Methylation marker combination, primer probe combination and kit for detecting gastric cancer and application of methylation marker combination, primer probe combination and kit

Abstract

The invention discloses a methylation marker combination for detecting gastric cancer, a corresponding primer probe combination, a kit and application thereof. The methylation marker combination related to the gastric cancer comprises a CpG methylation site cg08630279 of a ZNF569 gene, a CpG methylation site cg24773720 of a GHR gene and a CpG methylation site cg20622089 of a CNR1 gene, wherein the CpG methylation site cg08630279 of the ZNF569 gene, the CpG methylation site cg24773720 of the GHR gene and the CpG methylation site The methylation levels of the ZNF569, GHR and CNR1 sites are related to gastric cancer staging, and the ZNF569, GHR and CNR1 sites can be used as high-specificity markers for early screening of gastric cancer. A primer probe combination for detecting the marker combination comprises specific upstream primers, downstream primers and probes aiming at methylation sites of all markers. The kit comprises a primer probe combination, and also comprises a positive control, a negative control and a quality control reagent. Three-site combined detection is adopted, the detection sensitivity in a gastric cancer blood sample reaches 91% or above, the specificity reaches up to 100%, the kit is used for auxiliary screening, detection or molecular analysis related to gastric cancer and precancerous lesions of the gastric cancer, and a technical scheme high in sensitivity, high in specificity and good in repeatability is provided for clinical detection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and DNA detection technology, and relates to a methylation marker combination for detecting gastric cancer, a primer probe combination, a kit, and application of the marker combination and the kit in detecting gastric cancer. BACKGROUND

[0002] Gastric cancer (GC) is one of the malignant tumors with high morbidity and mortality worldwide. Although screening and treatment techniques have improved in recent years, most patients are in the middle and advanced stages when diagnosed, which seriously affects the treatment effect and survival rate.

[0003] Early lesions of gastric cancer often have no obvious specific symptoms, and the early detection rate is low. The 5-year survival rate of patients with stage 0 and stage I gastric cancer can exceed 90%, while the 5-year survival rate of patients with stage III and stage IV gastric cancer is less than 30%. Therefore, improving the early screening rate is the key to reducing the mortality of gastric cancer.

[0004] Currently, the main screening and diagnosis methods for gastric cancer include gastroscopy, histopathological detection, imaging examination, and tumor marker detection (such as CEA, CA19-9, CA72-4, etc.). Among them, gastroscopy combined with biopsy is still the gold standard for diagnosis, but it is complex, highly invasive, expensive, and has poor patient compliance, which is not conducive to large-scale screening. Although marker detection is simple, its sensitivity and specificity are limited, especially in early gastric cancer, and the detection rate is low. Imaging examinations such as CT or MRI also have difficulty in detecting early micro lesions.

[0005] With the development of molecular biology and epigenetics, molecular diagnostic methods based on DNA methylation detection have gradually become a research hotspot for early screening of tumors. DNA methylation is a common and stable epigenetic modification that usually occurs on the CpG island of the gene promoter region and can cause gene transcription silencing. Many studies have shown that tumor cells exhibit specific methylation abnormalities at an early stage of carcinogenesis, and can be released into peripheral blood, gastric juice, or blood DNA, thus serving as an ideal molecular marker for early screening of tumors.

[0006] Existing DNA methylation studies on gastric cancer have reported multiple candidate genes, including CDH1, RASSF1A, RNF180, SEPTIN9, etc. These markers have different degrees of hypermethylation in gastric cancer tissues, but the sensitivity of single gene detection is usually between 60% and 80%, and the specificity is about 80% to 90%, still with false negative and false positive problems. Some studies have attempted to combine multiple methylation sites for detection to improve the diagnostic accuracy, but a standardized detection system that can be promoted clinically has not yet been formed.

[0007] In addition, the currently approved or commercialized methylation detection products are mainly focused on colorectal cancer (such as the Cologuard kit approved by the US FDA, which screens blood DNA by detecting the methylation of NDRG4 and BMP3 genes), and there is no high-sensitivity, high-specificity multi-site combined methylation detection product for gastric cancer. Therefore, it is of great clinical significance and market value to develop a gastric cancer methylation detection system with good stability, high specificity and suitable for large-scale clinical screening.

[0008] To solve the problems of difficult balance between sensitivity and specificity, insufficient early screening coverage and the like in the prior art, it is necessary to develop a specific methylation marker combination for gastric cancer and a matching primer probe system, significantly improve the detection sensitivity and specificity through multi-site combined detection, realize early and high-precision detection of gastric cancer, and provide a reliable technical means for clinical screening and auxiliary diagnosis. SUMMARY

[0009] To solve the key problems of insufficient sensitivity, low specificity and poor compliance of traditional detection methods in early diagnosis of gastric cancer, the present application provides a new scheme for gastric cancer screening based on specific DNA methylation detection. By screening and verifying a combination of methylation sites specific to gastric cancer, a primer probe system with high specificity and high sensitivity is designed, and a detection kit suitable for clinical and large-scale screening is constructed, thereby realizing early and high-precision detection of gastric cancer.

