CN112410427A

CN112410427A - Detection method of tamoxifen metabolic marker and application thereof

Info

Publication number: CN112410427A
Application number: CN202011298778.7A
Authority: CN
Inventors: 黄韫平; 刘丹; 叶婷
Original assignee: Best Guangdong Precision Medical Technology Co Ltd
Current assignee: Best Guangdong Precision Medical Technology Co Ltd
Priority date: 2020-11-19
Filing date: 2020-11-19
Publication date: 2021-02-26

Abstract

The invention discloses a detection method of a tamoxifen metabolic marker and application thereof, wherein the tamoxifen metabolic marker is CYP2D6 x 5, and influence of polymorphism of the tamoxifen metabolic marker on tamoxifen metabolism is detected. The invention adopts a two-round amplification method to repeatedly amplify the same segment, and the second round of primers are combined in the first PCR product, so that the second PCR amplified segment is shorter than the first amplification, the amplification specificity is very high, the pollution of non-specific amplification caused by weak primer pairing specificity is basically avoided, the segment to be amplified is efficiently and accurately amplified, the CYP2D6 x 5 genotype detection is carried out, the conclusion can be rapidly given, and the clinical prediction of the curative effect of the tamoxifen is convenient.

Description

Detection method of tamoxifen metabolic marker and application thereof

Technical Field

The invention relates to a detection method of a tamoxifen metabolic marker and application thereof, belonging to the field of biology.

Background

The present situation of tamoxifen as a breast cancer marker

The auxiliary endocrine treatment is an important mode in the comprehensive treatment of the breast cancer, and the chemotherapy of the breast cancer is closely related to the hormone level in the body. Tamoxifen is a chemically synthesized non-steroidal anti-estrogen anti-cancer drug, is mainly used for clinically treating advanced breast cancer, and is listed as a first-line drug of a palliative therapy for the advanced breast cancer of menopausal women abroad. In the clinical treatment of breast cancer in China at present, estrogen receptors (PR) are still mainly used as the medication basis for endocrine treatment and prognosis evaluation of breast cancer, and the influence of genotypes on drug treatment is rarely considered.

Tamoxifen (TAM), also known as Tamoxifen, is an artificially synthesized non-cholesterol antiestrogen drug, and is a basic drug for endocrine adjuvant therapy of breast cancer. After the TAM is combined with an estrogen receptor, the three-dimensional space configuration of the receptor is induced and changed, the receptor is prevented from being combined with related reaction components of estrogen on DNA, the processes of transcription and the like cannot be normally carried out, the estrogen is prevented from playing a role, the growth of hormone-dependent breast tumor cells is inhibited, and the effect of treating breast cancer is achieved. Meanwhile, TAM is a prodrug, has low affinity with an Estrogen Receptor (ER) in vivo, and needs to be activated by P450 metabolism to generate 4-hydroxy-N-demethyltamoxifen (Endoxifen) and N-demethyltamoxifen to play a role, wherein the Endoxifen has the strongest therapeutic activity.

TAM is widely used for treating breast cancer patients, but when the same drug dose is taken by different patients, individual differences of drug effect and toxicity can be caused, and dangerous adverse reactions can be caused to a few patients. This individualized difference cannot be completely explained by liver and kidney function, age, lifestyle or combined medication and patient compliance, where genetic factors play an important determinant.

Second, CYP2D6 family introduction

Cytochrome CYP450 is a group of isozymes encoded by superfamily genes related to structure and function, widely exists in organisms in the nature, such as animals, plants, fungi and bacteria, has the main function of catalyzing the biotransformation of drugs, environmental chemicals and natural substances by means of metabolism modes such as oxidation, hyper-oxidation and reduction, and has the main function of transferring a single oxygen atom from external oxygen molecules into the metabolized substances. There are three members of the human CYP2D family of genes: CYP2D6, CYP2D7 and CYP2D8P genes. The three genes are arranged in sequence on chromosome 22. Each gene has 9 exons, and the length of the nucleotide sequence is 7 Kb. The CYP2D6 gene is a gene with complete function, can express a functional CYP2D6 protein, and is an enzyme playing an important role in human bodies. The CYP2D7 gene is 97% base similar to CYP2D6, and although it has TATA box, it has modified reading frame due to the insertion of a thymic sucking (T) at position 226 of exon I, so that translation cannot proceed normally, therefore, functional protein cannot be expressed. The CYP2D8P gene is a pseudogene, is a chimeric gene, consists of a part of CYP2D7AP and a part of CYP2D6, and although only 14 bases are different from CYP2D6, functional protein cannot be expressed.

