PCR (Polymerase Chain Reaction) primer probe combination for digestive tract tumor gene methylation detection and application of PCR primer probe combination

Abstract

The embodiment of the invention provides a PCR primer probe combination for digestive tract tumor gene methylation detection and application of the PCR primer probe combination. The PCR primer probe combination comprises a PCR primer probe combination for SGIP1 methylation detection, a PCR primer probe combination for CCNA1 methylation detection and a PCR primer probe combination for EYA4 methylation detection. According to the embodiment of the invention, a primer probe combination with a specific sequence is designed according to the gene methylation state of a specific site, a specific primer for detecting liver cancer, a specific primer for detecting esophageal cancer and a specific primer for detecting gastric cancer are integrated into the same kit, and the primers do not interfere with one another in a system and are compatible; through highly specific gene amplification, early diagnosis of liver cancer, esophageal cancer and gastric cancer is realized. The PCR primer probe combination provided by the embodiment of the invention can realize high-efficiency cooperative detection of specific primers of liver cancer, esophageal cancer and gastric cancer in a single kit.

Description

Technical Field

[0001] The disclosed embodiments relate to the field of gene detection technology, and specifically to a PCR primer-probe combination for detecting gene methylation in digestive tract tumors and its use. Background Art

[0002] Gastrointestinal tumors, including colon and stomach cancer, liver cancer, colorectal cancer, pancreatic cancer, and esophageal cancer, are currently among the most common malignancies. Taking pancreatic cancer as an example, relevant studies have shown that the free DNA molecules in the peripheral blood circulation of pancreatic cancer patients are closely correlated with the degree of pancreatic cancer progression. The more advanced the tumor progression, the more tumor-associated free DNA molecules are in the blood circulation. In pancreatic cancer, methylation can affect the expression of multiple genes, leading to abnormal cell proliferation, impaired differentiation, and blocked apoptosis, which may ultimately lead to tumor development.

[0003] In fact, multiple genes in gastrointestinal tumors exhibit abnormal methylation, which may contribute to tumor development and progression by affecting the function of key genes. Currently, in multi-gene combined testing, it is necessary to consider potential interference between multiple primer pairs (such as primer complementarity and competitive binding) to ensure uniform amplification efficiency across all genes. Simultaneously testing the methylation status of multiple genes increases experimental complexity and data analysis difficulties.

[0004] Therefore, how to achieve joint detection of multi-gene methylation status in gastrointestinal tumors is an urgent problem to be solved in this field. Summary of the Invention

[0005] The purpose of the embodiments of the present disclosure is to provide a PCR primer probe combination for detecting gene methylation in digestive tract tumors and its use.

[0006] 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 digestive tract tumors, the PCR primer-probe combination comprising: PCR primer-probe combinations for SGIP1 methylation detection include primer-probe combination 1, primer-probe combination 2, or primer-probe combination 3; 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; PCR primer-probe combinations for CCNA1 methylation detection, including primer-probe combination 4, primer-probe combination 5, or primer-probe combination 6; The primer-probe combination 4 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 primer-probe combination 5 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 primer-probe combination 6 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; PCR primer-probe combinations for EYA4 methylation detection, including primer-probe combination 7, primer-probe combination 8, or primer-probe combination 9; The primer-probe combination 7 includes an upstream primer as shown in SEQ ID NO.19, a downstream primer as shown in SEQ ID NO.20, and a fluorescent probe as shown in SEQ ID NO.21; The primer-probe combination 8 includes an upstream primer as shown in SEQ ID NO.22, a downstream primer as shown in SEQ ID NO.23, and a fluorescent probe as shown in SEQ ID NO.24; The primer-probe combination 9 includes an upstream primer as shown in SEQ ID NO.25, a downstream primer as shown in SEQ ID NO.26, and a fluorescent probe as shown in SEQ ID NO.27.

[0007] 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.28, a downstream primer as shown in SEQ ID NO.29, and a fluorescent probe as shown in SEQ ID NO.30; The internal standard primer-probe combination 2 includes an upstream primer as shown in SEQ ID NO.31, a downstream primer as shown in SEQ ID NO.32, and a fluorescent probe as shown in SEQ ID NO.33; The internal standard primer-probe combination 3 includes an upstream primer as shown in SEQ ID NO.34, a downstream primer as shown in SEQ ID NO.35, and a fluorescent probe as shown in SEQ ID NO.36.

[0008] 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.

[0009] 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.