[0010] The existing gastric cancer screening and auxiliary diagnosis methods have obvious deficiencies: gastroscopy as the gold standard is highly invasive and has poor compliance; the sensitivity and specificity of the markers are limited; and the detection rate of early lesions by imaging methods is not high. Although DNA methylation detection technology has been proven to be effective in several tumors, there is still a lack of mature detection products specifically for gastric cancer with a stable target combination. To achieve the above-mentioned purposes, the present application discloses a gastric cancer-specific methylation marker combination for detecting the methylation marker combination of gastric cancer, which is composed of target gene sequences or fragments containing the following CpG methylation sites:

[0011] Methylation marker 1: CpG methylation site cg08630279 of ZNF569 gene;

[0012] Methylation marker 2: CpG methylation site cg24773720 of GHR gene;

[0013] Methylation marker 3: CpG methylation site cg20622089 of CNR1 gene.

[0014] The methylation status of the above three sites has a significant correlation with the occurrence of gastric cancer, and their combined detection can significantly improve the detection sensitivity and specificity.

[0015] The cg08630279 site of the ZNF569 gene comprises the nucleotide sequence shown in SEQ ID NO. 16.

[0016] The cg24773720 site of the GHR gene comprises the nucleotide sequence shown in SEQ ID NO. 17.

[0017] The cg20622089 site of the CNR1 gene comprises the nucleotide sequence shown in SEQ ID NO. 18.

[0018] In another aspect, the present application discloses a primer probe combination for detecting the above-mentioned methylation marker combination, which comprises an upstream primer, a downstream primer and a probe for each methylation marker methylation site, wherein:

[0019] The upstream primer for the methylation marker 1 methylation site is ZNF569-FP as shown in SEQ ID NO. 1, the downstream primer is ZNF569-RP as shown in SEQ ID NO. 2, and the probe is ZNF569-P as shown in SEQ ID NO. 3;

[0020] The upstream primer for the methylation marker 2 methylation site is GHR-FP as shown in SEQ ID NO. 4, the downstream primer is GHR-RP as shown in SEQ ID NO. 5, and the probe is GHR-P as shown in SEQ ID NO. 6;

[0021] The upstream primer for the methylation marker 3 methylation site is CNR1-FP as shown in SEQ ID NO. 7, the downstream primer is CNR1-RP as shown in SEQ ID NO. 8, and the probe is CNR1-P as shown in SEQ ID NO. 9.

[0022] Further, the nucleotide sequences of the primers and the probes are designed according to the nucleotide sequences of the corresponding methylation sites of the target genes after bisulfite modification, and the bisulfite modified sequences are as follows:

[0023] The bisulfite modified sequence of the ZNF569 gene is as shown in SEQ ID NO. 10;

[0024] The bisulfite modified sequence of the GHR gene is as shown in SEQ ID NO. 11;

[0025] The bisulfite modified sequence of the CNR1 gene is as shown in SEQ ID NO. 12.

[0026] Further, the ZNF569 gene cg08630279 site in the methylation case is the 1st CG from the 5' end to the 3' end of the nucleotide sequence shown as SEQ ID NO. 3;

[0027] The GHR gene cg24773720 site in the methylation case is the 1st CG from the 5' end to the 3' end of the nucleotide sequence shown as SEQ ID NO. 6;

[0028] The CNR1 gene cg20622089 site in the methylation case is the 2nd CG from the 5' end to the 3' end of the nucleotide sequence shown as SEQ ID NO. 9.

[0029] The application also discloses a kit comprising the primer probe combination for detecting the methylation marker combination.

[0030] Further, the kit further comprises a positive control; the positive control comprises a plasmid containing a target gene methylation sequence after being modified by sulfite and / or a gastric cancer cell strain; the carrier plasmid is selected from pUC57, pUC57-Kan, pUC57-Simple, pUC57-mini, pUC18 or pUC19; the gastric cancer cell strain is selected from MKN-45, AGS, SGC-7901, BGC-823 or HGC-27, and the positive cell strain is from the American Type Culture Collection (ATCC); and the target gene methylation sequence after being modified by sulfite is as follows:

[0031] The ZNF569 gene sulfite modification sequence is shown as SEQ ID NO. 10;

[0032] The GHR gene sulfite modification sequence is shown as SEQ ID NO. 11;

[0033] The CNR1 gene sulfite modification sequence is shown as SEQ ID NO. 12.

[0034] Further, the kit further comprises a quality control gene primer pair and a quality control gene probe; the quality control gene is β-actin (β-actin), wherein the quality control gene primer pair is a quality control gene forward primer and a quality control gene reverse primer; and the primer and probe sequences are as follows:

[0035] The quality control gene forward primer (ACTB-FP) is shown as SEQ ID NO. 13;

[0036] The quality control gene reverse primer (ACTB-RP) is shown as SEQ ID NO. 14;

[0037] The quality control gene probe (ACTB-P) is shown as SEQ ID NO. 15.

[0038] In some embodiments, the reporter fluorescent group of the quality control gene probe and the reporter fluorescent group of the probe ZNF569-P, the probe GHR-P, the probe CNR1-P are each independently selected from one or more of FAM, VIC, NED, CY5, CY3, JOE, HEX; the reporter quencher group of the quality control gene probe and the reporter quencher group of the probe ZNF569-P, the probe GHR-P, the probe CNR1-P are each independently selected from one or more of MGB, BHQ1, BHQ2, TAMRA.

