A primer combination, reagent for gene methylation detection and its application

Abstract

The present invention discloses a primer combination, reagent and application for gene methylation detection, wherein the primer combination includes primer pair combinations shown by SEQ ID NO: 1 and SEQ ID NO: 2; and / or SEQ ID NO: 16 and SEQ ID NO: 17; and / or SEQ ID NO: 22 and SEQ ID NO: 23; based on this primer combination or a gene methylation detection reagent containing this primer combination, it can effectively distinguish breast tumor and non-breast tumor individuals, facilitating the provision of an effective breast tumor diagnosis strategy. Especially when jointly detecting corresponding genes, it has significant characteristics of high specificity and high sensitivity, which is beneficial to being actually put into clinical diagnosis, so that breast tumor patients can be diagnosed and treated early, facilitating the timely formulation of corresponding treatment strategies and improving the survival rate and survival period.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more particularly, to a primer combination for gene methylation detection, a reagent and its application. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women worldwide. According to the latest data surveyed by the International Agency for Research on Cancer (IARC) in 2018, the incidence rate of breast cancer among global women is 24.2%, ranking first among female cancers, and 52.9% of which occur in developing countries. Moreover, worldwide, the incidence rate of breast cancer has been showing an increasing trend in recent years. Therefore, the development of measures for breast cancer diagnosis, treatment, etc. becomes increasingly important.

[0003] Breast cancer belongs to a type of tumor that is easy to treat and control in the early stage. If it can be detected, diagnosed, and treated early, the prognosis is generally relatively good. Clinically, some breast cancers have achieved ideal treatment effects due to early detection, but most patients have missed the best treatment opportunity due to late detection. Therefore, the timely diagnosis of breast cancer is a key factor in dealing with breast cancer currently.

[0004] Currently, although the prior art has explored various aspects of breast cancer markers, the biomarkers and diagnostic strategies available for breast cancer diagnosis are still relatively scarce. The prior art urgently needs a biomarker or biomarker combination and a diagnostic strategy for breast cancer diagnosis to fill the gap in breast cancer diagnosis technology. Summary of the Invention

[0005] The present invention aims to overcome at least one of the above-mentioned deficiencies of the prior art, and provides a primer combination for gene methylation detection, a reagent and its application, so as to provide an effective diagnostic measure for breast cancer.

[0006] One object of the present invention is to provide a primer combination, and the primer combination is a primer pair combination shown in SEQ ID NO: 1 and SEQ ID NO: 2; and / or, SEQ ID NO: 16 and SEQ ID NO: 17; and / or, SEQ ID NO: 22 and SEQ ID NO: 23. In one or more embodiments of the present invention, the methylation detection of the corresponding gene can be effectively achieved through the above primer combination, and the detection results can distinguish breast cancer from healthy samples; and when the above primer combinations are used in combination, breast tumors can be diagnosed with high specificity. Further, the complementary sequences based on the above primer sequences are also convenient for obtaining amplification primer sequences that can capture the methylation of the corresponding gene.

[0007] Another object of the present invention is to provide a multi-gene methylation combined detection reagent, wherein the genes are CTNNA2, AXIN1 and RASSF1, and the reagent includes primers for detecting the methylation of each gene; further, the primers are primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 16 and SEQ ID NO: 17, and SEQ ID NO: 22 and SEQ ID NO: 23.

[0008] With the in-depth research in the field of molecular biology, it has been found that abnormal regulation other than DNA sequences is more common in the occurrence and development of tumors. This heritable regulation that does not depend on DNA sequence changes is called epigenetic modification, which mainly includes DNA methylation, histone modification, gene imprinting, and microRNA regulation, etc. As a potential molecular biomarker and drug target, epigenetics has been increasingly emphasized in the field of tumor prevention and treatment, and one of its main forms is DNA methylation. DNA methylation refers to the process in which an organism, under the catalysis of DNA methyltransferase (DNMT), uses S-adenosylmethionine (SAM) as a methyl donor to transfer a methyl group to a specific base. DNA methylation can occur at the C-5 position of cytosine, the N-6 position of adenine, the N-4 position of cytosine, or the N-7 position of guanine, etc. DNA methylation is an early event in tumorigenesis and plays an important role in the occurrence and development of tumors. And this application constructs corresponding primers and combined detection strategies for specific target genes to obtain a new diagnostic strategy.

