PCR (Polymerase Chain Reaction) primer probe combination for breast cancer gene methylation detection and application of PCR primer probe combination

Abstract

The embodiment of the invention provides a PCR primer probe combination for breast cancer gene methylation detection and application of the PCR primer probe combination. The PCR primer probe combination comprises a primer probe combination 1, a primer probe combination 2 or a primer probe combination 3 for detecting CDO1 gene methylation. According to the PCR primer probe combination for breast cancer gene methylation detection provided by the embodiment of the invention, the methylation state of a CDO1 gene promoter region can be rapidly and sensitively detected by designing the specific primer and the probe, so that a basis is provided for early screening, accurate detection and individualized treatment of breast cancer. Therefore, the embodiment of the invention has the advantages of high sensitivity, high specificity and simplicity and convenience in operation, and is suitable for clinical large-scale screening and diagnosis.

Description

Technical Field

[0001] The disclosed embodiments relate to the field of gene detection technology, and more particularly to a PCR primer-probe combination for detecting breast cancer gene methylation and its use. Background Art

[0002] Breast cancer is the uncontrolled proliferation of mammary epithelial cells in response to multiple carcinogens. Early symptoms often include breast lumps, nipple discharge, and axillary lymphadenopathy. In advanced stages, cancer cells can metastasize to distant organs, leading to multi-organ involvement. With advancements in medical care, breast cancer treatment is becoming more effective, and the earlier it is detected and treated, the higher the cure rate.

[0003] In recent years, with the advancement of molecular biology techniques, genetic testing has played a vital role in the diagnosis and treatment of breast cancer. The CDO1 (Cysteine Dioxygenase Type 1) gene is closely associated with tumorigenesis and progression, and the methylation status of its promoter region is closely correlated with the malignant potential of breast cancer. Related studies have shown that aberrant methylation of the CDO1 gene can lead to downregulation of its expression, thereby promoting the proliferation, invasion, and metastasis of breast cancer cells. Therefore, detecting the methylation status of the CDO1 gene is of great significance for the early diagnosis and prognostic assessment of breast cancer.

[0004] While traditional genetic testing methods such as methylation-specific PCR (MSP) and sequencing can detect the methylation status of the CDO1 gene, they are complex, time-consuming, and have low sensitivity. Fluorescence PCR, as a highly sensitive and specific molecular biological assay, has been widely used to detect gene methylation status. However, no CDO1 gene methylation detection method based on fluorescence PCR has been applied in the diagnosis of breast cancer.

[0005] Therefore, developing detection products targeting the methylation of the above-mentioned specific genes is of great significance for improving the diagnostic accuracy of breast cancer and guiding personalized treatment. Summary of the Invention

[0006] The purpose of the embodiments of the present disclosure is to provide a PCR primer probe combination for breast cancer gene methylation detection and its use.

[0007] To achieve the above objectives, the present disclosure proposes the following technical solutions: In a first aspect, the present disclosure provides a PCR primer-probe combination for detecting gene methylation in breast cancer, wherein the PCR primer-probe combination includes primer-probe combination 1, primer-probe combination 2, or primer-probe combination 3 for detecting CDO1 gene methylation; The primer-probe combination 1 includes an upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2, and a fluorescent probe as shown in SEQ ID NO.3; The primer-probe combination 2 includes an upstream primer as shown in SEQ ID NO.4, a downstream primer as shown in SEQ ID NO.5, and a fluorescent probe as shown in SEQ ID NO.6; The primer-probe combination 3 includes an upstream primer as shown in SEQ ID NO.7, a downstream primer as shown in SEQ ID NO.8, and a fluorescent probe as shown in SEQ ID NO.9.

[0008] As an embodiment, the PCR primer probe combination further includes an internal standard primer probe combination 1, an internal standard primer probe combination 2 or an internal standard primer probe combination 3 for detecting an internal reference gene; The internal standard primer-probe combination 1 includes an upstream primer as shown in SEQ ID NO.10, a downstream primer as shown in SEQ ID NO.11, and a fluorescent probe as shown in SEQ ID NO.12; The internal standard primer-probe combination 2 includes an upstream primer as shown in SEQ ID NO.13, a downstream primer as shown in SEQ ID NO.14, and a fluorescent probe as shown in SEQ ID NO.15; The internal standard primer-probe combination 3 includes an upstream primer as shown in SEQ ID NO.16, a downstream primer as shown in SEQ ID NO.17, and a fluorescent probe as shown in SEQ ID NO.18.

[0009] As an embodiment, the 5' end of the fluorescent probe comprises a fluorescent group; The fluorescent group is selected from FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY3 or CY5.

[0010] As an embodiment, the 3' end of the fluorescent probe comprises a quenching group; The quenching group is selected from BHQ-1, BHQ-2, BHQ-3, TAMRA or DABCYL.

