CN113549681A

CN113549681A - Detection kit for tamoxifen metabolic marker, detection method and application thereof

Info

Publication number: CN113549681A
Application number: CN202110671045.1A
Authority: CN
Inventors: 刘丹; 易倩春
Original assignee: Hunan Feisite Precision Medical Technology Co ltd
Current assignee: Hunan Feisite Precision Medical Technology Co ltd
Priority date: 2021-06-17
Filing date: 2021-06-17
Publication date: 2021-10-26

Abstract

The invention discloses a detection kit for tamoxifen metabolic markers, a detection method and application thereof, wherein the detection kit is used for detecting the gene polymorphism of the tamoxifen metabolic markers CYP2D6(G1846A) and CYP19A1(rs4646C > A), and comprises the following components: CYP2D6(G1846A) amplification primer, CYP2D6(G1846A) sequencing primer, CYP19A1(RS4646C > A) amplification primer, CYP19A1(RS4646C > A) sequencing primer and positive control. The invention adopts multiple RPA amplification and optimized pyrosequencing technology as a combination to detect the gene polymorphism related to tamoxifen curative effect and adverse reaction risk prediction, and the kit can simultaneously detect CYP2D6(G1846A) and CYP19A1(rs4646C > A) gene polymorphism and provides a gene angle suggestion for clinical personalized medication.

Description

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

Technical Field

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

Background

Tamoxifen competes with estrogen for binding to estrogen receptors, thereby inhibiting proliferation of breast cancer cells, and is widely applied to treatment of estrogen receptor positive breast cancer. Tamoxifen acts mainly through its active metabolites 4-hydroxytamoxifen and indoxifen, and its active products have over 100 times of activity in inhibiting cell proliferation as compared with tamoxifen. A decrease in CYP2D6 activity may result in a decrease in the efficacy of tamoxifen. Clinically, the treatment effect and the adverse reaction are obviously different even if a patient receives the same medicament with the same dosage. Such differences among individuals are associated with polymorphisms of metabolic enzymes encoding drugs and drug transporter genes, in addition to non-genetic factors such as age, organ function, drug interaction, and the like.

CYP2D6 is also known as isoquinoidine 4' -hydroxylase, an important member of the CYP second subfamily. The CYP2D6 activity in the population is manifested by a four-state distribution of strong (EM), Intermediate (IM), weak (PM) and ultra-strong (UM) metabolites. More than 70 genetic variations of the CYP2D6 gene have been found. Different mutation types have varying effects on enzyme activity and drug metabolism. Polymorphism in the absence of CYP2D6 enzyme activity can affect the in vivo metabolism of antipyrine, codeine, beta blockers such as metoprolol and carvedilol, chlorimipramine, nortriptyline, desipramine, doxepin, imipramine, maprotiline, opipramol, trimetmipramine, ondansetron, tramadol, tamoxifen, and the like, thereby affecting the curative effect and adverse reaction of these drugs, and clinical adjustment of dosage according to individual genotypes the US FDA recommends CYP2D6 genotype testing on estrogen receptor positive breast cancer patients before receiving tamoxifen therapy to ensure the curative effect of the drugs. The enzymes encoded by the CYP19a1 gene are members of the cytochrome P450 superfamily, which catalyse many reactions involved in drug metabolism, cholesterol, steroids and other lipid synthesis. The CYP19a1 protein, which is localized to the endoplasmic reticulum, also known as estrogen synthase, is the last step in catalyzing estrogen biosynthesis.

At present, there are many methods for detecting gene polymorphism, such as direct sequencing, chip method, high-resolution melting curve method, allele-specific amplification method, taqman fluorescence probe method, etc. 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 tamoxifen metabolic marker, and a detection method and application thereof.

In order to realize one of the above purposes, the technical scheme of the detection kit for the tamoxifen 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 the polymorphism of CYP2D6(G1846A) and CYP19A1(rs4646C > A), and comprises the following components: reagent 1 to reagent 5.

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

preferably, the sequence of the specific primer group of CYP2D6(G1846A) is shown in sequence tables SEQ ID NO 1-SEQ ID NO 2; the sequence of the specific primer group of CYP19A1(rs4646C > A) is shown in sequence tables SEQ ID NO. 3-SEQ ID NO. 4.

Preferably, the CYP2D6(G1846A) sequencing primer and the CYP19A1(rs4646C > A) sequencing primer are respectively shown as SEQ ID NO: 5-SEQ ID NO:6 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 CYP2D6(G1846A) sequencing primer is a to-be-detected sequence of CYP2D6(G1846A), and is shown as SEQ ID NO:7 in a sequence table; the sequencing region corresponding to the CYP19A1(rs4646C > A) sequencing primer is a sequence to be detected by CYP19A1(rs4646C > A), and is shown as a sequence table SEQ ID NO: 8.