[0009] In one or more embodiments of the present invention, through the aforementioned multi-gene methylation combined detection, breast tumor samples can be accurately detected. Compared with some existing markers, this application provides a marker combination with extremely high detection sensitivity and specificity for breast cancer, and this type of marker combination is expected to be truly used in the clinical detection of breast cancer. At the same time, primers and probes are one of the influencing factors for the marker combination to play a role, and the above-mentioned primers and the following probes provided in this application can enable the marker combination to play a role in specific detection.

[0010] By detecting the methylation levels of the promoter regions of the CTNNA2, AXIN1 and RASSF1 gene combinations, breast cancer specimens can be well distinguished from plasma cfDNA. This application uses methylation reagents for the above gene combinations to detect breast cancer, and both the detection sensitivity and specificity for breast cancer are relatively high, both reaching more than 90%.

[0011] Furthermore, it also includes probes for detecting the methylation of each gene. The probes are beneficial to the presentation and observation of detection results. Further, the probes are a combination of the sequences shown in SEQ ID NO: 3, SEQ ID NO: 18, and SEQ ID NO: 24. In one or more embodiments of the present invention, the above probes can cooperate with the corresponding gene primer combinations to facilitate the presentation of results. Further, in at least one embodiment of the present invention, the methylation detection of the corresponding gene is achieved through primers and / or probes obtained from the CpG islands of each gene. Further, the CpG islands are the CpG islands of each gene body, intergenic region, promoter region, and / or the region near the promoter region. In one or more embodiments of the present invention, the methylation detection reagent provided by the present invention includes primers and / or probes obtained from the CpG islands of the promoter region and the region near the promoter region of each gene in the gene combination of CTNNA2, AXIN1, and RASSF1.

[0012] Further, in one or more embodiments of the present invention, the methylation of the corresponding gene is detected by qMSP (quantitative methylation specific PCR) using the primers and / or probes.

[0013] Another object of the present invention is to provide a kit containing the aforementioned primer combination or the aforementioned reagent.

[0014] Further, the kit includes: a first container containing a primer combination for amplification; a second container containing a nucleic acid probe combination.

[0015] Further, the kit also includes common reagents in the kit, such as common conversion agents in qMSP, which are used to convert non-methylated cytosine bases into uracil, while methylated cytosine bases remain unchanged. There is no particular limitation on the conversion agent, and any reagent reported in the prior art that can achieve the conversion of cytosine to uracil can be used, such as one or several of hydrazine salts, bisulfite salts, and hydrogen sulfite salts (such as sodium metabisulfite, potassium bisulfite, cesium bisulfite, ammonium bisulfite, etc.). Another example is the commonly used DNA polymerase, dNTPs, Mg2+ ions, and buffer for amplifying CTNNA2, AXIN1, and / or RASSF1 genes.

[0016] Another object of the present invention is to provide the use of the aforementioned primer combination or the aforementioned reagent in the preparation of a reagent or kit for methylation detection; further, the use in the preparation of a reagent or kit for detecting breast tumors; further, the breast tumors include breast cancer and breast adenoma. In the present invention, "detection" in detecting breast tumors is the same as diagnosis, including not only the early diagnosis of breast tumors, but also the diagnosis of the middle and late stages of breast tumors, and also includes breast tumor screening, risk assessment, prognosis, disease identification, diagnosis of disease stages and selection of therapeutic targets.

[0017] The application of the breast tumor marker combination CTNNA2, AXIN1 and RASSF1 makes the early diagnosis of breast tumors possible. When it is determined that the genes methylated in cancer cells are methylated in clinically or morphologically normal-appearing cells, this indicates that the normal-appearing cells are developing towards cancer. Therefore, breast cancer can be diagnosed at an early stage by detecting the methylation of the breast tumor-specific CTNNA2, AXIN1 and RASSF1 gene combination in normal-appearing cells. Among them, early diagnosis refers to the possibility of detecting cancer before its occurrence and metastasis, preferably before morphological changes in tissues or cells can be observed.