[0010] In a second aspect, an embodiment of the present disclosure provides a digestive tract tumor gene methylation detection kit, wherein the detection kit includes the PCR primer probe combination described in the first aspect.

[0011] In a third aspect, the present disclosure provides a non-disease diagnostic method for detecting gene methylation in digestive tract tumors, using the detection kit described in the second 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.

[0012] 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.

[0013] In a fourth aspect, the embodiments of the present disclosure propose the use of the PCR primer-probe combination described in the first aspect in preparing a kit for detecting digestive tract tumors.

[0014] In a fifth aspect, the present disclosure provides for the use of a reagent for detecting methylation of the SGIP1, CCNA1, and EYA4 genes in the preparation of a digestive tract tumor detection product, wherein the reagent comprises a PCR primer-probe combination, wherein the PCR primer-probe combination comprises: PCR primer-probe combinations for SGIP1 methylation detection include primer-probe combination 1, primer-probe combination 2, or primer-probe combination 3; 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; PCR primer-probe combinations for CCNA1 methylation detection, including primer-probe combination 4, primer-probe combination 5, or primer-probe combination 6; The primer-probe combination 4 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 primer-probe combination 5 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 primer-probe combination 6 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; PCR primer-probe combinations for EYA4 methylation detection, including primer-probe combination 7, primer-probe combination 8, or primer-probe combination 9; The primer-probe combination 7 includes an upstream primer as shown in SEQ ID NO.19, a downstream primer as shown in SEQ ID NO.20, and a fluorescent probe as shown in SEQ ID NO.21; The primer-probe combination 8 includes an upstream primer as shown in SEQ ID NO.22, a downstream primer as shown in SEQ ID NO.23, and a fluorescent probe as shown in SEQ ID NO.24; The primer-probe combination 9 includes an upstream primer as shown in SEQ ID NO.25, a downstream primer as shown in SEQ ID NO.26, and a fluorescent probe as shown in SEQ ID NO.27.

[0015] As an embodiment, the SGIP1, CCNA1 and EYA4 gene methylation are used as biomarkers.

[0016] 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 gastrointestinal tumors. By designing specific primers and probes, the PCR primer-probe combination can rapidly and sensitively detect the methylation status of the promoter regions of the SGIP1, CCNA1, and EYA4 genes, thereby providing a basis for early screening, accurate detection, and individualized treatment of gastrointestinal tumors.

[0017] The disclosed embodiments design primer-probe combinations with specific sequences for the gene methylation status of specific sites (SGIP1, CCNA1, and EYA4), and integrate specific primers for detecting liver cancer, specific primers for detecting esophageal cancer, and specific primers for detecting gastric cancer into the same kit. The above primers do not interfere with each other in the system (no template competition, primer dimers, or cross-binding will occur) and are compatible. Through highly specific gene amplification, the uniformity of the amplification efficiency of each gene is effectively guaranteed, thereby achieving early diagnosis, efficacy monitoring, and prognosis evaluation of liver cancer, esophageal cancer, and gastric cancer.

[0018] In summary, the PCR primer-probe combination provided in the embodiments of the present disclosure has the advantages of high sensitivity, high specificity and simple operation. It can realize the efficient collaborative detection of specific primers for liver cancer, esophageal cancer and gastric cancer in a single kit, ensuring the accuracy and clinical reliability of the test results, and is suitable for large-scale clinical screening and diagnosis.

[0019] 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

[0020] Figure 1 A schematic diagram of the ROC curve for SGIP1 methylation detection is shown; Figure 2 Shown is a schematic diagram of the ROC curve for CCNA1 methylation detection; Figure 3 A schematic diagram of the ROC curve for EYA4 methylation detection is shown. DETAILED DESCRIPTION

[0021] 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.

[0022] 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.

[0023] 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.

[0024] The following describes in detail the PCR primer and probe combination for detecting gene methylation in digestive tract tumors and its use in this embodiment.

[0025] First, the PCR primer-probe combination for detecting gene methylation in digestive tract tumors according to the first aspect of this embodiment is described.