[0011] In a second aspect, the embodiments of the present disclosure provide a breast cancer gene methylation detection kit, which includes the PCR primer probe combination described in the first aspect.

[0012] In a third aspect, the present disclosure provides a non-disease diagnostic method for detecting breast cancer gene methylation, using the detection kit described in the first aspect. The detection method comprises: Extract free DNA to obtain the sample DNA to be tested; The DNA of the sample to be tested is subjected to bisulfite conversion treatment to obtain converted DNA; Using the converted DNA as a template, a fluorescent PCR amplification reaction is performed using a PCR primer probe combination, and the fluorescent signal is detected to determine the result.

[0013] As an embodiment, the procedure of the fluorescent PCR amplification reaction is: React at 94-96°C for 4-8 minutes, and cycle 1-2 times; React at 94-96°C for 12-20 seconds, then at 58-68°C for 25-35 seconds, for 18-22 cycles; The reaction was carried out at 94-96°C for 8-15 s, and at 55-65°C for 25-35 s, and the cycle was repeated 38-42 times, and fluorescence was collected.

[0014] In a fourth aspect, embodiments of the present disclosure provide for use of the PCR primer-probe combination described in the first aspect in preparing a kit for detecting breast cancer.

[0015] In a fifth aspect, embodiments of the present disclosure provide for use of a reagent for detecting CDO1 gene methylation in preparing a breast cancer diagnostic product, wherein the reagent comprises a PCR primer-probe combination, wherein the PCR primer-probe combination comprises primer-probe combination 1, primer-probe combination 2, or primer-probe combination 3 for detecting CDO1 gene methylation; The primer-probe combination 1 includes an upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2, and a fluorescent probe as shown in SEQ ID NO.3; The primer-probe combination 2 includes an upstream primer as shown in SEQ ID NO.4, a downstream primer as shown in SEQ ID NO.5, and a fluorescent probe as shown in SEQ ID NO.6; The primer-probe combination 3 includes an upstream primer as shown in SEQ ID NO.7, a downstream primer as shown in SEQ ID NO.8, and a fluorescent probe as shown in SEQ ID NO.9.

[0016] In one embodiment, the CDO1 gene methylation is used as a biomarker.

[0017] Compared with the prior art, the embodiments of the present disclosure have at least the following beneficial effects: The disclosed embodiments provide a PCR primer-probe combination for detecting gene methylation in breast cancer. By designing specific primers and probes, this combination can rapidly and sensitively detect the methylation status of the CDO1 gene promoter region, thereby providing a basis for early screening, accurate detection, and personalized treatment of breast cancer. As such, the disclosed embodiments offer the advantages of high sensitivity, high specificity, and ease of use, making them suitable for large-scale clinical screening and diagnosis.

[0018] Additional aspects and advantages of the present disclosure will be set forth in part in the following description, will become apparent from the following description, or may be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A schematic diagram of the ROC curve of Example 1 is shown. DETAILED DESCRIPTION

[0020] The following will be combined with the embodiments of the present disclosure and the accompanying drawings to clearly and completely describe the technical solutions in the embodiments. Obviously, the embodiments described below are only some of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present disclosure.

[0021] It will be understood that when used in this specification and the appended claims, the terms “comprises” and “comprising” indicate the presence of described features, integers, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0022] It should also be understood that the terms used in this specification of the disclosed embodiments are only for the purpose of describing specific embodiments and are not intended to limit the disclosed embodiments. As used in the specification of the disclosed embodiments and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise.

[0023] The following describes in detail the PCR primer-probe combination for detecting breast cancer gene methylation and its application in this embodiment.

[0024] First, the PCR primer-probe combination for detecting breast cancer gene methylation according to the first aspect of this embodiment is described.

[0025] PCR primer probe combination for breast cancer gene methylation detection In the prior art, the CDO1 (Cysteine Dioxygenase Type 1) gene, a gene closely associated with tumorigenesis and progression, is used in the detection and diagnosis of breast cancer. The methylation status of its promoter region is closely correlated with the malignant potential of breast cancer. Existing studies have shown that aberrant methylation of the CDO1 gene can lead to downregulation of its expression, thereby promoting the proliferation, invasion, and metastasis of breast cancer cells. The enzyme encoded by the CDO1 gene may be involved in cell cycle regulation. When its expression level is reduced, cell cycle regulation may be disrupted, leading to uncontrolled cell proliferation. Furthermore, in addition to enhancing proliferation, downregulation of CDO1 gene expression may also promote the invasion and metastasis of breast cancer cells. This is because the CDO1 gene may be involved in regulating processes such as cell adhesion, migration, and invasion. When its expression level is reduced, the regulation of these processes may be disrupted, making breast cancer cells more likely to detach from the primary site and metastasize to other sites.