Human CYP2D6 is an important metabolic enzyme of cytochrome P450, the content of the important metabolic enzyme accounts for the fifth place in human liver CYP450 enzyme system, about ten types of medicines metabolized by CYP2D6 exist, more than 80 medicines account for 18.8% of clinically important medicines, and the main medicines are as follows: beta receptor blockers, alpha receptor blockers, antiarrhythmics, analgesics, 5-HT antagonists, antipsychotics, antitussives and antiasthmatics, hypoglycemic agents, tricyclic antidepressants and other antidepressants. And simultaneously participate in the catalytic metabolism of other exogenous and endogenous compounds. 95% of environmental carcinogens are indirect (pro) carcinogens, and many exogenous compounds are catalyzed by liver cytochrome P450 to form light electron intermediates, which damage DNA and thus initiate carcinogenesis or mutagenic processes.

The CYP2D6 gene is located on 22 # autosome and is recessive inheritance, and has more than 70 variant alleles, thereby causing the obvious individual difference of the organism on the curative effect of the medicament. CYP2D6 consists of 497 amino acids with 100 allelic variations. These variations can produce differential changes in the enzymatic activity in vivo, which are classified into 4 types: slow metabolism (PM), Intermediate Metabolism (IM), fast metabolism (EM) and ultra-fast metabolism (UM). The PM allele has no CYP2D6 enzyme activity and no protein expression, the relevant alleles are: 3/' 4/' 5/' 6; IM allele CYP2D6 enzyme activity was low and could be impaired; normal enzymatic activity of EM allele CYP2D 6; the UM allele CYP2D6 enzyme activity was highest.

The incidence of the CYP2D6 metabolic phenotype varies from race to race and from region to region. Frequency of PM type: 15% of Nielia, 12% of Canadian, 9% of English, 1.5% of Egypt and 1% of Chinese. In addition, 1-2% of caucasians are UM, and 29% of Heiphora nigra are UM. Because of this difference, people with different genetic backgrounds have different tolerance doses for some drugs.

Current situation of taking tamoxifen as breast cancer marker

TAM is widely used for treating breast cancer patients, but when the same drug dose is taken by different patients, individual differences of drug effect and toxicity can be caused, and dangerous adverse reactions can be caused to a few patients. This individualized difference cannot be completely explained by liver and kidney function, age, lifestyle or combined medication and patient compliance, where genetic factors play an important determinant. Fourth, the influence of genetic factors on tamoxifen metabolism

The genetic polymorphism of CYP2D6 has been shown to have an effect on the metabolism of tamoxifen and is a key enzyme catalyzing TAM to produce Endoxifen. Since the detection of 1 or a few polymorphic sites alone cannot determine the CYP2D6 genotype or the CYP2D6 active metabolic pattern, many commercially available kits evaluate the active metabolic pattern after detecting multiple sites. For example, CYP2D6 Gene polymorphism detection kit of Luminks trade Co., Ltd

The BaiO genotype detection chip kit and the like of Shanghai Baiao science and technology Limited all detect various genotypes. However, due to the difference of the genotype region and species, such multi-site detection is not completely applicable to Asians.

Through statistical arrangement, the polymorphic sites with the CYP2D6 frequency of more than 1% in Asian population have: 2:2850C > T (normal function), 5: gene deletion (no function), 10:100C > T (reduced function), 36:100C > T and partial fragment of CYP2D7 (no function), 41:2850C > T and 2988A (reduced function). Rs1065852, also known as CYP2D6 x 10(c.100c > T), is the most common mutation in asian populations, with mutation frequencies of 30-50% in chinese. Retrospective studies in china have shown that female breast cancer patients carrying the CYP2D6 x 10 allele have lower plasma concentrations of 4-hydroxy-tamoxifen, which inhibits the treatment of TAM. Another study of 72 patients with early stage breast cancer treated with TAM showed that homozygous mutants with T/T genotype were a negative prognostic factor for young breast cancer patients using TAM. In addition to CYP2D6 x 10, another mutation with a higher frequency for chinese was CYP2D6 x 5, which was an allelic deletion. The deletion results in deletion of the CYP2D6 gene, thereby failing to produce CYP2D6 enzyme to metabolize TAM.