[0039] Further, the kit further comprises a negative control; the negative control is normal human genomic DNA, or DNA of a negative cell line 293T (from ATCC).

[0040] Further, the kit detection result is determined by qPCR analysis result of the primer probe combination:

[0041] Quality control effectiveness: Ct of β-actin ≤ 36, indicating that the DNA template loading amount is within the allowable range, and the result is reliable; Ct > 36, indicating that the DNA template loading amount is not within the allowable range, and the result is not reliable;

[0042] Site positive determination: Ct of ZNF569, GHR and CNR1 single site ≤ 40 is determined as methylation positive, and Ct > 40 is determined as negative;

[0043] Joint determination: any site is methylation positive, which is determined as gastric cancer positive, and three sites are all negative, which is determined as gastric cancer negative.

[0044] Further, the qPCR is real-time fluorescence qPCR, and the reaction conditions are as follows: 96℃ pre-denaturation for 3 minutes; 15 cycles of 95℃ denaturation for 15 seconds, 70℃ annealing and extension for 20 seconds, 64℃ annealing and extension for 20 seconds, and 72℃ extension for 10 seconds; 35 cycles of 95℃ denaturation for 15 seconds, 70℃ annealing and extension for 20 seconds, 60℃ annealing and extension for 34 seconds, and 72℃ extension for 10 seconds; and 35 cycles of detecting fluorescence signal during annealing.

[0045] Preferably, the final concentration of each primer in the qPCR is 0.2-0.3 μM, and the final concentration of the probe is 0.2 μM.

[0046] Preferably, the amplification system of the qPCR is as follows: 2×Premix Ex Taq TM (Probe qPCR) 20 μL, 50×Rox II 0.8 μL, final concentration of each primer 0.2-0.3 μM, final concentration of the probe 0.2 μM, DNA template > 10 ng, and water is added to 40 μL.

[0047] Further, other reagents required for the qPCR reaction, such as polymerase, buffer, dNTP.

[0048] Preferably, the polymerase is 2x Premix Ex Taq TM (Probe qPCR); the buffer is 50x Rox II. 2x Premix Ex Taq TM (Probe qPCR) contains dNTP.

[0049] The CpG methylation site cg08630279 of the ZNF569 gene, the CpG methylation site cg24773720 of the GHR gene and the CpG methylation site cg20622089 of the CNR1 gene of the present application have not been reported to be used for the detection of gastric cancer at the same time, and in the multi-tumor comparison, it is shown that the methylation is high in gastric cancer, but the methylation degree is low in other tumors (pancreatic cancer, colorectal cancer, lung squamous cell carcinoma, lung adenocarcinoma and esophageal cancer, etc.), indicating that the site combination is specific to gastric cancer. Therefore, the present application provides a new DNA methylation marker for a gastric cancer detection kit.

[0050] The kit of the present application is a patient's peripheral blood, and adopts a fluorescence qPCR detection technology, the result is intuitive to detect the change in the qPCR process, and the whole detection process is optimized, the detection speed is fast, the steps are simple, and compared with the existing method, the sensitivity is good, and the specificity is obviously superior, the problem that the sensitivity and specificity are difficult to balance in the prior art and the early screening coverage is insufficient is solved, and the kit is more suitable for early screening of gastric cancer.

[0051] The present application also relates to the use of the methylation marker combination of the present application in the preparation of a primer probe or methylation detection reagent for detecting gastric cancer, wherein the gastric cancer-related methylation marker comprises the CpG methylation site cg08630279 of the ZNF569 gene, the CpG methylation site cg24773720 of the GHR gene and the CpG methylation site cg20622089 of the CNR1 gene; the primer probe combination is used for detecting the gastric cancer-related methylation marker.

[0052] The present application also relates to the use of the kit of the present application in the preparation of a molecular analysis product for detecting gastric cancer screening, detection, diagnosis or prognosis evaluation.

[0053] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0054] 1. Innovative three-site combination, significantly improving detection performance.

[0055] The application firstly proposes that the CpG methylation site cg08630279 of the ZNF569 gene, the CpG methylation site cg24773720 of the GHR gene and the CpG methylation site cg20622089 of the CNR1 gene are jointly applied to gastric cancer detection. The three sites all show high methylation in gastric cancer samples, and the methylation degree is lower in normal tissues and other tumors. Through database sample analysis and clinical sample verification, the sensitivity of the three-site joint detection is significantly improved, among which the detection sensitivity for gastric cancer is improved to 91.57%, and the detection sensitivity for precancerous lesions reaches 80%; the specificity all reaches 100%, which is obviously better than the existing single-point (such as CDH1, RASSF1A) or double-site (such as RNF180+SEPTIN9) detection scheme, and solves the problem that the sensitivity and specificity are difficult to balance in the prior art.

[0056] 2. Simple operation.

[0057] The detection sample source of the application is peripheral blood DNA, which is convenient and safe to take, has high patient compliance, and is suitable for large-scale screening. The whole detection process only needs one reaction system to complete the three-site joint detection, the detection process is standardized and has high repeatability, and is suitable for clinical laboratory and primary medical institutions.

[0058] 3. Stable detection system, reliable results.

