.

7. The detection kit for the methotrexate metabolic marker according to claim 1, which further comprises an amplification buffer, 18mM magnesium acetate, dNTPS, strand displacement DNA polymerase, single-stranded DNA binding protein, recombinase binding to single-stranded nucleic acid and trehalose.

8. The kit for detecting a methotrexate metabolic marker according to claim 1, wherein the positive control comprises ABCB1(C3435T), MTHFR (C677T), SLCO1B1(c.1865+4846T > C) hybrid genomic DNA with the concentration of 20 ng/ul.

9. A detection kit using the methotrexate metabolic marker according to any one of claims 1 to 8, wherein the detection method comprises the following steps:

a. amplifying the amplification reaction solution and 5ul of genome DNA to be detected by adopting multiple RPA amplification;

b. binding the binding solution (containing the microbeads) with the amplification product;

c. treating the denatured liquid to obtain a single-chain product;

d. adding a washing buffer solution for rinsing;

e. adding a sequencing enzyme and a sequencing substrate to each sequencing tube;

f. taking an 8-calandria, and sequentially adding dATP, dTTP, dGTP, dCTP, ABCB1(C3435T) sequencing primer, MTHFR (C677T) sequencing primer, SLCO1B1(c.1865+4846T > C) sequencing primer, ddGTP and ddCTP from one round smooth end to the flat end;

g. pyrosequencing;

h. determining the genotypes of the ABCB1(C3435T) site, MTHFR (C677T) site, SLCO1B1(c.1865+4846T > C) site methotrexate drug dose and adverse reaction site.

10. The application of the detection kit for the methotrexate metabolic marker according to any one of claims 1 to 8, wherein the detection kit is used for in vitro detection of ABCB1(C3435T), MTHFR (C677T) and SLCO1B1(c.1865+4846T > C) gene polymorphisms in a sample to be detected.

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