[0026] Therefore, detecting the methylation status of the CDO1 gene is of great significance for the early diagnosis and prognosis evaluation of breast cancer.

[0027] In view of this, this embodiment proposes a PCR primer probe combination for detecting breast cancer gene methylation, wherein the PCR primer probe combination includes primer probe combination 1, primer probe combination 2, or primer probe combination 3 for detecting CDO1 gene methylation; The primer-probe combination 1 includes an upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2, and a fluorescent probe as shown in SEQ ID NO.3; The primer-probe combination 2 includes an upstream primer as shown in SEQ ID NO.4, a downstream primer as shown in SEQ ID NO.5, and a fluorescent probe as shown in SEQ ID NO.6; The primer-probe combination 3 includes an upstream primer as shown in SEQ ID NO.7, a downstream primer as shown in SEQ ID NO.8, and a fluorescent probe as shown in SEQ ID NO.9.

[0028] It will be understood that the gene methylation detection site of this embodiment includes CDO1, wherein CDO1 gene methylation serves as a biomarker; this embodiment targets the CDO1 specific methylation detection site, and this embodiment designs a specific PCR primer probe combination (primer probe combination 1 or primer probe combination 2 or primer probe combination 3) for detecting CDO1 gene methylation. This PCR primer probe combination, by designing specific primers and probes, can rapidly and sensitively detect the methylation status of the CDO1 gene promoter region, thereby providing a basis for early screening, accurate detection, and personalized treatment of breast cancer.

[0029] It can be understood that the primer-probe combination 1, primer-probe combination 2, or primer-probe combination 3 used in this embodiment can achieve the purpose of quickly and sensitively detecting the methylation status of the CDO1 gene promoter region.

[0030] In the following examples, the upstream primer of primer-probe combination 1 is denoted as CDO1-F1, the downstream primer is denoted as CDO1-R1, and the fluorescent probe is denoted as CDO1-P1. The upstream primer of primer-probe combination 2 is denoted as CDO1-F2, the downstream primer is denoted as CDO1-R2, and the fluorescent probe is denoted as CDO1-P2. The upstream primer of primer-probe combination 3 is denoted as CDO1-F3, the downstream primer is denoted as CDO1-R3, and the fluorescent probe is denoted as CDO1-P3.

[0031] In one embodiment, the 5' end of the fluorescent probe comprises a fluorescent group; the fluorescent group is selected from FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY3 or CY5.

[0032] The 3' end of the fluorescent probe comprises a quenching group; the quenching group is selected from BHQ-1, BHQ-2, BHQ-3, TAMRA or DABCYL.

[0033] It is understood that when the fluorescent probe is intact, the fluorescent group at the 5' end is restricted by the quenching group at the 3' end, preventing detection of fluorescence. However, when the fluorescent probe binds to the target DNA template, the fluorescent group at the 5' end is released and emits fluorescence. Therefore, this embodiment can detect the methylation status of the CDO1 gene by measuring the fluorescence intensity in the reaction system.

[0034] Specifically, the PCR primer-probe combination provided in this embodiment is used in combination with bisulfite modification during the actual detection process; first, this embodiment uses bisulfite to convert unmethylated C bases (cytosine) into U bases (uracil), which are then converted into T bases during the PCR amplification process, while the methylated C bases remain unchanged, thereby distinguishing between methylated and unmethylated C bases.

[0035] At the same time, using a TaqMan probe and a fluorescent quantitative PCR instrument as a platform, the probe specifically anneals and binds to the complementary sequence between the forward and reverse primers during the PCR reaction. When the probe is intact, the fluorophore emits only weak fluorescence due to energy resonance transfer. After the specific probe binds to the corresponding sequence, DNA polymerase activates its 5' to 3' exonuclease activity, cleaving the reporter fluorophore, freeing it from the quenching effect of the 3' end quencher fluorophore, resulting in fluorescence.

[0036] Therefore, this embodiment can detect the methylation status of the CDO1 gene according to the detected fluorescence intensity, and based on the methylation status of the CDO1 gene promoter region, it can provide a basis for early screening, accurate detection and personalized treatment of breast cancer.

[0037] In summary, this embodiment has the advantages of high sensitivity, high specificity and simple operation, and is suitable for large-scale clinical screening and diagnosis.

[0038] As an embodiment, the PCR primer probe combination further includes an internal standard primer probe combination 1, an internal standard primer probe combination 2 or an internal standard primer probe combination 3 for detecting an internal reference gene; The internal standard primer-probe combination 1 includes an upstream primer as shown in SEQ ID NO.10, a downstream primer as shown in SEQ ID NO.11, and a fluorescent probe as shown in SEQ ID NO.12; The internal standard primer-probe combination 2 includes an upstream primer as shown in SEQ ID NO.13, a downstream primer as shown in SEQ ID NO.14, and a fluorescent probe as shown in SEQ ID NO.15; The internal standard primer-probe combination 3 includes an upstream primer as shown in SEQ ID NO.16, a downstream primer as shown in SEQ ID NO.17, and a fluorescent probe as shown in SEQ ID NO.18.