[0059] The application adopts the fluorescence quantitative PCR (qPCR) technology based on TaqMan probe, has strong amplification specificity and high sensitivity. By setting beta-actin (ACTB) as an internal reference quality control gene, the DNA quality of the sample and the effectiveness of the reaction system can be accurately judged, and the accuracy and repeatability of the detection results are ensured.

[0060] 4. Good experimental repeatability, high clinical application value.

[0061] The three-site joint detection system provided by the application can effectively identify early gastric cancer, and provides a detection method with high sensitivity, high specificity, low cost and good repeatability for early screening of gastric cancer, and can be widely applied to clinical screening, auxiliary diagnosis and postoperative recurrence monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0062] The concept, specific structure and generated technical effects of the application will be further described below in combination with the drawings, so as to fully understand the purpose, characteristics and effects of the application.

[0063] Figure 1 ROC curve of joint detection of cg24773720 (GHR), cg08630279 (ZNF569) and cg20622089 (CNR1).

[0064] Figure 2 ROC curve of combined detection of cg24773720 (GHR), cg14142713 (ZNF569) and cg20622089 (CNR1).

[0065] Figure 3 ROC curve of combined detection of cg24773720 (GHR), cg08630279 (ZNF569) and cg14186641 (CNR1).

[0066] Figure 4 ROC curve of combined detection of cg24773720 (GHR), cg14142713 (ZNF569) and cg14186641 (CNR1).

[0067] Figure 5 Differential analysis of ZNF569 locus in cancer tissue (T) and para-cancer tissue (N).

[0068] Figure 6 Differential analysis of GHR locus in cancer tissue (T) and para-cancer tissue (N).

[0069] Figure 7 Differential analysis of CNR1 locus in cancer tissue (T) and para-cancer tissue (N). DETAILED DESCRIPTION

[0070] The application will be further described with some non-limiting examples in connection with the accompanying drawings. It should be understood, however, that these descriptions are merely examples and are not intended to limit the scope of the application. In addition, in the following description, descriptions of well-known and commonly used technologies and methods are omitted to avoid unnecessary confusion of the concept of the application.

[0071] Screening process of Example 1 loci

[0072] The focus and difficulty of the application is to find three loci that can ensure screening specificity and improve detection sensitivity. In order to determine such a locus combination, the methylation number of nearly 13000 cases of 33 tumor types was used in the database during the development process. The specific steps are as follows:

[0073] 1.1 We selected 450 samples (415 tumor samples and 35 normal samples) from the Stomach adenocarcinoma (STAD) database of The Cancer Genome Atlas (TCGA) database. We performed a Wilcoxon rank-sum test on 485,578 loci, setting threshold conditions: p-value < 0.01, mean β ≥ 0.2, and Δβ ≥ 0.2. 14,053 loci met these threshold conditions.

[0074] 1.2 Differential analysis was performed on sample data (111 normal samples) from the population blood methylation database (GSE33651 and GSE54129), and sites with methylation difference values ​​greater than 25%, i.e., 2145 sites, were retained.

[0075] 1.3 CpG sites (cytosine-phosphate-guanine sites, i.e., sites in the DNA sequence immediately following guanine after cytosine) with high methylation levels, associated with key genes of interest, were selected from 450 TCGA database samples and sorted in descending order of β difference (Δβ) ≥ 0.5, β difference (Δβ) ≥ 0.3, and β difference (Δβ) ≥ 0.2. DNA methylation in vertebrates generally occurs at CpG sites.

[0076] 1.4 The 16 loci selected in step 1.3 were combined and sorted in descending order according to the β difference (Δβ) ≥ 0.3. Four combinations with a frequency greater than 95% were selected (see Table 1), and ROC curves of these four combinations were plotted using data from the Cancer Genome Atlas database (see Table 1). Figure 1 , Figure 2 , Figure 3 and Figure 4 The results were consistent with those in Table 1. Further analysis of cell, tissue, and blood samples was conducted to select the most sensitive and specific locus combinations. The screening process for the four combinations in Table 1 is described in Example 2 and Comparative Example 1. Finally, the optimal combination—Combination 1 in Table 1 (cg08630279 locus of ZNF569 gene, cg24773720 locus of GHR gene, and cg20622089 locus of CNR1 gene)—was selected for further research.

[0077] Table 1. Combinations with a frequency greater than 95% after being sorted in descending order.

[0078]

[0079] Example 2 Primer and probe design and screening

[0080] The application respectively takes the constructed plasmid containing the methylation site nucleotide sequence of ZNF569, GHR and CNR1 gene sulfite modification and the DNA of positive cell strain MKN-45 as templates, constructs a ZNF569, GHR and CNR1 gene methylation real-time fluorescence qPCR detection system, takes VIC, FAM and NED as fluorescence signal detection objects, and realizes rapid and accurate detection through the optimization combination of ZNF569, GHR and CNR1 gene methylation primers and the optimization of the detection system of the fluorescence probe. The specific steps are as follows:

[0081] The plasmid is a synthetic plasmid obtained by inserting the artificial designed target gene sequence fragment into a molecular cloning vector plasmid by Shanghai Shenghua Biotechnology Engineering Co., Ltd. The target gene sequence includes the methylation site nucleotide sequence of the ZNF569 gene after sulfite modification, the methylation site nucleotide sequence of the GHR gene after sulfite modification and the methylation site nucleotide sequence of the CNR1 gene after sulfite modification.