Preferably, CYP2D6(G1846A) and CYP19A1(rs4646C > A) share a dispensing instruction as shown in sequence Listing SEQ ID NO: 9. "ddT" in SEQ ID NO 9 indicates that the last base ddTTP added to the CYP2D6(G1846A) sequencing reaction terminates the sequencing reaction. The "-" in SEQ ID NO 9 indicates that the addition of the reagent was suspended for 3 min. During this pause CYP19A1(rs4646C > A) sequencing primers will be added.

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

preferably, the reagent 2 comprises: CYP2D6(G1846A) rear primer (0.32uM), CYP2D6(G1846A) rear primer (0.32uM), CYP19A1(rs4646C > A) front primer (0.32uM), CYP19A1(rs4646C > A) 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 to single-stranded nucleic acid (4.8 ng/. mu.L), trehalose (0.2%); the simultaneous amplification of CYP2D6(G1846A)/CYP19A1(rs4646C > A) 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 CYP2D6(G1846A) and CYP19A1(rs4646C > A) hybrid type genomic DNA with the 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 the tamoxifen curative effect and adverse reaction risk 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, a CYP2D6(G1846A) sequencing primer, a CYP19A1(rs4646C > A) sequencing primer and ddTTP from the round and 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 CYP2D6(G1846A) site and the CYP19A1(rs4646C > A) site.

The invention also discloses a kit for predicting the curative effect and adverse reaction risk of tamoxifen and application of the method, wherein the detection kit is used for detecting CYP2D6(G1846A) and CYP19A1(rs4646C > A) so as to guide the prediction of the curative effect and adverse reaction risk of tamoxifen from a gene level.

The invention uses Recombinase Polymerase Amplification (RPA) to amplify CYP2D6(G1846A) and CYP19A1(rs4646C > A) in one tube, and uses streptavidin to capture the target single-stranded DNA. After washing, adding a CYP2D6(G1846A) sequencing primer and a sequencing raw material to perform pyrosequencing, and adding ddTTP to the last base to terminate the sequencing reaction. Then adding CYP19A1(rs4646C > A) sequencing primer and corresponding dNTP for sequencing. The sequencing of two sites is carried out in sequence by one treatment, so that the operation time is reduced and the sequencing flux is improved.

Compared with the prior art, the kit provided by the invention detects the gene polymorphism related to tamoxifen curative effect and adverse reaction risk prediction by taking multiple RPA amplification and optimized pyrosequencing technologies as a combination, can simultaneously detect CYP2D6(G1846A) and CYP19A1(rs4646C > A) gene polymorphisms, and provides a gene angle suggestion for clinical personalized medication.

Drawings

FIG. 1 is an exemplary graph of the CC-type sequencing result of CYP2D6(G1846A) GG/CYP19A1(rs4646C > A) provided by the present invention;

FIG. 2 is a diagram showing an example of the sequencing result of CYP2D6(G1846A) GA/CYP19A1(rs4646C > A) CA type provided by the present invention;

FIG. 3 is an exemplary graph of the sequencing results of CYP2D6(G1846A) AA/CYP19A1(rs4646C > A) AA provided by the present invention.

Detailed Description

The following embodiments are provided to further detail and fully illustrate the detection kit for tamoxifen metabolic markers, the detection method thereof, and the 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 CYP2D6(G1846A) and CYP19A1(rs4646C > A) 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 final concentration of each component of the reagent 2 is as follows: CYP2D6(G1846A) pre-primer (0.32uM), CYP2D6(G1846A) post-primer (0.32uM), CYP19A1(rs4646C > A) pre-primer (0.32uM), CYP19A1(rs4646C > A) 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), recombinase binding to single-stranded nucleic acid (4.8 ng/. mu.L), trehalose (0.2%);

composition (I)	Volume (ul)
		Recombinase binding single-stranded nucleic acid (100 ng/. mu.L)	1.2
Single-stranded DNA binding protein (100 ng/. mu.L)	0.8
		Strand Displacement DNA polymerase (100 ng/. mu.L)	0.3
dNTPs(25mM)	0.3
		CYP2D6(G1846A) Pre-primer (20. mu.M)	0.4
CYP2D6(G1846A) rear primer (20. mu.M)	0.4
		CYP19A1(rs4646C>A) Front primer (20. mu.M)	0.4
CYP19A1(rs4646C>A) Rear primer (20. mu.M)	0.4
		Trehalose (20%)	0.25

After the preparation is finished, 98 ul/tube is subpackaged and freeze-dried.