[0039] In the following examples, the upstream primer of internal standard primer probe combination 1 is denoted as GAPDH-F1, the downstream primer is denoted as GAPDH-R1, and the fluorescent probe is denoted as GAPDH-P1; the upstream primer of internal standard primer probe combination 2 is denoted as GAPDH-F2, the downstream primer is denoted as GAPDH-R2, and the fluorescent probe is denoted as GAPDH-P2; the upstream primer of internal standard primer probe combination 3 is denoted as GAPDH-F3, the downstream primer is denoted as GAPDH-R3, and the fluorescent probe is denoted as GAPDH-P3.

[0040] In this embodiment, upper and lower primers and a fluorescent probe with the nucleotide sequence described above are designed for the internal reference gene GAPDH. The upper and lower primers and fluorescent probe provided in this embodiment can monitor the sample extraction and PCR amplification process, promptly identify problems that may arise in different links of the entire process, and monitor possible inhibitory factors.

[0041] The detection genes and corresponding detection channels of this embodiment are shown in Table 1 below.

[0042] Table 1:

[0043] Next, the detection kit according to the second aspect of this embodiment will be described.

[0044] Detection kit The breast cancer gene methylation detection kit of this embodiment includes the PCR primer probe combination described in the first aspect.

[0045] The kit of this embodiment is used to detect breast cancer, specifically to detect the methylation level of the CDO1 gene in a biological sample; The sample can be selected from the group consisting of: histological sections, tissue biopsies, paraffin-embedded tissues, body fluids, surgical resection samples, isolated blood cells, cells isolated from blood, and any combination thereof.

[0046] As a preferred embodiment, the body fluid is selected from the group consisting of whole blood, serum, plasma, and any combination thereof.

[0047] It should be noted that the test results of the aforementioned test kits do not constitute a specific disease diagnosis and should not be used as the sole criterion for disease assessment. A comprehensive assessment of the condition must be made in conjunction with the patient's clinical presentation and other test indicators. For example, if the test result is positive, a comprehensive consideration of the patient's clinical presentation, medical history, imaging examinations, pathological examinations, and the results of the breast cancer test kit is necessary to make an accurate assessment of the condition and formulate an appropriate treatment plan.

[0048] As an embodiment, the detection kit generally further includes PCR reaction reagents (or PCR mixture).

[0049] The PCR reaction reagents include DNA polymerase, buffer and dNTPs, the content of the DNA polymerase is 3% to 6%, the content of the buffer is 30% to 40%, and the content of the dNTPs is 15% to 30%, based on the total volume of the PCR reaction reagents.

[0050] Typically, in addition to DNA polymerase, buffer, and dNTPs, PCR reaction reagents are dissolved and mixed using pure water. The PCR reaction reagents can be selected from existing conventional PCR reaction reagents. Specifically, the PCR reaction reagent (PCR mixture) in this embodiment is an amplification premix purchased from Takara Biotechnology (Dalian) Co., Ltd., item number R007A.

[0051] As an embodiment, the detection kit generally further includes a negative control and a positive control; for example, the positive control is bisulfite-modified fully methylated human genomic DNA, and the negative control is bisulfite-modified unmethylated human genomic DNA.

[0052] Understandably, negative quality control products will produce negative results, while positive quality control products will produce positive results. Negative quality control products can eliminate false positive results caused by reagent or environmental contamination; positive quality control products can monitor the performance of reagents or whether personnel operations are normal, thereby eliminating false negative results.

[0053] Typically, in order to facilitate medical staff in extracting samples and performing bisulfite conversion on the samples, the kit will also typically include nucleic acid extraction or purification reagents and bisulfite conversion agents.

[0054] Illustratively, the nucleic acid extraction or purification reagent can be the nucleic acid extraction or purification reagent of Anhui Medical Technology Co., Ltd. (medical device registration number: Anhui Wuxixibei 20220023 or Anhui Wuxixibei 20190002) to extract the DNA of each sample.

[0055] The bisulfite conversion agent can use the nucleic acid extraction or purification reagent (centrifugal column type) produced by Anhui Dajian Medical Technology Co., Ltd. (product registration number: Wanwu Xiebei 20200003).

[0056] The specific operations of the above reagents can be found in the corresponding instructions and will not be described in detail here.

[0057] For example, in the kit of this embodiment, the final concentration and dosage of each component are shown in Tables 2 and 3 below.

[0058] Table 2: PCR primer probe mixture

[0059] Table 3: PCR MIX

[0060] In summary, based on the PCR primer-probe combination of the first aspect, the kit of this embodiment can rapidly and sensitively detect the methylation status of the CDO1 gene promoter region by designing specific primers and probes, thereby providing a basis for early screening, accurate detection, and personalized treatment of breast cancer.