[0082] The ZNF569 gene nucleotide sequence (SEQ ID NO. 16, containing the cg08630279 site) is as follows:

[0083] GGCGCGGGCTGGGACAGAGGCGGCACTGAGGCCGGCGCTGTCGGTGGCTGAGAGCGCCACAAGTCTCGGTCCGTTACACCAGGGGCGACGCTTCCCAGAGGCCCCCGCGGCTCACCCGGGCGGGACTGGCTTCACTGTTTGCGCGTCCTGAGAAGCAGACCACGGTGTTCCAGGGCTCACAGCTCCGCGCAGGGGAGCTCAGCCTAGGTTTTGCACGAGCGGCCTCCCGCGAGCCCAGCTCTGAGAGATTGGGAGCGCAACTTGGTGCTGAGAAGAATCGAATCGTTCCGCTGCTTCCTGCGCCGAACTACATTTCCCAGAGGCCTTCGCGGCCCTACTTCCCCGACCCCACCTGCAAAAAGGCACTTCCTTCTTACAGTCGGCTGGAGCTGCAGGTTCGCAAGGTCCGTGAAGGAAGCCCGGCTCAGTGTGTTTAGTTCCGTCTTTTTGGGTCTCTACCTGAGGCTAGAATACAGCGGGGTGAAGGTGGTGAGAGCTCCAGGGCCAGTGAAGGCCGGGTCAGCTGGAGCCTTAAGGTCCTGACAGCGGCTAGAAGAGGGAGGAGTGACGTAGTGTGACTGTATGTGTGAAACTTCGGCCAT

[0084] GHR gene nucleotide sequence (SEQ ID NO. 17, containing the cg24773720 site):

[0085] CCTTGCGAAGAAGTTGTTTTCTGCTGGTGGGTTGTTGTAACCCAATCTAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCTGGAAGTTGGTGAGGGCGGCAGGGAGTTGCGGGCGACAGACGAACCATCACACTCTGGCGTCTGCTCTGGCCCGCGAGTAGTGTACGTGGAGGGGTTTACTCCGGAGACAGTTTTGTTAAAGTCATAAAAGTTTTGCTAGTGTGTTTCTGTTTGCCATCATCTGCCTGGCTGCGGCAGGAACTGCCGAGGCTGCTGCTGTTGCGCGGGGAAGAATCCCCGGCAGCGCGACTGGAGAGACTGGGGAGGTCGAGCTGTGCGCGTGGACACAGCGCGCAGAGCGCGCGGTCTTTTGCGCGTTTGTGCGGGCCGCAGCCGCACGTTGGCACCGATGGAACTGGGGTCAGTAGAGTGACAGCCACCAGTCCGCATGAACTGGGGTAAGTGGAAATTGTGGCGAGCCGACCTCCCCCAGCTTTTGACACACTAGTGGTTGTAAAATCAACCAGGCTTAAAGTTTTGACAGAACTGCCAGAGGCTGCGGGTCAATGGGGTGGCCGCGTGTC

[0086] CNR1 gene nucleotide sequence (SEQ ID NO. 18, containing the cg20622089 locus):

[0087] CTCGGGACAGAAGCTCCCTTGGCACCTCTCGCCCAGCTCAGGGGCTGGTTGTCCGCTAGAACGAAATATCCCCCACTGACTACGGAGAGCTCTGCAGGGAGCCGAGGCCCCCGCCCGGGCCAAGGGAAGGCGCTGGCGCCGCGGGAGACAAGAAGAGGCGGAGGCGGAAAAGAAGTGGAGAAGGAAGGGGTGGCAGAGGGAGTAGCGTGCGGGAGGCGGCGCCGGCGCCGGGCTGCTGGCGAGGCGGGGTGGGGTGGAGTGGGGTGTGCCTCCGGCGGGCGGTCAGCAAGTCAGTCCGTCCGAGCGCCGGCGTCCCGGTCTCCAGCGCCCGCCCGCGACAGCGACCGGGACTGGCGCCCCGCTGCTCCGACCGCCGGCGAGCCTCGCCCCTTCCCAGGCTCTTCACTGGGTCCTCCCGCGAGCCCCGCCTGGCCCGGCGTGGGGCCGCCCGTCTCCGCCAGCCCGGGCGCCCGTCGCCTCGTCCCGCTCGCGCAGTCCCTGCCGCTCCCTCCGCTCGCGCTGTCTCTGGCTCCCTCTCGCTCCAGTCCCATTTATGAAGCGCGGTCCCATCACGTGTTAATGAGCCTTTGTCCTGCCCTGGC

[0088] ZNF569 gene after bisulfite modification of methylation site nucleotide sequence (SEQ ID NO. 10, ZNF569-S-L (cg08630279) contains cg08630279 site):

[0089] ZNF569-S-L (cg08630279):