Example 2 detection of Pyrophosphoric acid

The apparatus used in the present invention is as follows: thermostats, pyrosequencing instruments (Wuhan Firster 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℃	25min	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, a CYP2D6(G1846A) sequencing primer, a CYP19A1(rs4646C > A) sequencing primer and ddTTP 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.

8) Pyrosequencing; the sequencing 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 result of the positive control is CYP2D6(G1846A) and CYP19A1(rs4646C > A).

2) Criteria for determination of results

In the DNA sequencing peak map of CYP2D6(G1846A),

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;

the frequency of 40% to G is less than or equal to 60%, and the frequency of 40% to A is less than or equal to 60%, which is GA type;

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;

b. CyP19A1(rs4646C > A) DNA sequencing peak map,

the frequency of C is not less than 90 percent, the frequency of A is not less than 10 percent, and the model is CC;

the frequency of 40% to C is 60% and the frequency of 40% to A is 60%, this is CA type;

the frequency of A is larger than or equal to 90 percent, the frequency of C is smaller than or equal to 10 percent, and the product is AA type.

Example 3 correlation between gene detection results and tamoxifen efficacy and adverse reaction risk prediction

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.

Sequence listing

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Claims

1. A detection kit for tamoxifen metabolic markers is characterized in that the detection kit is used for detecting the gene polymorphism of the tamoxifen metabolic markers CYP2D 6G 1846A and CYP19A1rs4646C > A, and the kit comprises the following components: CYP2D 6G 1846A amplification primer, CYP2D 6G 1846A sequencing primer, CYP19A1RS4646C > A amplification primer, CYP19A1RS4646C > A sequencing primer and positive control.

2. The detection kit for the tamoxifen metabolic marker according to claim 1, wherein the CYP2D 6G 1846A amplification primer is shown in sequence tables SEQ ID NO 1-2.

3. The detection kit for the tamoxifen metabolic marker according to claim 1, wherein the CYP19A1RS4646C > A amplification primer is shown in sequence table SEQ ID NO 3-4.

4. The detection kit for the tamoxifen metabolic marker according to claim 1, wherein the sequencing primer CYP2D 6G 1846A is shown in sequence table SEQ ID NO:5, and CYP19A1RS4646C > A is shown in sequence table SEQ ID NO: 6.

5. The detection kit for a tamoxifen metabolism marker according to claim 1, wherein the detection kit for a statin therapeutic effect prediction according to claim 1, wherein the sequencing primer is a conjugate of agarose gel particles and amino-labeled DNA sequences.

6. A test kit for a tamoxifen metabolic marker according to claim 1, wherein said test kit for statin therapeutic effect prediction according to claim 1 further comprises amplification buffer, 18mM magnesium acetate, dNTPS, strand displacement DNA polymerase, single stranded DNA binding protein, recombinase binding to single stranded nucleic acid and trehalose.

7. The detection kit for the tamoxifen metabolism marker according to claim 6, wherein the detection kit for the statin curative effect prediction according to claim 1 is characterized in that the final concentrations of the components in the kit are as follows: 0.32uM for each primer before and after CYP2D 6G 1846A amplification, 0.32uM for each primer before and after CYP19A1RS4646C > A amplification, 0.3mM of dNTPS, 1.2 ng/muL of strand displacement DNA polymerase, 3.2 ng/muL of single-stranded DNA binding protein, 4.8 ng/muL of recombinase for binding single-stranded nucleic acid and 0.2% of trehalose.

8. The tamoxifen metabolism marker detection kit of claim 1, wherein the positive control comprises CYP2D 6G 1846A and CYP19A1RS4646C > A heterozygous genomic DNA at a concentration of 20 ng/ul.

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

b. combining 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;

f. taking an 8-row pipe, and sequentially adding dATP, dTTP, dGTP, dCTP, dTTP and dTTP from one round smooth end to the flat end,

CYP2D 6G 1846A sequencing primer, CYP19A1rs4646C > A sequencing primer, ddTTP;

g. pyrosequencing;

h. determining the genotypes of the CYP2D 6G 1846A site and the CYP19A1rs4646C > A site.

10. Use of the tamoxifen metabolic marker detection kit according to any one of claims 1 to 8, wherein the detection kit is used for in vitro detection of CYP2D 6G 1846A and CYP19A1RS4646C > A gene polymorphisms in a sample to be detected.