[0061] The following describes the detection method according to the third aspect of this embodiment.

[0062] Detection method This embodiment is based on the detection kit of the second aspect and further describes the detection method of this embodiment. The detection method includes: (1) extracting free DNA to obtain the sample DNA to be tested; (2) The DNA of the sample to be tested is subjected to bisulfite conversion treatment to obtain converted DNA (also called Bis-DNA); (3) Using the converted DNA as a template, a fluorescent PCR amplification reaction is performed using a PCR primer probe combination, and the fluorescent signal is detected to determine the result.

[0063] It is understandable that the detection method of this embodiment does not involve specific disease diagnosis and treatment. As mentioned above, the test results cannot be used as the only criterion for disease evaluation. A comprehensive evaluation of the disease must be conducted in combination with the patient's clinical manifestations and other test indicators.

[0064] The following further describes the relevant steps in the detection method of this embodiment.

[0065] In step (1), the biological sample (DNA sample) can be whole blood, serum, plasma, or any combination thereof; taking plasma sample as an example, it can adopt the sampling operation currently known in the art.

[0066] After sampling, an existing DNA extraction kit or nucleic acid extraction reagent can be used to extract the DNA of the sample to obtain the sample DNA to be tested.

[0067] For example, the nucleic acid extraction or purification reagents of Anhui Medical Technology Co., Ltd. (medical device registration number: Wanwu Xiebei 20220023 or Wanwu Xiebei 20190002) can be used to extract the DNA of each sample. For specific operations, please refer to the kit instructions.

[0068] Next, the DNA of the sample to be tested extracted in the above steps is subjected to bisulfite conversion and purification.

[0069] Step (2) converts the DNA of the sample to be tested into bisulfite to obtain converted DNA, namely Bis-DNA.

[0070] The bisulfite conversion treatment can be performed using a nucleic acid extraction or purification reagent (centrifugal column type) produced by Anhui Dajian Medical Technology Co., Ltd. (Product Registration Number: Wanwu Xiebei 20200003). For specific procedures, refer to the kit instructions. After obtaining Bis-DNA, store it at -20°C.

[0071] In step (2), bisulfite is used to convert unmethylated C bases (cytosine) into U bases (uracil), which are then converted into T bases during PCR amplification, while methylated C bases remain unchanged, thus distinguishing between methylated and unmethylated C bases.

[0072] Next, after obtaining Bis-DNA, a detection kit is used to perform detection through PCR reaction.

[0073] In step (3), a TaqMan probe is used, and a fluorescent quantitative PCR instrument is used as a platform. During the PCR reaction, the probe can specifically anneal and bind to the complementary sequence between the forward primer and the reverse primer. When the probe is present in its intact form, the fluorescent group only emits weak fluorescence due to energy resonance transfer. After the specific probe binds to the corresponding sequence, the DNA polymerase exerts its 5' to 3' exonuclease activity, cleaving the reporter fluorescent group, freeing it from the quenching effect of the 3' end quenching fluorescent group, thereby emitting fluorescence.

[0074] Wherein, in step (3), the procedure of the fluorescent PCR amplification reaction is: React at 94-96°C for 4-8 minutes, and cycle 1-2 times; React at 94-96°C for 12-20 seconds, then at 58-68°C for 25-35 seconds, for 18-22 cycles; The reaction was carried out at 94-96°C for 8-15 s, and at 55-65°C for 25-35 s, and the cycle was repeated 38-42 times, and fluorescence was collected.

[0075] Preferably, the procedure of the fluorescent PCR amplification reaction is: The reaction was carried out at 95°C for 5 min, and the cycle was repeated once; The reaction was carried out at 95°C for 15 seconds and at 64°C for 30 seconds, and the cycle was repeated 20 times. The reaction was carried out at 95°C for 10 s and at 58°C for 31 s, and the cycle was repeated 40 times, and fluorescence was collected.

[0076] In summary, the detection method of this embodiment can detect the methylation status of the CDO1 gene based on the detected fluorescence intensity. Based on the methylation status of the CDO1 gene promoter region, it can provide a basis for early screening, accurate detection and personalized treatment of breast cancer.

[0077] Next, the purpose of the fourth aspect in this embodiment will be described.

[0078] use This embodiment also proposes the use of the PCR primer-probe combination described in the first aspect in preparing a kit for detecting breast cancer.

[0079] As described in the first aspect, the gene methylation detection site of this embodiment includes CDO1. Targeting the CDO1 specific methylation detection site, this embodiment designs a specific PCR primer-probe combination for detecting CDO1 gene methylation. This PCR primer-probe combination, by designing specific primers and probes, can rapidly and sensitively detect the methylation status of the CDO1 gene promoter region, thereby providing a basis for early screening, accurate detection, and personalized treatment of breast cancer.