[0090] GGCGCGGGTTGGGATAGAGGCGGTATTGAGGTCGGCGTTGTCGGTGGTTGAGAGCGTTATAAGTTTCGGTTCGTTATATTAGGGGCGACGTTTTTTAGAGGTTTTCGCGGTTTATTCGGGCGGGATTGGTTTTATTGTTTGCGCGTTTTGAGAAGTAGATTACGGTGTTTTAGGGTTTATAGTTTCGCGTAGGGGAGTTTAGTTTAGGTTTTGTACGAGCGGTTTTTCGCGAGTTTAGTTTTGAGAGATTGGGAGCGTAATTTGGTGTTGAGAAGAATCGAATCGTTTCGTTGTTTTTTGCGTCGAATTATATTTTTTAGAGGTTTTCGCGGTTTTATTTTTTCGATTTTATTTGTAAAAAGGTATTTTTTTTTTATAGTCGGTTGGAGTTGTAGGTTCGTAAGGTTCGTGAAGGAAGTTCGGTTTAGTGTGTTTAGTTTCGTTTTTTTGGGTTTTTATTTGAGGTTAGAATATAGCGGGGTGAAGGTGGTGAGAGTTTTAGGGTTAGTGAAGGTCGGGTTAGTTGGAGTTTTAAGGTTTTGATAGCGGTTAGAAGAGGGAGGAGTGACGTAGTGTGATTGTATGTGTGAAATTTCGGTTAT

[0091] Methylation site nucleotide sequence of GHR gene after bisulfite modification (SEQ ID NO. 11, GHR-S-L (cg24773720) contains cg24773720 site):

[0092] GHR-S-L (cg24773720):

[0093] TTTTGCGAAGAAGTTGTTTTTTGTTGGTGGGTTGTTGTAATTTAATTTAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTGGAAGTTGGTGAGGGCGGTAGGGAGTTGCGGGCGATAGACGAATTATTATATTTTGGCGTTTGTTTTGGTTCGCGAGTAGTGTACGTGGAGGGGTTTATTTCGGAGATAGTTTTGTTAAAGTTATAAAAGTTTTGTTAGTGTGTTTTTGTTTGTTATTATTTGTTTGGTTGCGGTAGGAATTGTCGAGGTTGTTGTTGTTGCGCGGGGAAGAATTTTCGGTAGCGCGATTGGAGAGATTGGGGAGGTCGAGTTGTGCGCGTGGATATAGCGCGTAGAGCGCGCGGTTTTTTGCGCGTTTGTGCGGGTCGTAGTCGTACGTTGGTATCGATGGAATTGGGGTTAGTAGAGTGATAGTTATTAGTTCGTATGAATTGGGGTAAGTGGAAATTGTGGCGAGTCGATTTTTTTTAGTTTTTGATATATTAGTGGTTGTAAAATTAATTAGGTTTAAAGTTTTGATAGAATTGTTAGAGGTTGCGGGTTAATGGGGTGGTCGCGTGTT

[0094] CNR1 gene after methylation site modified by bisulfite nucleotide sequence (SEQ ID NO. 12, CNR1-S-L (cg20622089) contains cg20622089 site):

[0095] CNR1-S-L (cg20622089):

[0096] TTCGGGATAGAAGTTTTTTTGGTATTTTTCGTTTAGTTTAGGGGTTGGTTGTTCGTTAGAACGAAATATTTTTTATTGATTACGGAGAGTTTTGTAGGGAGTCGAGGTTTTCGTTCGGGTTAAGGGAAGGCGTTGGCGTCGCGGGAGATAAGAAGAGGCGGAGGCGGAAAAGAAGTGGAGAAGGAAGGGGTGGTAGAGGGAGTAGCGTGCGGGAGGCGGCGTCGGCGTCGGGTTGTTGGCGAGGCGGGGTGGGGTGGAGTGGGGTGTGTTTTCGGCGGGCGGTTAGTAAGTTAGTTCGTTCGAGCGTCGGCGTTTCGGTTTTTAGCGTTCGTTCGCGATAGCGATCGGGATTGGCGTTTCGTTGTTTCGATCGTCGGCGAGTTTCGTTTTTTTTTAGGTTTTTTATTGGGTTTTTTCGCGAGTTTCGTTTGGTTCGGCGTGGGGTCGTTCGTTTTCGTTAGTTCGGGCGTTCGTCGTTTCGTTTCGTTCGCGTAGTTTTTGTCGTTTTTTTCGTTCGCGTTGTTTTTGGTTTTTTTTCGTTTTAGTTTTATTTATGAAGCGCGGTTTTATTACGTGTTAATGAGTTTTTGTTTTGTTTTGGT

[0097] The molecular cloning vector plasmid used pUC57.

[0098] The positive cell line MKN-45 was from the American Type Culture Collection (ATCC).

[0099] 1. For detection of methylation of ZNF569, GHR and CNR1 genes, 26 groups of primer probe combinations were designed using Primer5 software. For positive cell lines, tissues and blood (such as Examples 2, 3 and Comparative Example 1), actin (Actin) was used as a quality control gene (or referred to as an internal reference gene), wherein primer Actin-FP and Actin-RP were used as quality control gene primers, and probe Actin-P was used as a quality control gene probe for quality control. Commonly used actin generally includes beta actin (β-actin, ACTB), but is not limited to β-actin, and can also be alpha actin, gamma actin, etc. In this embodiment, ACTB was used as an example to illustrate the quality control gene.