[0018] In addition to the early diagnosis of breast tumors, the primer combination, reagent / kits of the present invention are also expected to be used for breast tumor screening, risk assessment, prognostic diagnosis, disease identification, diagnosis of disease stages and screening of therapeutic targets. In one or more embodiments of the present invention, there are significant differences in the methylation levels of the above marker combination before and after breast tumor resection. Based on the above marker combination, it is beneficial for the prognosis of breast tumor treatment. For example, if the methylation level is still high after resection, there is a potential risk of recurrence. Similarly, the primer combination, reagent / kits of the present invention are also expected to be applied to prediction, that is, when the methylation level of the above marker combination in an individual shows an upward trend within a certain period of time, it is possible to develop into a breast tumor, and the detection of the above marker combination can play a predictive role.

[0019] Further, the test sample to be detected is a tissue, body fluid or excretory sample. The tissue includes breast tissue; the body fluid includes blood, plasma, serum, extracellular fluid, tissue fluid, lymph fluid, cerebrospinal fluid or aqueous humor; the excretions include sputum, urine, saliva or feces. Further still, in one or more embodiments of the present invention, the test sample is a plasma sample.

[0020] In one or more embodiments of the present invention, by using the above primer combination and the multi-gene methylation combined detection reagent, breast cancer can be clearly distinguished, and it has high specificity and sensitivity.

[0021] Another object of the present invention is to provide the use of primers and / or probes for detecting AXIN1 gene methylation in the preparation of reagents or kits for detecting breast cancer; further, the primers for detecting AXIN1 gene methylation include the primer pair combination shown in SEQ ID NO: 16 and SEQ ID NO: 17; the probe for detecting AXIN1 gene methylation includes the sequence shown in SEQ ID NO: 18. In one or more embodiments of the present invention, it is found that the detection of AXIN1 methylation can distinguish breast cancer from healthy samples, and based on the above primers and probes, the discrimination effect is obvious. That is, methylated AXIN1 can exist as a breast cancer biomarker, and the diagnosis of breast cancer can be facilitated by detecting this biomarker.

[0022] Another object of the present invention is to provide a gene methylation detection reagent, which includes primers for detecting the methylation of CTNNA2, AXIN1 or RASSF1 genes; in one or more embodiments of the present invention, the methylation of CTNNA2, AXIN1 or RASSF1 genes can be detected to distinguish breast cancer from non-tumor samples; the detection reagent for the corresponding gene is beneficial to be applied in the clinical diagnosis of breast cancer; the primers for detecting the methylation of CTNNA2 gene are the primer pair combination shown in SEQ ID NO: 1 and SEQ ID NO: 2; the primers for detecting the methylation of RASSF1 gene are the primer pair combination shown in SEQ ID NO: 22 and SEQ ID NO: 23; further, it includes primers for detecting the methylation of AXIN1 gene; and / or, the primers for detecting the methylation of AXIN1 gene are the primer pair combination shown in SEQ ID NO: 16 and SEQ ID NO: 17.

[0023] Further, it also includes probes for detecting the methylation of CTNNA2, AXIN1 or RASSF1 genes; further, the probe for detecting the methylation of CTNNA2 gene includes the sequence shown in SEQ ID NO: 3; and / or, the probe for detecting the methylation of AXIN1 gene includes the sequence shown in SEQ ID NO: 18; and / or, the probe for detecting the methylation of RASSF1 gene includes the sequence shown in SEQ ID NO: 24.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: Compared with the existing markers for detecting breast cancer, the technical solutions of the primers, nucleic acid probes, and the gene combination of CTNNA2, AXIN1, and RASSF1 provided by the present invention can detect breast cancer with extremely high sensitivity and specificity. In the detection solution provided by the present invention, the detection sensitivity and specificity for breast cancer are both higher than 90%. Moreover, in one technical solution provided by the present application, the methylation of the gene combination of CTNNA2, AXIN1, and RASSF1 is jointly detected. This method can achieve the joint detection of multiple genes, greatly reducing the complexity of the experiment and improving the detection efficiency. At the same time, for the methylation detection reagent containing the target genes CTNNA2, AXIN1, and RASSF1 gene combination, the extraction and detection processes are very convenient, and it can accurately judge breast cancer and normal specimens; the methylation detection kit based on this gene combination is expected to be used in breast cancer gene detection kits and serve clinical detection of breast cancer. It should be noted that in one technical solution provided by the present application, the reagent / kits detect and diagnose cancer through methylation levels. At the same time, more and more studies have confirmed that methylation changes are early events in the process of tumorigenesis. Therefore, based on the gene methylation diagnosis strategy provided by the present application, detecting abnormal methylation is more likely to detect early lesions, so as to facilitate timely adoption of corresponding treatment strategies and improve the treatment effect. Generally, based on the primer combination of the present application or the gene methylation detection reagent containing this primer combination, it can effectively distinguish breast tumor and non-breast tumor individuals, facilitating the provision of an effective breast tumor diagnosis strategy. Especially when jointly detecting the corresponding genes, for breast cancer belonging to malignant tumors, it has significant high specificity and high sensitivity detection characteristics, with the highest specificity up to more than 95% and the highest sensitivity up to more than 90%. Based on the primers and corresponding reagents provided by the present application, it is beneficial for actual clinical diagnosis, enabling early diagnosis and early treatment of breast cancer patients; facilitating the timely formulation of corresponding treatment strategies and improving the survival rate and survival period of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the amplification curve of primer pair C1 in Example 1;