[0026] PCR primer and probe combinations for gene methylation detection in digestive tract tumors In the prior art, there are methylation abnormalities in multiple genes in gastrointestinal tumors. These abnormal methylations may participate in the occurrence and development of tumors by affecting the functions of key genes. In multi-gene joint detection, adding multiple pairs of primers and probes (such as three genes) in the same reaction system greatly increases the risk of primer dimer formation, nonspecific amplification, and mutual interference between different primers / probes (such as competitive inhibition), resulting in decreased amplification efficiency, decreased sensitivity, or false positive / false negative results. That is, in the detection process, it is necessary to consider the possible mutual interference between multiple pairs of primers to ensure the uniformity of the amplification efficiency of each gene. Detecting the methylation status of multiple genes at the same time will increase the complexity of the experiment and the difficulty of data analysis.

[0027] For example, combined detection requires the simultaneous amplification of multiple genes, and primers may compete for resources, resulting in differences in amplification efficiency, which will cause deviations in the quantitative results based on Ct values, thereby affecting the accurate assessment of gene expression levels; for example, genes with low amplification efficiency may not be effectively detected, resulting in false-negative results; genes with high amplification efficiency may mask the signals of other genes, resulting in false-positive results.

[0028] In view of this, this embodiment proposes a PCR primer probe combination for detecting gene methylation in digestive tract tumors. Specifically, this embodiment is to jointly detect the methylation status of SGIP1, CCNA1 and EYA4 genes.

[0029] The PCR primer probe combination provided in this embodiment includes: (1) PCR primer-probe combination for SGIP1 methylation detection, including primer-probe combination 1, primer-probe combination 2, or primer-probe combination 3; 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) PCR primer-probe combinations for CCNA1 methylation detection, including primer-probe combination 4, primer-probe combination 5, or primer-probe combination 6; The primer-probe combination 4 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 primer-probe combination 5 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 primer-probe combination 6 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) PCR primer-probe combinations for EYA4 methylation detection, including primer-probe combination 7, primer-probe combination 8, or primer-probe combination 9; The primer-probe combination 7 includes an upstream primer as shown in SEQ ID NO.19, a downstream primer as shown in SEQ ID NO.20, and a fluorescent probe as shown in SEQ ID NO.21; The primer-probe combination 8 includes an upstream primer as shown in SEQ ID NO.22, a downstream primer as shown in SEQ ID NO.23, and a fluorescent probe as shown in SEQ ID NO.24; The primer-probe combination 9 includes an upstream primer as shown in SEQ ID NO.25, a downstream primer as shown in SEQ ID NO.26, and a fluorescent probe as shown in SEQ ID NO.27.

[0030] It can be understood that the PCR primer probe combination of this embodiment includes a PCR primer probe combination for SGIP1 methylation detection (primer probe combination 1, primer probe combination 2 or primer probe combination 3), a PCR primer probe combination for CCNA1 methylation detection (primer probe combination 4, primer probe combination 5 or primer probe combination 6) and a PCR primer probe combination for EYA4 methylation detection (primer probe combination 7, primer probe combination 8 or primer probe combination 9); wherein, any one of primer probe combination 1, primer probe combination 2 or primer probe combination 3 can be used in combination with any one of primer probe combination 4, primer probe combination 5 or primer probe combination 6, and any one of primer probe combination 7, primer probe combination 8 or primer probe combination 9 to simultaneously and rapidly and sensitively detect the methylation status of the promoter regions of the SGIP1, CCNA1 and EYA4 genes.

[0031] For example, primer-probe combination 1, primer-probe combination 4, and primer-probe combination 7 are simultaneously integrated into the same kit for detection.

[0032] The gene methylation detection sites in this embodiment include SGIP1, CCNA1 and EYA4, among which SGIP1, CCNA1 and EYA4 gene methylation serve as biomarkers; this embodiment designs specific PCR primer probe combinations for detecting SGIP1, CCNA1 and EYA4 gene methylation for the specific methylation detection sites of SGIP1, CCNA1 and EYA4. By designing specific primers and probes, this PCR primer probe combination can quickly and sensitively detect the methylation status of the SGIP1, CCNA1 and EYA4 gene promoter regions, thereby providing a basis for early screening, accurate detection and personalized treatment of gastrointestinal tumors.

[0033] Among them, the SGIP1 detection site is used to specifically detect liver cancer, the CCNA1 detection site is used to specifically detect gastric cancer, and the EYA4 detection site is used to specifically detect esophageal cancer.

[0034] 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.

[0035] 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.

[0036] 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 example can detect the methylation status of the SGIP1, CCNA1, and EYA4 genes by measuring the fluorescence intensity in the reaction system.

[0037] 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.

[0038] 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.