[0100] Quality control gene forward primer (ACTB-FP): 5'-GGTGTTTAAGATAGTGTTGTGGGTG-3' (SEQ ID NO. 13)

[0101] Quality control gene reverse primer (ACTB-RP): 5'-CACACTCCAAAACCGCTTTACA-3' (SEQ ID NO. 14)

[0102] Quality control gene probe (ACTB-P) is: 5'-ACCTCATAACCTTATCACAC-3' (SEQ ID NO. 15)

[0103] 2. 2 x 10 3 Copy the synthesized plasmid as a template, use different primer probe combinations according to the following amplification system for real-time fluorescence qPCR amplification (total volume 40 μL):

[0104] 2 x Premix Ex Taq TM (Probe qPCR) 20 μL, Rox II (50 x) 0.8 μL, each primer final concentration 0.2-0.3 μM, probe final concentration 0.2 μM, DNA template, and water to 40 μL.

[0105] Among them, the 2 x Premix Ex Taq TM (Probe qPCR) and Rox II (50 x) used in the real-time fluorescence qPCR amplification come from TAKARA company.

[0106] The real-time fluorescence qPCR reaction conditions are:

[0107] 96℃ pre-denaturation for 3 minutes; 15 cycles: 95℃ denaturation for 15 seconds, 70℃ annealing and extension for 20 seconds, 64℃ annealing and extension for 20 seconds, 72℃ extension for 10 seconds; 35 cycles, 95℃ denaturation for 15 seconds, 70℃ annealing and extension for 20 seconds, 60℃ annealing and extension for 34 seconds, 72℃ extension for 10 seconds; and 35 cycles of annealing, detecting fluorescence signal.

[0108] The primer probe combination and the detection results are shown in Table 2. Preferably, one set of GHR primer probe combination (combination 1), one set of ZNF569 primer probe combination (combination 19) and one set of CNR1 primer probe combination (combination 2) are used for the second round of probe primer screening in positive cell strains.

[0109] Table 2 primer probe combination

[0110]

[0111]

[0112] 3. Using 10 ng of DNA of positive cell line MKN-45 as a template, qPCR amplification was performed using the primer probe combination of ZNF569, the primer probe combination of GHR and the primer probe combination of CNR1 preferred in the previous step, and the qPCR system and reaction conditions were the same as those in the previous step.

[0113] The primer probe combination and detection results are shown in Table 3. The combination 19 (ZNF569-FP2, ZNF569-RP2 and ZNF569-P2) of ZNF569 was highly methylated in the positive cell lines of gastric cancer, and the amplification curve of the primer probe combination was "S" type, and the fluorescence signal was strong. The combination 1 (GHR-FP1, GHR-RP1 and GHR-P1) of GHR was highly methylated in the positive cell lines of gastric cancer, and the amplification curve of the primer probe combination was "S" type, and the fluorescence signal was strong. The combination 2 (CNR1-FP2, CNR1-RP1 and CNR1-P1) of CNR1 was highly methylated in the positive cell lines of gastric cancer, and the amplification curve of the primer probe combination was "S" type, and the fluorescence signal was strong. And the primer sequences are as follows:

[0114] Preferred probe primers of ZNF569:

[0115] ZNF569-FP2: 5'-AGTTTAGTTTTGAGAGATTGGGAGC-3' (SEQ ID NO. 1)

[0116] ZNF569-RP2: 5'-AATAAAACCGCGAAAACCTCT-3' (SEQ ID NO. 2)

[0117] ZNF569-P2: (SEQ ID NO. 3, the cg08630279 site is circled with a box)

[0118] Preferred probe primers of GHR:

[0119] GHR-FP1: 5'-TTGTTTGGTTGCGGTAGGAA-3' (SEQ ID NO. 4)

[0120] GHR-RP1: 5'-CTCCCCAATCTCTCCAATCG-3' (SEQ ID NO. 5)

[0121] GHR-P1: (SEQ ID NO. 6, the cg24773720 site is circled with a box)

[0122] Preferred probe primers of CNR1:

[0123] CNR1-FP2: 5'-GGGTGGAGTGGGGTGTGTTT-3' (SEQ ID NO. 7)

[0124] CNR1-RP1: 5'-CTATCGCGAACGAACGCTAA-3' (SEQ ID NO. 8)

[0125] CNR1-P1: (SEQ ID NO. 9, cg20622089 locus circled with a box)

[0126] The other sequences in Table 2 are as follows:

[0127] ZNF569-FP1: 5'-GGTGTTGAGAAGAATCGAATCG-3' (SEQ ID NO. 19)

[0128] ZNF569-FP3: 5'-GGGAGCGTAATTTGGTGTTGAG-3' (SEQ ID NO. 20)

[0129] ZNF569-FP4: 5'-CGCGAGTTTAGTTTTGAGAGATTGG-3' (SEQ ID NO. 21)

[0130] ZNF569-RP1: 5'-CCTACAACTCCAACCGACTATA-3' (SEQ ID NO. 22)

[0131] ZNF569-RP3: 5'-TATAGTCGGTTGGAGTTGTAGG-3' (SEQ ID NO. 23)

[0132] ZNF569-RP4: 5'-CTTCACGAACCTTACGAACCTAC-3' (SEQ ID NO. 24)

[0133] ZNF569-P1: 5'-TTGTTTTTTGCGTCGAA-3' (SEQ ID NO. 25)