[0026] Figure 2 It is the amplification curve of primer pair C2 in Example 1;

[0027] Figure 3 It is the amplification curve of primer pair C3 in Example 1;

[0028] Figure 4 It is the amplification curve of primer pair A1 in Example 2;

[0029] Figure 5 It is the amplification curve of primer pair A2 in Example 2;

[0030] Figure 6 It is the amplification curve of primer pair A3 in Example 2;

[0031] Figure 7 It is the amplification curve of primer pair R1 in Example 3;

[0032] Figure 8 It is the amplification curve of primer pair R2 in Example 3;

[0033] Figure 9 It is the amplification curve of primer pair R3 in Example 3;

[0034] Figure 10 It is the ROC curve for the combined detection of breast cancer and breast adenoma using the CTNNA2, AXIN1, and RASSF1 genes in 733 whole blood specimens in Example 4;

[0035] Figure 11 It is the number of positive cases in the methylation test before and after surgery for 25 breast cancer patients in Example 5. Detailed implementation manners

[0036] It should be noted that the following detailed descriptions are all illustrative and are intended to provide further explanations of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0037] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary implementation manners according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0038] The present invention will now be further described in conjunction with specific examples. The following examples are only for explaining the present invention and do not constitute a limitation to the present invention. The test samples and test procedures used in the following examples include the following contents (if the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions or the conditions recommended by reagent companies; the reagents, consumables, etc. used in the following examples can be obtained from commercial sources without special instructions).

[0039] Example 1

[0040] Design three pairs of primers for CTNNA2 according to Table 1 and study the specificity of amplification of the three pairs of primers.

[0041] Table 1 Primers and probes for CTNNA2

[0042]

[0043] In this example and the following examples, FP is the upstream primer, RP is the downstream primer, and probe is the probe.

[0044] Use the above 3 pairs of primer-probes to detect the DNA of plasma samples from 3 breast cancer patients and the DNA of plasma samples from 3 normal individuals respectively, and analyze the amplification specificity.

[0045] The corresponding results of groups C1, C2, and C3 are respectively as Figure 1 、 2 、3 show that the amplification specificity of the CTNNA2 primer-probe combination C1 is better; while the difference in the amplification curves of positive and negative samples of the other two groups of primer-probes is small, and positive and negative samples cannot be effectively distinguished.

[0046] Example 2

[0047] Design three pairs of primers for AXIN1 according to Table 2 and study the amplification specificity of the three pairs of primers.

[0048] Table 2 Primers and probes for AXIN1

[0049]

[0050] Use the above 3 pairs of primer-probes to detect the DNA of plasma samples from 3 breast cancer patients and the DNA of plasma samples from 3 normal individuals respectively, and analyze the amplification specificity.

[0051] The corresponding results of groups A1, A2, and A3 are respectively as Figure 4 、 5 、6 show that the amplification specificity of the AXIN1 primer-probe combination A3 is better; while the difference in the amplification curves of positive and negative samples of the other two groups of primer-probes is small, and positive and negative samples cannot be effectively distinguished.

[0052] Example 3

[0053] Design three pairs of primers for RASSF1 according to Table 3 and study the amplification specificity of the three pairs of primers.