[0080] Therefore, this embodiment can prepare a detection kit for detecting the methylation status of the CDO1 gene based on the PCR primer-probe combination described in the first aspect; the CDO1 gene methylation detection kit has the advantages of high sensitivity and high specificity in detecting breast cancer.

[0081] Next, the use of the reagent for detecting CDO1 gene methylation according to the fifth aspect of this embodiment in preparing a breast cancer diagnosis product will be described.

[0082] Use of a reagent for detecting CDO1 gene methylation in the preparation of breast cancer diagnostic products This embodiment further provides the use of a reagent for detecting CDO1 gene methylation in preparing a breast cancer diagnostic product, wherein the reagent includes a PCR primer-probe combination, wherein the PCR primer-probe combination includes primer-probe combination 1, primer-probe combination 2, or primer-probe combination 3 for detecting CDO1 gene methylation; The primer-probe combination 1 includes an upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2, and a fluorescent probe as shown in SEQ ID NO.3; The primer-probe combination 2 includes an upstream primer as shown in SEQ ID NO.4, a downstream primer as shown in SEQ ID NO.5, and a fluorescent probe as shown in SEQ ID NO.6; The primer-probe combination 3 includes an upstream primer as shown in SEQ ID NO.7, a downstream primer as shown in SEQ ID NO.8, and a fluorescent probe as shown in SEQ ID NO.9.

[0083] It will be appreciated that this embodiment uses CDO1 gene methylation as a biomarker. Targeting the CDO1 specific methylation detection site, this embodiment designs a specific PCR primer-probe combination for detecting CDO1 gene methylation. This PCR primer-probe combination, through the design of specific primers and probes, can rapidly and sensitively detect the methylation status of the CDO1 gene promoter region.

[0084] In combination with the first aspect, this embodiment can detect the methylation status of the CDO1 gene based on the detected fluorescence intensity. Based on the methylation status of the CDO1 gene promoter region, it can provide a basis for early screening, accurate detection and personalized treatment of breast cancer.

[0085] The following is a further explanation of the result analysis method and quality control standards of the detection method.

[0086] Result Analysis This study analyzed the methylation level of the CDO1 gene based on the amplification curve and threshold cycle number (Ct value) of fluorescent PCR. A higher methylation level indicates lower CDO1 gene expression and higher malignant potential of breast cancer.

[0087] Baseline setting: The baseline can be automatically output by the instrument, or manually adjusted according to the instrument's instructions.

[0088] Threshold setting: It should be set at the exponential amplification stage of the fluorescence signal. The fluorescence curve above the threshold should have a typical "S" curve.

[0089] Positive Criteria: A total of 95 clinical samples, including 28 cases and 67 controls, were tested. Receiver-operating characteristic (ROC) curve analysis was performed using SPSS software. When the CUT-OFF value of the CDO1 gene methylation reagent was 7, the Youden index reached a maximum of 0.8481, indicating a sensitivity of 89.29% and a specificity of 95.52%.

[0090] Kit validity: (1) Negative control: The target gene ROX channel test has no amplification curve or has an amplification curve, but the Ct value is greater than 20; the internal standard gene CY5 channel test has an amplification curve and the Ct value is ≤20.

[0091] (2) Positive control: ROX alone is amplified, or both ROX and CY5 channels are amplified (showing a typical S-shaped curve) and the Ct value of the ROX channel is ≤30.

[0092] The above requirements must be met simultaneously in the same experiment; otherwise, the experiment will be invalid and must be repeated.

[0093] The present disclosure is further described below with reference to the following examples. It should be understood that these examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure.

[0094] In the following examples, all materials, reagents and instruments used can be purchased from commercial sources unless otherwise specified.

[0095] The present disclosure will be further described below with reference to specific embodiments.

[0096] Example 1 In this example, we screened for breast cancer-associated hypermethylated genes by collecting literature. We then designed a specific PCR primer-probe combination for detecting CDO1 gene methylation, targeting the CDO1 specific methylation detection site. Specific primers and probes were also designed for the internal reference gene GAPDH. The specific PCR primer-probe combination and the internal reference gene internal standard primer-probe combination are shown in Table 4 below.

[0097] Table 4:

[0098] The primer probes designed above were used to detect the methylation status of the CDO1 gene in cancer tissue samples. In this example, plasma was used as the sample to be tested.

[0099] The specific detection steps include: (1) Extraction of sample DNA 95 serum samples (including 28 cases in the case group and 67 cases in the negative control group) and DNA test samples (plasma) were collected, and high-purity DNA was extracted using nucleic acid extraction or purification reagents produced by Anhui Dajian Medical Technology Co., Ltd. (product registration number: Wanwu Xiebei 20220023).