In conclusion, most of the existing kits are CYP2D6 gene detection kits, multiple sites need to be detected, the detection period is long, the cost is high, and the detection as the tamoxifen metabolic marker is high in accuracy but not beneficial to clinical popularization. The selection of metabolic markers is preferably representative and of a smaller number of mutation types. At present, the existing patents disclose the polymorphism detection of CYP2D6 × 10, for example, chinese patent CN108070659A discloses the application of an SNP marker in predicting the curative effect of TAM assisted endocrine therapy on breast cancer patients, wherein the gene polymorphism site of CYP2D6 × 10 is used as a detection target, and the genotype of rs1080989 site is detected to predict the curative effect of TAM assisted therapy on breast cancer patients. Another Chinese patent CN104862308A also discloses a kit for guiding the administration of tamoxifen, wherein the C > T mutation (c.100C > T) of the 100 th base of CYP2D6 gene and the deletion (also called 775delA mutation) of the base A are used as detection sites. The two kits mainly take the gene polymorphism site of CYP2D6 x 10 as a detection target, but the gene polymorphism site of CYP2D6 x 10 can only weaken the function of CYP2D6 gene, and can not determine whether the mutant is in a slow metabolism type or not. Therefore, merely detecting the genetic polymorphism of CYP2D6 x 10 is not a marker for determining the metabolic rate of tamoxifen.

Among the CYP2D6 × 5 genotypes, there may be found: *5/*5: slow metabolism PM,/5/2: normal metabolism EM, # 5/# 10: medium metabolic EM, # 5/# 36: slow metabolism PM, 5/' 41: medium metabolizing EM, therefore, detecting x 5 alone, one can determine if it is of the x 5/x 5 slow metabolizing type. And whether the detected tamoxifen is slow metabolism or not is judged, and the judgment on the metabolic capability of the tamoxifen has a direct guiding effect.

Disclosure of Invention

In view of the above problems in the prior art, the present invention aims to provide a method for detecting a tamoxifen metabolic marker using CYP2D6 × 5 as the tamoxifen metabolic marker, and applications thereof.

In order to realize the purpose, the technical scheme of the method for detecting the tamoxifen metabolic marker adopted by the invention is as follows:

the tamoxifen metabolic marker is CYP2D6 x 5, the influence of polymorphism on tamoxifen metabolism is detected, the detection method adopts two rounds of PCR reactions to amplify target fragments, wherein a first round of fragments to be amplified is longer than a second round of fragments to be detected and contains the second round of fragments to be amplified, the second round of amplification is carried out by taking a first round of amplification products as a template, and a first round of amplification primer in the detection method is shown as SEQ ID NO: 1-2, wherein the second round amplification primer is shown as a sequence table SEQ ID NO: 3 to 4.

The primer sequence provided in the detection method is only the most basic primer sequence, the primer sequence can be modified according to different final detection means in the detection process, such as adding a fluorescent label, a biotin label, a nano microsphere, a magnetic bead and the like into the primer, the modification method can adopt a conventional method in the field, and the modification methods are considered to fall into the protection scope of the invention.

The detection sample can be selected from a blood sample or an oral swab, the gene screening has no pain and no side effect, the genetic basis can be rapidly and accurately provided for clinical medication of tamoxifen, and the clinical medication and prognosis tracking are facilitated.

Preferably, the amplification product of the present invention can be subjected to Pyrosequencing to rapidly determine whether a mutation has occurred, wherein the sequencing is performed by referring to the method steps in "Determination of CYP2D6 Gene Copy Number by Pyrosequencing" (PMID: 15650034). Sequencing takes CYP2D7 or CYP2D8 as a control reference, and no absorption peak of a corresponding site appears in a sequencing result, thus indicating that the CYP2D6 gene has no copy.

Preferably, the DNA template in the detection method is selected from the group consisting of the full sequence or partial sequence of the CYP2D6 x 5 gene comprising the first round of amplification. Since mutations in CYP2D6 × 5 result in deletion of CYP2D6 gene, amplification of the entire sequence is not required for detection, as long as the first round of amplified fragments are included in the extracted genome.

Preferably, in the detection method, compared with the first round and the second round amplification systems, the volume of the second round PCR amplification system is at least 2 times of that of the first round PCR amplification system, the primer concentration is changed in an equal proportion, but the template addition amount is the same. The amplification system can ensure the complete performance of the second round of PCR amplification.