[0054] Table 3 Primers and probes for RASSF1

[0055]

[0056]

[0057] Use the above 3 pairs of primer-probes to detect the DNA of plasma samples from 3 breast cancer patients and the DNA of plasma samples from 3 normal individuals respectively, and analyze the amplification specificity.

[0058] The corresponding results of groups R1, R2, and R3 are respectively asFigure 7 , 8 As shown in Figure 9, the result shows that the amplification specificity of the RASSF1 primer-probe combination R2 is better; while for the other two groups of primer-probe amplification curves, the difference between the positive and negative samples is small, and the positive and negative samples cannot be effectively distinguished.

[0059] Example 4

[0060] Select 733 whole blood specimens (301 breast cancer cases, 231 breast adenoma cases, 201 non-tumor individuals, all confirmed by B-ultrasound or pathology), and perform centrifugation to separate plasma and extract plasma-free DNA.

[0061] The technical solution is as follows:

[0062] (1) Collect the whole blood of normal people and breast cancer patients with pathological results, centrifuge to separate the plasma, and extract plasma-free DNA according to the operation method of QIAamp Circulating Nucleic Acid Kit (Qiagen).

[0063] (2) Use the EZ DNA Methylation Kit (Zymo Research) to perform bisulfite conversion treatment on the DNA fragments in the previous step according to the method in the instruction manual. The final eluate of 20 μl is used for qMSP detection, and the methylation levels of the CTNNA2, AXIN1, and RASSF1 gene combinations in the specimens are judged according to the Ct values.

[0064] The qMSP reaction system of this example specifically includes the following materials:

[0065] The reaction system is 30 μl: 1.52 μl of nuclease-free water, 6 μl of 5×Colorless GoTaq Flexi Buffer, 5 μl of MgCl2 (25 mM), 1 μl of dNTPs (10 mM), 0.6 μl of GoTaq Hot Start polymerase, 0.05 μl of ACTB-FP (100 μM), 0.05 μl of ACTB-RP (100 μM), 0.03 μl of ACTB-Probe (100 μM), 0.1 μl of CTNNA2-FP (100 μM), 0.1 μl of CTNNA2-RP (100 μM), 0.05 μl of CTNNA2-Probe (100 μM), 0.1 μl of AXIN1-FP (100 μM), 0.1 μl of AXIN1-RP (100 μM), 0.05 μl of AXIN1-Probe (100 μM), 0.1 μl of RASSF1-FP (100 μM), 0.1 μl of RASSF1-RP (100 μM), 0.05 μl of RASSF1-Probe (100 μM), and 10 μl of DNA. The reaction procedure is: 95°C for 5 min, (95°C for 15 s, 58°C for 30 s, 72°C for 30 s) × 45 Cycles, 40°C for 30 s.

[0066] In the PCR reaction, ACTB is used as an internal reference gene to calculate the Ct values of the combined detection of CTNNA2, AXIN1, and RASSF1 genes, and the optimal positive judgment value is calculated according to the ROC curve to judge the methylation level in the specimen.

[0067] The methylation sites of CTNNA2, AXIN1, and RASSF1 genes are mainly located in the CpG islands near the promoter region.

[0068] In this example, PCR probes labeled with the same fluorescent group are used, so the total methylation level of these genes can be conveniently detected through one fluorescent channel, without the need to detect each gene with a different fluorescent channel, reducing the complexity of the experiment.

[0069] The primer and probe sequences of this example are as follows:

[0070] The qMSP primer and probe sequences of the CTNNA2 gene:

[0071] SEQ ID NO: 1: 5’-TTTGTTCGTTTTCGTATTC-3’

[0072] SEQ ID NO: 2: 5’-CGCGTTACCCTACGAACG-3’

[0073] SEQ ID NO: 3: 5'-TTTTTCGGGTTTCGCGATTTC-3'

[0074] qMSP primer and probe sequences for the AXIN1 gene:

[0075] SEQ ID NO: 16: 5'-GTTTTTATTCGTTCGTTTC-3'

[0076] SEQ ID NO: 17: 5'-CGAAAAACGATCGTAACCG-3'

[0077] SEQ ID NO: 18: 5'-TTTCGGGTCGATTTCGGTCGGTT-3'

[0078] qMSP primer and probe sequences for the RASSF1 gene:

[0079] SEQ ID NO: 22: 5'-CGCGTTGGTACGTTTTAGTC-3'

[0080] SEQ ID NO: 23: 5'-CGAACCCAACCGAACCATATCG-3'

[0081] SEQ ID NO: 24: 5'-TTTAGCGCGTTTAGCGGGTGTTAG-3'

[0082] Based on the results of these 733 whole blood specimens, the ROC curve for the combined detection of breast cancer and adenoma using this biomarker combination was plotted using IBM SPSS statistics 23 software, and the results are as Figure 10 shown.