[0039] It can be understood that in this embodiment, a primer-probe combination with a specific sequence is designed for the gene methylation status of specific sites (SGIP1, CCNA1 and EYA4), and specific primers for detecting liver cancer, specific primers for detecting esophageal cancer, and specific primers for detecting gastric cancer are integrated into the same kit. The above primers do not interfere with each other in the system (no template competition, primer dimers or cross-binding will occur) and are compatible. Through highly specific gene amplification, the uniformity of the amplification efficiency of each gene is effectively guaranteed, and at the same time, early diagnosis, efficacy monitoring and prognosis evaluation of liver cancer, esophageal cancer and gastric cancer can be achieved.

[0040] Specifically, this embodiment, by designing a primer-probe combination with specific sequences for the gene methylation status of specific loci, enables single-tube detection, effectively avoiding the problem of false positives caused by PCR product contamination. The PCR primer-probe combination provided in this example enables fluorescent PCR amplification, improving detection throughput while also offering high sensitivity, good specificity, simple operation, and prevention of cross-contamination. This embodiment can detect the methylation status of the SGIP1, CCNA1, and EYA4 genes based on the detected fluorescence intensity. Based on the methylation status of the SGIP1, CCNA1, and EYA4 gene promoter regions, this can provide a basis for early screening, accurate detection, and personalized treatment of gastrointestinal tumors.

[0041] In summary, the PCR primer-probe combination provided in the embodiments of the present disclosure has the advantages of high sensitivity, high specificity and simple operation. It can realize the efficient collaborative detection of specific primers for liver cancer, esophageal cancer and gastric cancer in a single kit, ensuring the accuracy and clinical reliability of the test results, and is suitable for large-scale clinical screening and diagnosis.

[0042] 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.28, a downstream primer as shown in SEQ ID NO.29, and a fluorescent probe as shown in SEQ ID NO.30; The internal standard primer-probe combination 2 includes an upstream primer as shown in SEQ ID NO.31, a downstream primer as shown in SEQ ID NO.32, and a fluorescent probe as shown in SEQ ID NO.33; The internal standard primer-probe combination 3 includes an upstream primer as shown in SEQ ID NO.34, a downstream primer as shown in SEQ ID NO.35, and a fluorescent probe as shown in SEQ ID NO.36.

[0043] 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.

[0044] Specifically, the detection genes and corresponding detection channels of this embodiment are shown in Table 1 below.

[0045] Table 1:

[0046] Next, the detection kit according to the second aspect of this embodiment will be described.

[0047] Detection kit The digestive tract tumor gene methylation detection kit of this embodiment includes the PCR primer probe combination described in the first aspect.

[0048] The kit of this embodiment is used to detect digestive tract tumors, and specifically can detect the methylation levels of SGIP1, CCNA1, and EYA4 genes in biological samples.

[0049] 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.

[0050] As a preferred embodiment, the body fluid is selected from the group consisting of whole blood, serum, plasma, and any combination thereof.

[0051] It should be noted that the test results of the aforementioned test kits do not involve 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 gastrointestinal tumor test kit is required to make an accurate disease assessment and formulate an appropriate treatment plan.

[0052] As an embodiment, the detection kit generally further includes a PCR reaction reagent (or PCR MIX); The PCR reaction reagents include dNTPs, DNA polymerase and 10×PCR Buffe, the content of the DNA polymerase is 3% to 6%, the content of the 10×PCR Buffe is 30% to 40%, and the content of the dNTPs is 15% to 30%, based on the total volume of the PCR reaction reagents.

[0053] Typically, in addition to DNA polymerase, 10× PCR 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.

[0054] For example, in every 10.5 uL of PCR reaction reagent, the content of DNA polymerase is 0.5 uL / 10.5 uL, the content of 10×PCRBuffe is 4 uL / 10.5 uL, the content of dNTPs is 3 uL / 10.5 uL, and the content of pure water is 3 uL / 10.5 uL.

[0055] As an embodiment, the detection kit generally further includes a negative control and a positive control; for example, the positive control is a methylation-positive nucleic acid of each gene, and the negative control is a methylation-negative nucleic acid of each gene.

[0056] It is understandable that the negative control test result is negative and the positive control test result is positive. Negative quality control can eliminate false positive results caused by reagent or environmental contamination; positive quality control can monitor the performance of reagents or whether personnel operation is normal to eliminate false negative results.

[0057] 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.