Preferably, in order to ensure that the two rounds of amplification reactions can be completely performed, the denaturation time of the second round of PCR amplification system is prolonged, so that the template double strand can be conveniently and fully opened, the annealing temperature is slightly higher than that of the first round, but the number of cycles can be properly reduced due to the short fragments, and the specificity of the amplified product is ensured.

As a preferred embodiment, the reaction procedure of the two-round amplification system is specifically as follows: the first round of PCR amplification system is 25 μ L, the extracted genome DNA is used as a template, and the reaction procedure is 94 ℃ for 2 min; 15sec at 94 ℃, 20sec at 50 ℃ and 2min at 72 ℃ for 35 cycles, 10min at 72 ℃ and heat preservation at 4 ℃;

the second round of PCR amplification system is shown in Table 2 below, the reaction system is 50 μ L, the first round product is used as a template, and the reaction procedure is 94 ℃ for 4 min; 15sec at 94 ℃, 20sec at 55 ℃ and 2min at 72 ℃ for 30 cycles, 10min at 72 ℃ and heat preservation at 4 ℃.

As a preferred embodiment, Primix Taq enzyme is used in both PCR reactions, 10. mu.L is added in the first round and 25. mu.L is added in the second round.

The amplification product can be directly detected qualitatively by agarose gel electrophoresis, and can also be detected by DNA in other modes. When the sample is subjected to agarose gel electrophoresis, the bands of the CYP2D 6X 5 wild-type homozygote are bright and clear, the CYP2D 6X 5 heterozygote has bands, but the bands emit light weakly and are thin, and the CYP2D 6X 5 mutant homozygote cannot generate bands.

The invention also aims to provide application of the tamoxifen metabolic marker, and application of the primer specific to the metabolic marker in preparation of a kit for detecting tamoxifen metabolism.

The invention also provides a kit, which comprises a first round amplification primer and necessary reagents thereof, and a second round amplification primer and necessary reagents thereof.

Preferably, the DNA polymerase in the reagents necessary for amplification is Primix Taq enzyme.

Preferably, the kit further comprises a DNA recovery reagent.

The PharmGKB database detects CYP2D6 x 5/' 5 slow metabolism based on the medication guidelines for patients with CYP2D6 slow metabolism, with the following clinical significance:

1) tricyclic antidepressants (TCAs): TCAs should be avoided. Alternative drugs not metabolized by CYP2D6 are contemplated; if TCAs have to be used, a reduction of 50% of the starting dose is considered; dose adjustments are guided by therapeutic drug monitoring;

2) codeine: codeine should be avoided due to potential non-therapeutic effects;

3) tamoxifen: increased likelihood of recurrence, no event-decreased survival without recurrence;

4) paroxetine: an alternative drug is selected which is not primarily metabolised by CYP2D6, if paroxetine must be used, taking into account a 50% reduction in the initial dose.

Therefore, the CYP2D6 detection method and the detection kit in the application can also be used for the detection of tricyclic antidepressant, codeine and paroxetine before clinical medication, and are not limited to the medication detection of tamoxifen.

Compared with the prior art, the method adopts a two-round amplification method to repeatedly amplify the same segment, the second round of primers are combined in the first PCR product, so that the second PCR amplified segment is shorter than the first amplification, and because the probability of primer pairing and amplification on the wrong segment is extremely low, the two-round amplification can well correct the wrong segment generated in the first amplification, the amplification specificity is very high, and the pollution of non-specific amplification caused by the weak specificity of primer pairing is basically avoided. The fragment to be amplified is efficiently and accurately amplified, the CYP2D6 x 5 gene is detected by pyrosequencing, a conclusion can be quickly given, clinical prediction of the curative effect of tamoxifen is facilitated, sampling is convenient, detection is efficient, quick and low in cost, and economic and physical burdens of patients are relieved.

Drawings

FIG. 1 is a diagram showing the results of gel electrophoresis of PCR products according to the present invention;

FIG. 2 is a diagram showing pyrosequencing results of different sequencing primers of the present invention;

FIG. 3 is a diagram showing pyrosequencing results of different sequencing primers of the present invention.

Detailed Description

The following examples are provided to illustrate the method for detecting tamoxifen metabolic markers and the application thereof in further detail and in a complete way. 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.