[0100] The specific steps are as follows: (a) Take a 1.5 mL centrifuge tube, add 200 μL of the sample to be tested and 20 μL of pancreatic lipase, vortex and shake thoroughly, and then let it stand at room temperature for 5 minutes; (b) Add 20 μL of proteinase K and 360 μL of lysis buffer to the centrifuge tube, vortex and mix thoroughly, centrifuge briefly, and place in a 70°C water bath for 10 min. (c) Briefly centrifuge, add 200 μL of pre-chilled isopropanol to the centrifuge tube, vortex to mix thoroughly, briefly centrifuge to remove droplets on the inner wall of the tube cap, and let stand at -20°C for 5 min; (d) Add the solution and flocculent precipitate from step c to an adsorption column (the adsorption column is placed in a collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and reuse the collection tube; (e) Add 600 μL of pre-cooled rinse solution I to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste solution; (f) Add 600 μL of pre-cooled rinse buffer II to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste solution. (g) Place the adsorption column in a clean 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 3 min. Discard the centrifuge tube and waste liquid. (h) Place the adsorption column in a clean 1.5 mL centrifuge tube, open the tube cap, and air dry for 3 min. (i) Add 100 μL of elution buffer dropwise to the middle of the column, incubate at room temperature for 3 min, centrifuge at 12,000 rpm for 2 min, collect the DNA sample into a centrifuge tube, and store at -20°C.

[0101] (2) Bisulfite conversion and purification Next, the DNA extracted in the above step is subjected to bisulfite conversion and purification to obtain Bis-DNA.

[0102] In this embodiment, the bisulfite conversion treatment was performed using a nucleic acid extraction or purification reagent (centrifugal column type) produced by Anhui Dajian Medical Technology Co., Ltd. (Product Registration Number: Wanwu Xiebei 20200003); the specific steps are as follows: (a) Place 45 μL of the DNA sample to be tested in a new 1.5 mL centrifuge tube, add 5 μL of conversion buffer, and incubate in a metal bath at 37°C for 15 min. (b) After incubation, add 100 μL of the pre-prepared conversion solution to each sample, mix thoroughly, centrifuge briefly, and incubate in a metal bath at 60°C in the dark for 12–16 h. (c) The sample was placed on ice (0-4°C) and incubated for 10 min; (d) Place the adsorption column in the collection tube and add 400 μL of binding solution to the adsorption column; (e) Add the sample from step c to the adsorption column (containing the binding solution), cap the tube tightly, invert it several times to mix, let it stand for three minutes, centrifuge it at full speed (14000 rpm) for 30 seconds, and discard the waste liquid; (f) Add 100 μL of rinse solution to the adsorption column, centrifuge at full speed for 30 s, and discard the waste solution; (g) Add 200 μL of desulfurization solution to the adsorption column, incubate at room temperature (25°C) for 20 min, then centrifuge at full speed for 30 s and discard the waste liquid; (h) Add 200 μL of rinse solution to the adsorption column and centrifuge at full speed for 30 s. Repeat by adding 200 μL of rinse solution and centrifuging at full speed for 30 s. Discard the waste solution and the collection tube. (i) Place the adsorption column in a 1.5 mL sterile centrifuge tube. Add 30 μL of eluent dropwise to the middle of the adsorption membrane to elute the transformed DNA. Centrifuge at full speed for 1 min to collect the Bis-DNA and store at -20°C.

[0103] Next, after obtaining Bis-DNA, a fluorescent PCR amplification reaction was performed using primer probe combination 1 as an exemplary PCR primer probe combination and bisulfite-converted Bis-DNA as a template. PCR reaction reagents, a PCR primer probe combination for CDO1 gene methylation, and an internal standard primer probe combination for an internal reference gene were added.

[0104] The PCR reaction system is shown in Table 5.

[0105] Table 5: PCR reaction system

[0106] Note: The above PCR reaction reagents are purchased from Takara Biotechnology (Dalian) Co., Ltd., product number R007A amplification premix.

[0107] The reaction system in Table 5 was thoroughly mixed, centrifuged briefly, and aliquoted into 0.2 mL PCR reaction tubes at 20 μL per tube. The PCR reaction tubes were placed in the corresponding fluorescent PCR detector (ABI 7500 fluorescent PCR amplifier), and the order of sample placement was recorded. Methylation quantitative fluorescent PCR amplification detection was then performed.

[0108] The procedure of the fluorescent PCR amplification reaction in this embodiment is: Table 6: PCR amplification reaction program

[0109] Test results In this example, when the negative quality control product and the positive quality control product meet the validity judgment of the kit, the statistical analysis results are as follows: Table 7 and Figure 1 shown.

[0110] Table 7: ROC curve analysis results

[0111] Figure 1 The ROC curve diagram of this embodiment is shown, combined with Table 7 and Figure 1 The test results show that the sensitivity of the test kit of this embodiment is 89.2%, and the specificity is as high as 95.52%.