[0058] Illustratively, the nucleic acid extraction or purification reagent can use the "Nucleic Acid Extraction or Purification Reagent (Plasma Serum)" produced by Anhui Dajian Medical Technology Co., Ltd. (Medical Device Registration Certificate Number: Wanwu Xiebei 20220023). The operation steps are shown in the kit instructions.

[0059] The bisulfite conversion agent can use the "Nucleic Acid Extraction or Purification Reagent (Centrifugal Column Type)" produced by Anhui Dajian Medical Technology Co., Ltd. (Medical Device Registration Certificate Number: Wanwu Xiebei 20200003). The operation steps are shown in the kit instructions.

[0060] It is understood that the specific operations of the above reagents can be found in the corresponding instructions, and will not be described in detail here.

[0061] For example, in the kit of this embodiment, the final concentration and dosage of each component are shown in Tables 2 and 3 below.

[0062] Table 2: PCR primer probe mixture

[0063] Table 3: PCR MIX

[0064] In summary, based on the PCR primer-probe combination of the first aspect, the kit of this embodiment can quickly and sensitively detect the methylation status of the promoter regions of the SGIP1, CCNA1, and EYA4 genes by designing specific primers and probes, thereby providing a basis for early screening, accurate detection, and personalized treatment of gastrointestinal tumors.

[0065] The following describes the detection method according to the third aspect of this embodiment.

[0066] 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.

[0067] 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.

[0068] The following further describes the relevant steps in the detection method of this embodiment.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] Next, the DNA of the sample to be tested extracted in the above steps is subjected to bisulfite conversion and purification.

[0073] Step (2) converts the DNA of the sample to be tested into bisulfite to obtain converted DNA, namely Bis-DNA.

[0074] Among them, the bisulfite conversion treatment can be carried out by using the nucleic acid extraction or purification reagent (centrifugal column type) produced by Anhui Dajian Medical Technology Co., Ltd. (product registration number: Wanwu Xiebei 20200003).

[0075] 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.

[0076] Next, after obtaining Bis-DNA, a detection kit is used to perform detection through PCR reaction.

[0077] 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.

[0078] 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.

[0079] 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, for 20 cycles; 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.

[0080] It should be noted that during the above sampling process: (1) Use a sterile syringe to collect 3-5 ml of the subject's venous blood into a 5 ml disposable sterile coagulation tube. Immediately after whole blood collection, let it stand at room temperature for 30-60 minutes to precipitate the serum. Perform two low-temperature centrifugations as soon as possible to obtain 1.5-2.0 ml of the subject's serum sample. The processed serum sample should be stored in a refrigerator at 2-8°C for no more than 3 days. The serum sample can be stored at -20±5°C for no more than 4 weeks and below -70°C for 24 months. Avoid repeated freezing and thawing, and the number of repeated freezing and thawing should not exceed 3 times.

[0081] (2) The extracted nucleic acid samples should be tested as soon as possible, and stored at -20±5℃ for no more than 14 days. The number of freeze-thaw cycles should not exceed 3 times.

[0082] (3) Samples treated with bisulfite should be tested as soon as possible, at -20 ± 5°C for no more than 3 days, and avoid repeated freezing and thawing.

[0083] The storage conditions of the samples are as follows: no more than 24 hours at room temperature; 3 months in a refrigerator at -20℃±5℃; 12 months in a refrigerator at -70℃ or below.

[0084] In summary, the detection method of this embodiment can detect the methylation status of the SGIP1, CCNA1, and EYA4 genes based on the detected fluorescence intensity. Based on the methylation status of the promoter regions of the SGIP1, CCNA1, and EYA4 genes, it can provide a basis for early screening, accurate detection, and personalized treatment of gastrointestinal tumors.

[0085] Next, the purpose of the fourth aspect in this embodiment will be described.

[0086] use This embodiment also proposes the use of the PCR primer-probe combination described in the first aspect in preparing a kit for detecting digestive tract tumors.

[0087] As described in the first aspect, the gene methylation detection sites of this embodiment include SGIP1, CCNA1 and EYA4. Targeting the specific methylation detection sites of SGIP1, CCNA1 and EYA4, this embodiment designs a specific PCR primer probe combination for detecting methylation of the SGIP1, CCNA1 and EYA4 genes. This PCR primer probe combination can quickly and sensitively detect the methylation status of the promoter regions of the SGIP1, CCNA1 and EYA4 genes by designing specific primers and probes, thereby providing a basis for early screening, accurate detection and individualized treatment of gastrointestinal tumors.