The tamoxifen metabolic marker detection kit in the embodiment takes CYP2D6 x 5 as a detection index to detect whether the gene mutation occurs, and the kit mainly comprises a genome amplification primer, an amplification reagent and necessary materials for clinical sampling detection. The following describes the genotyping of CYP2D6 x 5 using the core-amplification primer in the kit as an example.

CYP2D6 x 5 is deletion mutation of the whole gene, and the deletion can result in deletion of CYP2D6 enzyme, so that tamoxifen cannot be catalyzed, metabolic disorder is caused, and medication failure is caused. CYP2D6 × 5 has a complicated molecular mechanism, which has prevented the development of its clinical application. However, when the tamoxifen is used as a metabolic marker, only the fact that whether the tamoxifen is a wild type, a heterozygote or a mutant type needs to be detected.

In this example, the presence or absence of the CYP2D6 × 5 gene mutation was detected by a post amplification assay, in which the DNA of the subject was collected first.

Extraction of DNA

And (3) extracting the genome DNA in the blood sample of the tested person by adopting a DNA extraction kit, and detecting the concentration and purity after extraction to ensure that A260/280 is 1.8-2.0 for later use.

Second, design of primers

Primers were designed based on the sequence of CYP2D6 gene (Reference NG _008376) recorded at NCBI, two pairs of amplification primers were designed using Primer Premier 5, and they were synthesized by Shanghai Biotechnology engineering service, Inc. The two pairs of amplification primers correspond to two PCR reactions, the second round of fragments to be amplified are contained in the first round of fragments to be amplified, and the sequences of the primers are shown in a sequence table SEQ ID NO: 1 to 4, F1 and F2 are Forward primers, and R1 and R2 are Reverse primers. The primer sequences are specifically shown in the following table 1:

TABLE 1

Third, PCR reaction and result identification

The amplification primers are shown in the table 1 above, the amplification system is shown in the table 2 below, the first round of PCR amplification system is 25 μ L, the extracted genomic DNA is used as a template, and the reaction procedure is 94 ℃ for 2 min; at 94 ℃ for 15sec, at 50 ℃ for 20sec, at 72 ℃ for 2min, for 35 cycles, at 72 ℃ for 10min, and at 4 ℃.

The two PCR reaction systems are shown in Table 2 below:

TABLE 2

The PCR product was purified and then electrophoresed through 1% agarose gel, the loading buffer contained the tracer dye, the loading amount was the same for each well, and the electrophoresis results are shown in FIG. 1. Bands of about 1000kb and around 250kb are clearly visible in the figure, and the specificity is high, wherein a band 1 is a DNAmarker, a band 2 is an amplification product of the first round, a band 3 is an amplification product of the second round, and a band 4 is a negative control. The amplification system is stable and effective, and the two pairs of primer pairs can be used for detecting CYP2D6 x 5.

Fourthly, sequencing the amplified product

The amplified products were sequenced by Pyrosequencing, the sequencing was performed by the method steps of "Determination of CYP2D6 Gene Copy Number by Pyrosequencing" (PMID: 15650034), and Pyrosequencing was performed with CYP2D7 Gene as reference, and the sequencing results are shown in FIGS. 2-3, while FIG. 2 is different from the sequencing primer of FIG. 3, but the peak pattern in FIGS. 2 and 3 shows that CYP2D6 is absent and 0 XD 6 indicates that CYP2D6 Gene has no Copy.

Fifth, clinical sampling detection

Taking a breast cancer patient in a third hospital in Wuhan city as a sampling group, performing blood drawing test on a patient with 271 list receiving Tamoxifen (TAM) treatment alone, wherein the administration dose is 20mg/D, drawing blood after 2h of administration, and detecting the blood concentration of tamoxifen and the CYP2D6 x 5 gene mutation condition in blood. And after DNA is extracted, carrying out two rounds of PCR amplification under the conditions, carrying out 1% agarose gel electrophoresis on the amplification product, and counting the result after electrophoresis of the amplification product.