[0083] The results showed that for the combined detection of breast cancer by methylation of the CTNNA2, AXIN1, and RASSF1 genes, the Youden index was calculated, and the optimal positive judgment value was determined to be Ct 36.0. When the specificity was 95.7%, the sensitivity for breast cancer was as high as 90.2%, the area under the ROC curve for detecting breast cancer was 0.940, and the CI was 0.908 - 0.973; for the combined detection of breast adenoma by methylation of the CTNNA2, AXIN1, and RASSF1 genes, the positive judgment value was Ct 36.0. When the specificity was 90.9%, the sensitivity for breast adenoma was as high as 80.3%, the area under the ROC curve for detecting breast adenoma was 0.873, and the CI was 0.829 - 0.916.

[0084] Example 5

[0085] Twenty-five patients diagnosed with breast cancer were selected, and whole blood specimens of the patients were collected before surgery and 3 - 6 months after surgery. A total of 50 whole blood specimens before and after surgery were detected using the biomarker combination (CTNNA2, AXIN1, and RASSF1 genes) of the present invention, and the methylation levels of the corresponding genes in the biomarker combination in plasma before and after surgery were compared.

[0086] The detection process and result judgment criteria of the biomarker combination were the same as those in Example 1. When the Ct value of the target gene was ≤ 36, it was judged as positive; when Ct > 36, it was judged as negative. The results were as Figure 11 shown.

[0087] The results showed that the test results of these 25 breast cancer patients were all positive before surgery. After the surgery, the plasma specimens were tested again, and the results were all negative. This indicates that after surgical resection of the tumor, the methylation level of this biomarker combination decreased significantly, suggesting that this biomarker combination can also be used for postoperative follow-up monitoring of breast cancer patients.

[0088] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the technical solutions of the present invention, rather than limitations on the specific implementation manners of the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the claims of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. Use of a reagent for combined detection of multiple gene methylation in the preparation of a reagent or kit for detecting breast tumors, characterized in that, the genes are CTNNA2, AXIN1 and RASSF1; the reagent for combined detection of multiple gene methylation includes primers and probes for methylation detection of the genes; the primers include primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 16 and SEQ ID NO: 17, and SEQ ID NO: 22 and SEQ ID NO: 23; the probes include combinations of sequences shown in SEQ ID NO: 3, SEQ ID NO: 18, and SEQ ID NO:

24.

2. The use according to claim 1, characterized in that, the kit includes: a first container containing a primer combination for amplification; a second container containing a nucleic acid probe combination.

3. The use according to claim 2, characterized in that, The kit further includes a converting agent, DNA polymerase, dNTPs, Mg 2+ ions and a buffer solution; the converter is used to convert all non-methylated cytosine bases into uracil; the DNA polymerase is used to amplify the CTNNA2, AXIN1 and RASSF1 genes; the buffer is used to maintain the activity of the DNA polymerase.

4. The use according to claim 3, characterized in that, the converter is one or more of hydrazine salts, bisulfite salts and hydrogen sulfite salts.

5. The use according to any one of claims 1-4, characterized in that, the breast tumors include breast cancer and breast adenoma.

6. Use of the reagent for combined detection of multiple gene methylation according to any one of claims 2-4 in the preparation of a reagent or kit for detecting breast tumors, characterized in that, the kit is used for detecting gene methylation in breast tumors, and the detection steps include: (1) Sample DNA extraction and purification; (2) Use the kit according to any one of claims 2-4 to perform qMSP detection on the sample DNA, and judge the methylation level of the CTNNA2, AXIN1 and RASSF1 gene combination in the sample DNA according to the Ct value.

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