[0088] Therefore, this embodiment can prepare a detection kit for detecting the methylation status of SGIP1, CCNA1 and EYA4 genes based on the PCR primer probe combination described in the first aspect; the SGIP1, CCNA1 and EYA4 gene methylation detection kit has the advantages of high sensitivity and high specificity in detecting gastrointestinal tumors.

[0089] Next, the use of the reagent for detecting methylation of SGIP1, CCNA1 and EYA4 genes according to the fifth aspect of this embodiment in preparing a product for detecting digestive tract tumors will be described.

[0090] Use of reagents for detecting SGIP1, CCNA1, and EYA4 gene methylation in the preparation of digestive tract tumor detection products This embodiment further proposes the use of a reagent for detecting methylation of SGIP1, CCNA1, and EYA4 genes in the preparation of a digestive tract tumor diagnosis product, wherein the reagent comprises a PCR primer-probe combination, and the PCR primer-probe combination comprises: PCR primer-probe combinations for SGIP1 methylation detection include primer-probe combination 1, primer-probe combination 2, or primer-probe combination 3; 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; PCR primer-probe combinations for CCNA1 methylation detection, including primer-probe combination 4, primer-probe combination 5, or primer-probe combination 6; The primer-probe combination 4 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 primer-probe combination 5 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 primer-probe combination 6 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; PCR primer-probe combinations for EYA4 methylation detection, including primer-probe combination 7, primer-probe combination 8, or primer-probe combination 9; The primer-probe combination 7 includes an upstream primer as shown in SEQ ID NO.19, a downstream primer as shown in SEQ ID NO.20, and a fluorescent probe as shown in SEQ ID NO.21; The primer-probe combination 8 includes an upstream primer as shown in SEQ ID NO.22, a downstream primer as shown in SEQ ID NO.23, and a fluorescent probe as shown in SEQ ID NO.24; The primer-probe combination 9 includes an upstream primer as shown in SEQ ID NO.25, a downstream primer as shown in SEQ ID NO.26, and a fluorescent probe as shown in SEQ ID NO.27.

[0091] It can be understood that this embodiment uses methylation of the SGIP1, CCNA1, and EYA4 genes as biomarkers. Targeting the specific methylation detection sites of SGIP1, CCNA1, and EYA4, this embodiment designs a specific PCR primer-probe combination for detecting methylation of the SGIP1, CCNA1, and EYA4 genes. This PCR primer-probe combination, by designing specific primers and probes, can rapidly and sensitively detect the methylation status of the promoter regions of the SGIP1, CCNA1, and EYA4 genes.

[0092] In combination with the first aspect, in this embodiment, a primer probe combination with a specific sequence is designed for the gene methylation status of specific sites (SGIP1, CCNA1 and EYA4), and specific primers for detecting liver cancer, specific primers for detecting esophageal cancer, and specific primers for detecting gastric cancer are integrated into the same kit. The above primers do not interfere with each other in the system (no template competition, primer dimers or cross-binding will occur) and are compatible. Through highly specific gene amplification, the uniformity of the amplification efficiency of each gene is effectively guaranteed, thereby realizing early diagnosis, efficacy monitoring and prognosis evaluation of liver cancer, esophageal cancer and gastric cancer.

[0093] The following is a further explanation of the result analysis method and quality control standards of the detection method.

[0094] Result Analysis This study analyzed the methylation levels of the SGIP1, CCNA1, and EYA4 genes based on the amplification curves and threshold cycle numbers (Ct values) of fluorescent PCR. A higher methylation level indicates lower expression of the SGIP1, CCNA1, and EYA4 genes and a higher malignant potential of the digestive tract tumor.

[0095] Threshold setting: You can set the threshold automatically according to the instrument output, or manually adjust the baseline according to the instrument's instructions. Set the threshold to the linear portion of the fluorescence logarithmic plot and read the Ct value from the software. If the gene is not amplified, the Ct value is set to 40.

[0096] Test kit validity determination: If the kit controls meet the criteria listed in Table 4 and the test sample is assayed in the same PCR reaction along with the negative and positive controls, the PCR reaction is considered valid.

[0097] Table 4: Test kit validity determination

[0098] Determination of test effectiveness: If the internal standard gene Ct value is ≤20 and the amplification curve is S-shaped, analysis can continue. If the internal standard gene Ct value is >20 or no amplification curve is observed, but the target gene Ct value is ≤36, analysis can continue (possibly due to inhibition of target gene amplification by the internal standard gene). If the internal standard gene Ct value is >20 or no amplification curve is observed, and the target gene is not amplified, or amplified but with a Ct value >36, analysis cannot be continued and testing must be repeated.