[0112] In summary, this example uses CDO1 gene methylation as a biomarker. Targeting the specific CDO1 methylation detection site, this example designs a specific PCR primer-probe combination for detecting CDO1 gene methylation. By designing specific primers and probes, this PCR primer-probe combination can rapidly and sensitively detect the methylation status of the CDO1 gene promoter region, thereby providing a basis for early screening, accurate detection, and personalized treatment of breast cancer.

[0113] The technical solutions provided by the embodiments of the present disclosure are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present disclosure. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present disclosure. At the same time, for those skilled in the art, according to the embodiments of the present disclosure, there may be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present disclosure.

Claims

1. A PCR primer-probe combination for detecting breast cancer gene methylation, characterized in that: The PCR primer-probe combination includes primer-probe combination 1, primer-probe combination 2 or primer-probe combination 3 for detecting CDO1 gene methylation; The primer-probe combination 1 includes an upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2, and a fluorescent probe as shown in SEQ ID NO.3; The primer-probe combination 2 includes an upstream primer as shown in SEQ ID NO.4, a downstream primer as shown in SEQ ID NO.5, and a fluorescent probe as shown in SEQ ID NO.6; The primer-probe combination 3 includes an upstream primer as shown in SEQ ID NO.7, a downstream primer as shown in SEQ ID NO.8, and a fluorescent probe as shown in SEQ ID NO.

9.

2. The PCR primer-probe combination according to claim 1, characterized in that: The PCR primer probe combination further includes an internal standard primer probe combination 1, an internal standard primer probe combination 2 or an internal standard primer probe combination 3 for detecting an internal reference gene; The internal standard primer-probe combination 1 includes an upstream primer as shown in SEQ ID NO.10, a downstream primer as shown in SEQ ID NO.11, and a fluorescent probe as shown in SEQ ID NO.12; The internal standard primer-probe combination 2 includes an upstream primer as shown in SEQ ID NO.13, a downstream primer as shown in SEQ ID NO.14, and a fluorescent probe as shown in SEQ ID NO.15; The internal standard primer-probe combination 3 includes an upstream primer as shown in SEQ ID NO.16, a downstream primer as shown in SEQ ID NO.17, and a fluorescent probe as shown in SEQ ID NO.

18.

3. The PCR primer-probe combination according to claim 1, characterized in that: The 5' end of the fluorescent probe contains a fluorescent group; The fluorescent group is selected from FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY3 or CY5.

4. The PCR primer-probe combination according to claim 1, characterized in that: The 3' end of the fluorescent probe contains a quenching group; The quenching group is selected from BHQ-1, BHQ-2, BHQ-3, TAMRA or DABCYL.

5. A breast cancer gene methylation detection kit, characterized in that: The detection kit comprises the PCR primer-probe combination according to any one of claims 1 to 4.

6. A non-disease diagnostic method for detecting breast cancer gene methylation, using the detection kit according to claim 5, characterized in that: The detection method comprises: Extract free DNA to obtain the sample DNA to be tested; The DNA of the sample to be tested is subjected to bisulfite conversion treatment to obtain converted DNA; Using the converted DNA as a template, a fluorescent PCR amplification reaction is performed using a PCR primer probe combination, and the fluorescent signal is detected to determine the result.

7. The detection method according to claim 6, characterized in that The procedure of the fluorescent PCR amplification reaction is: React at 94-96°C for 4-8 minutes, and cycle 1-2 times; React at 94-96°C for 12-20 seconds, then at 58-68°C for 25-35 seconds, for 18-22 cycles; The reaction was carried out at 94-96°C for 8-15 s, and at 55-65°C for 25-35 s, and the cycle was repeated 38-42 times, and fluorescence was collected.

8. Use of the PCR primer-probe combination according to any one of claims 1 to 4 in preparing a kit for detecting breast cancer.

9. Use of a reagent for detecting CDO1 gene methylation in the preparation of a breast cancer diagnostic product, characterized in that: The reagents include a PCR primer-probe combination, wherein the PCR primer-probe combination includes primer-probe combination 1, primer-probe combination 2, or primer-probe combination 3 for detecting CDO1 gene methylation; The primer-probe combination 1 includes an upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2, and a fluorescent probe as shown in SEQ ID NO.3; The primer-probe combination 2 includes an upstream primer as shown in SEQ ID NO.4, a downstream primer as shown in SEQ ID NO.5, and a fluorescent probe as shown in SEQ ID NO.6; The primer-probe combination 3 includes an upstream primer as shown in SEQ ID NO.7, a downstream primer as shown in SEQ ID NO.8, and a fluorescent probe as shown in SEQ ID NO.

9.

10. The use according to claim 9, characterized in that The CDO1 gene methylation is used as a biomarker.

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