The electrophoresis result is obvious under an ultraviolet lamp, the band of the CYP2D 6X 5 wild type is clear and bright, the band of the CYP2D 6X 5 heterozygote is dark, and the band of the CYP2D 6X 5 mutant is not. The amplified products are simultaneously subjected to sequencing detection so as to confirm the accuracy of the agarose gel electrophoresis result. The statistical results are shown in the following table 3:

TABLE 3

	Number of cases	TAM mean plasma concentration (ng/mL)	Average blood Endoxifen concentration (ng/mL)
				Wild type	115	195.2±88.6	220.2±61.4
Heterozygote	68	244±90.7	215.7±100.2
				Mutant forms	34	358±38.4	35.9±4.1

As can be seen from table 3, the mutants of CYP2D6 × 5 in breast cancer patients in wuhan city were small and accounted for 15.6% of the total number of spot-checks, but the mutants had a large effect on the metabolism of tamoxifen, the metabolic capacity of tamoxifen was very poor in the mutated patients, and the recurrence probability was easily increased by using only tamoxifen. Heterozygotes account for 27.1 percent of the number of the people to be randomly checked, have influence on the metabolic capability of the tamoxifen, still can have due curative effect if the tamoxifen is necessary to be used, and have lower recurrence risk. The wild type is a common normal genotype, can be used for treating by administration according to a normal dose, and has low recurrence risk.

The difference between the above experimental results has statistical significance, and the statistical results still have statistical significance in excluding the environmental factors related to breast cancer, such as pregnancy history, obesity, alcoholism and familial factors. For example, according to the influence of CYP2D6 x 5 gene polymorphism on breast cancer, after tamoxifen is used for treatment, the treatment effect is still poor, and other genetic influence factors and environmental factors are suggested to be screened.

The experimental results can only represent the experiment, and as the CYP2D6 x 5 gene has differences in different nations in different regions and breast cancer is related to various genetic factors and environmental factors, the condition of the patient should be considered as comprehensively as possible when tamoxifen is used for treatment, and a treatment scheme is formulated.

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

1. A method for detecting a tamoxifen metabolic marker is characterized by comprising the following steps: the tamoxifen metabolic marker is CYP2D6 x 5, the influence of polymorphism on tamoxifen metabolism is detected, the detection method adopts two rounds of PCR reactions to amplify target fragments, wherein a first round of fragments to be amplified is longer than a second round of fragments to be detected and contains the second round of fragments to be amplified, the second round of amplification is carried out by taking a first round of amplification products as a template, and a first round of amplification primer in the detection method is shown as SEQ ID NO: 1-2, wherein the second round amplification primer is shown as a sequence table SEQ ID NO: 3 to 4.

2. The method for detecting a tamoxifen metabolic marker according to claim 1, wherein: and sequencing the amplification product by adopting a pyrosequencing method.

3. The method for detecting a tamoxifen metabolic marker according to claim 2, wherein: sequencing controls were CYP2D7 or CYP2D 8.

4. The method for detecting a tamoxifen metabolic marker according to claim 1, wherein: the volume of the second round amplification system is 1.5-2.5 times of that of the first round amplification system, the addition amount of the amplification primers is changed in equal proportion, and the addition amount of the templates is the same.

5. The method for detecting a tamoxifen metabolic marker according to claim 2, wherein: the first round of PCR amplification system is 25 μ L, and the second round of PCR amplification system is 50 μ L.

6. The method for detecting the tamoxifen metabolic marker according to claim 1, wherein the first round of amplification reaction procedure is as follows: taking the extracted genome DNA as a template, and carrying out treatment at 94 ℃ for 2 min; at 94 ℃ for 15sec, at 50 ℃ for 20sec, at 72 ℃ for 2min, for 35 cycles, at 72 ℃ for 10min, and at 4 ℃.

7. The method for detecting the tamoxifen metabolic marker according to claim 1, wherein the second round of amplification reaction procedure is as follows: taking the first round product as a template, and keeping the temperature at 94 ℃ for 4 min; 15sec at 94 ℃, 20sec at 55 ℃ and 2min at 72 ℃ for 30 cycles, 10min at 72 ℃ and heat preservation at 4 ℃.

8. The method for detecting a tamoxifen metabolic marker according to claim 1, wherein: primix Taq enzyme was used in both PCR runs, 10. mu.L was added in the first run and 25. mu.L in the second run.

9. The application of a tamoxifen metabolic marker is characterized in that: the specific primer of the metabolic marker is applied to the preparation of a kit for tamoxifen metabolic detection.

10. A tamoxifen metabolism marker detection kit is characterized in that: the kit comprises a first round amplification primer, a second round amplification primer and DNA polymerase.