[0099] Test result determination: Please refer to Table 5 for the test result judgment.

[0100] Table 5: Test result judgment

[0101] 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.

[0102] In the following examples, all materials, reagents and instruments used can be purchased from commercial sources unless otherwise specified.

[0103] The present disclosure will be further described below with reference to specific embodiments.

[0104] Example 1 In this example, we screened for hypermethylated genes associated with digestive tract tumors by collecting literature. We then designed specific PCR primer-probe combinations for detecting methylation of the SGIP1, CCNA1, and EYA4 genes, targeting specific methylation detection sites. Specific primers and probes were also designed for the internal reference gene GAPDH. The specific PCR primer-probe combinations and internal reference gene internal standard primer-probe combinations are shown in Table 6 below.

[0105] Table 6:

[0106] The primer probes designed above were used to detect the methylation status of SGIP1, CCNA1, and EYA4 genes in cancer tissue samples. In this example, plasma was used as the sample to be tested (64 samples from benign lesions, 64 samples from liver cancer patients, 64 samples from esophageal cancer patients, and 64 samples from gastric cancer patients).

[0107] The specific detection steps include: (1) Extraction of sample DNA Take the DNA sample to be tested (plasma) and use the nucleic acid extraction or purification reagent produced by Anhui Dajian Medical Technology Co., Ltd. (product registration number: Wanwu Xiebei 20220023) to extract high-purity DNA.

[0108] 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 13,000 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 13,000 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 13,000 rpm for 1 min, and discard the waste liquid; (g) Place the adsorption column in a clean 1.5 mL centrifuge tube and centrifuge at 13,000 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 13,000 rpm for 2 min. Collect the DNA sample into a centrifuge tube and store at -20°C.

[0109] (2) Bisulfite conversion and purification Next, the DNA extracted in the above step is subjected to bisulfite conversion and purification to obtain Bis-DNA.

[0110] 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 50°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, centrifuge at full speed (14,000 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.

[0111] Next, after obtaining Bis-DNA, the bisulfite-converted Bis-DNA was used as a template, and PCR reaction reagents, PCR primer probe combinations for methylation of the SGIP1, CCNA1, and EYA4 genes, and an internal standard primer probe combination for an internal reference gene (shown in Table 6) were added to perform a fluorescent PCR amplification reaction.

[0112] The PCR reaction system is shown in Table 7.

[0113] Table 7: PCR reaction system

[0114] The specific composition of PCR MIX is shown in Table 3.

[0115] The reaction system in Table 7 was thoroughly mixed, centrifuged briefly, and aliquoted into 0.2 mL PCR reaction tubes at a volume of 22 μL per tube. The PCR reaction tubes were placed in a 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.

[0116] The procedure of the above fluorescent PCR amplification reaction is shown in Table 8.

[0117] Table 8: PCR reaction program

[0118] Test results In this example, 256 samples were tested, including 64 samples of benign lesions, 64 samples of liver cancer patients, 64 samples of esophageal cancer patients, and 64 samples of gastric cancer patients, under the condition that the negative control and positive control met the validity judgment of the kit. The sensitivity and specificity of the above gene methylation detection results were analyzed. The comparison of the detection results with the clinical results is shown in Tables 9 to 11 and Figure 1-Figure 3 .

[0119] Among them, Table 9 shows the SGIP1 methylation test results, Figure 1 Figure 10 shows the ROC curve of SGIP1 methylation detection; Table 11 shows the CCNA1 methylation test results. Figure 2 Figure 11 shows the ROC curve for CCNA1 methylation detection; Table 12 shows the EYA4 methylation test results. Figure 3 A schematic diagram of the ROC curve for EYA4 methylation detection is shown.

[0120] Table 9: SGIP1 methylation test results

[0121] Table 10: CCNA1 methylation test results

[0122] Table 11: EYA4 methylation test results

[0123] Combined with Table 9-Table 11 and Figure 1-Figure 3 The test results show that in this embodiment, a primer probe combination with a specific sequence is designed for the gene methylation status of specific sites (SGIP1, CCNA1 and EYA4), and specific primers for detecting liver cancer, specific primers for detecting esophageal cancer, and specific primers for detecting gastric cancer are integrated into the same kit. The above primers do not interfere with each other in the system (no template competition, primer dimers or cross-binding will occur) and are compatible. Through highly specific gene amplification, the uniformity of the amplification efficiency of each gene is effectively guaranteed.

[0124] Specifically, the specificity of the methylation detection results of SGIP1, CCNA1 and EYA4 genes were all greater than 92%, and the sensitivity was greater than 84%; among them, the specificity of the CCNA1 methylation detection results was greater than 95%; the sensitivity of the EYA4 and CCNA1 gene methylation detection results were both greater than 87%.

[0125] Therefore, the specific primers provided in this example ensure the uniformity of the amplification efficiency of each gene through highly specific gene amplification in the system, establish a reliable and efficient multi-gene methylation joint detection system, and can quickly and sensitively detect the methylation status of the promoter regions of the SGIP1, CCNA1 and EYA4 genes, thereby providing a basis for early screening, accurate detection and individualized treatment of gastrointestinal tumors (liver cancer, gastric cancer and esophageal cancer).

[0126] 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 gene methylation in digestive tract tumors, characterized in that: The PCR primer probe combination includes: PCR primer-probe combinations for SGIP1 methylation detection include primer-probe combination 1, primer-probe combination 2, or primer-probe combination 3; 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; PCR primer-probe combinations for CCNA1 methylation detection, including primer-probe combination 4, primer-probe combination 5, or primer-probe combination 6; The primer-probe combination 4 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 primer-probe combination 5 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 primer-probe combination 6 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; PCR primer-probe combinations for EYA4 methylation detection, including primer-probe combination 7, primer-probe combination 8, or primer-probe combination 9; The primer-probe combination 7 includes an upstream primer as shown in SEQ ID NO.19, a downstream primer as shown in SEQ ID NO.20, and a fluorescent probe as shown in SEQ ID NO.21; The primer-probe combination 8 includes an upstream primer as shown in SEQ ID NO.22, a downstream primer as shown in SEQ ID NO.23, and a fluorescent probe as shown in SEQ ID NO.24; The primer-probe combination 9 includes an upstream primer as shown in SEQ ID NO.25, a downstream primer as shown in SEQ ID NO.26, and a fluorescent probe as shown in SEQ ID NO.

27.

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.28, a downstream primer as shown in SEQ ID NO.29, and a fluorescent probe as shown in SEQ ID NO.30; The internal standard primer-probe combination 2 includes an upstream primer as shown in SEQ ID NO.31, a downstream primer as shown in SEQ ID NO.32, and a fluorescent probe as shown in SEQ ID NO.33; The internal standard primer-probe combination 3 includes an upstream primer as shown in SEQ ID NO.34, a downstream primer as shown in SEQ ID NO.35, and a fluorescent probe as shown in SEQ ID NO.

36.

3. The PCR primer-probe combination according to claim 1 or 2, 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 or 2, 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 digestive tract tumor 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 digestive tract tumor 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 digestive tract tumors.

9. Use of a reagent for detecting methylation of SGIP1, CCNA1 and EYA4 genes in the preparation of a digestive tract tumor detection product, characterized in that: The reagents include a PCR primer-probe combination, and the PCR primer-probe combination includes: PCR primer-probe combinations for SGIP1 methylation detection include primer-probe combination 1, primer-probe combination 2, or primer-probe combination 3; 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; PCR primer-probe combinations for CCNA1 methylation detection, including primer-probe combination 4, primer-probe combination 5, or primer-probe combination 6; The primer-probe combination 4 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 primer-probe combination 5 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 primer-probe combination 6 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; PCR primer-probe combinations for EYA4 methylation detection, including primer-probe combination 7, primer-probe combination 8, or primer-probe combination 9; The primer-probe combination 7 includes an upstream primer as shown in SEQ ID NO.19, a downstream primer as shown in SEQ ID NO.20, and a fluorescent probe as shown in SEQ ID NO.21; The primer-probe combination 8 includes an upstream primer as shown in SEQ ID NO.22, a downstream primer as shown in SEQ ID NO.23, and a fluorescent probe as shown in SEQ ID NO.24; The primer-probe combination 9 includes an upstream primer as shown in SEQ ID NO.25, a downstream primer as shown in SEQ ID NO.26, and a fluorescent probe as shown in SEQ ID NO.

27.

10. The use according to claim 9, characterized in that The SGIP1, CCNA1 and EYA4 gene methylation serve as biomarkers.

Images