[0134] GHR-FP2: 5'-GGTTGCGGTAGGAATTGTCGAGG-3' (SEQ ID NO. 26)

[0135] GHR-FP3: 5'-GGTTGCGGTAGGAATTGTCGAGG-3' (SEQ ID NO. 27)

[0136] GHR-RP2: 5'-CAATCTCTCCAATCGCGCTACCGA-3' (SEQ ID NO. 28)

[0137] GHR-RP3: 5'-CTCGACCTCCCCAATCTCTCC-3' (SEQ ID NO. 29)

[0138] GHR-RP4: 5'-CGCACAACTCGACCTCCCCAATC-3' (SEQ ID NO. 30)

[0139] GHR-RP5: 5'-CCACGCGCACAACTCGACCTCC-3' (SEQ ID NO. 31)

[0140] GHR-RP6: 5'-CTATATCCACGCGCACAACTCGACC-3' (SEQ ID NO. 32)

[0141] GHR-RP7: 5'-CGCTATATCCACGCGCACAACTCG-3' (SEQ ID NO. 33)

[0142] GHR-P2: 5'-GTTGTTGTTGCGCGGGGA-3' (SEQ ID NO. 34)

[0143] CNR1-FP1: 5'-GTGGAGTGGGGTGTGTTTTCGGC-3' (SEQ ID NO. 35)

[0144] CNR1-FP3: 5'-GTCGGGTTGTTGGCGAGGCGG-3' (SEQ ID NO. 36)

[0145] CNR1-RP2: 5'-CGAACGCTAAAAACCGAAACG-3' (SEQ ID NO. 37)

[0146] CNR1-RP3: 5'-CGCGAACGAACGCTAAAAACCG-3' (SEQ ID NO. 38)

[0147] CNR1-RP4: 5'-CGATCGCTATCGCGAACGAACGC-3' (SEQ ID NO. 39)

[0148] CNR1-RP5: 5'-CGAAACGCCAATCCCGATCGCTATCG-3' (SEQ ID NO. 40)

[0149] CNR1-RP6: 5'-CAACGAAACGCCAATCCCGATCG-3' (SEQ ID NO. 41)

[0150] CNR1-RP7: 5'-CGATCGAAACAACGAAACGCCAATC-3' (SEQ ID NO. 42)

[0151] CNR1-P2: 5'-AGTTCGTTCGAGCGTCG-3' (SEQ ID NO. 43)

[0152] Table 3 Probe and primer combination and screening results in positive cell lines

[0153]

[0154] 4. Sensitivity analysis: The plasmid template was diluted from 10000 copies to 5 copies, and then detected respectively. The results showed that the fluorescence PCR method of the application had high sensitivity, and the primer detection corresponded to the plasmid sample, and 5 copies / 40 microliters could be detected (as shown in Table 4).

[0155] Table 4 Plasmid gradient dilution results

[0156] Plasmid copy number ZNF569 GHR CNR1 1 x 10 4 ]] 25.12 26.23 26.53 1 x 10 3 ]] 27.21 27.35 27.65 1 x 10 2 ]] 29.34 29.43 29.76 50 30.09 30.56 30.87 25 31.34 31.42 31.69 10 32.26 32.28 32.89 5 34.68 34.56 35.09 0 No Ct No Ct No Ct

[0157] Example 3 Tissue and blood sample detection

[0158] 1. Taking tissue as sample

[0159] Gastric cancer and paracancerous tissue specimens removed by surgery or gastroscopy were selected, and the methylation levels of GHR, ZNF569 and CNR1 genes were quantitatively detected. The specimens were 26 pairs of gastric cancer and paracancerous paired tissues. The cancer tissues and the paired paracancerous tissue samples were detected by using the preferred primer probe combination and real-time fluorescence qPCR reaction system in the application.

[0160] Step 1, sample processing, DNA extraction and transformation:

[0161] The DNA of tissue cells was extracted by using a cell DNA extraction kit (purchased from QIAGEN), and the specific operation was referred to the kit instruction manual.

[0162] Step 2, bisulfite modification:

[0163] The extracted cell DNA was subjected to bisulfite modification by using an EZDNA methylation kit (purchased from ZYMORESEARCH), and the specific operation was referred to the kit instruction manual.

[0164] Step 3, qPCR amplification was performed according to the following amplification system (total volume 40 μL)

[0165] 2x Premix Ex Taq TM (Probe qPCR) 20 μL, Rox II (50x) 0.8 μL, final concentration of each primer 0.2-0.3 μM, final concentration of probe 0.2 μM, DNA template > 10 ng, and water to 40 μL. The cell DNA after sulfite modification obtained in step 2 was used as a template.

[0166] 2x Premix Ex Taq in qPCR amplification TM (Probe qPCR) and Rox II (50x) from TAKARA company.

[0167] The real-time fluorescence qPCR reaction condition is:

[0168] 96 °C pre-denaturation for 3 minutes; 15 cycles: 95 °C denaturation for 15 seconds, 70 °C annealing and extension for 20 seconds, 64 °C annealing and extension for 20 seconds, 72 °C extension for 10 seconds; 35 cycles, 95 °C denaturation for 15 seconds, 70 °C annealing and extension for 20 seconds, 60 °C annealing and extension for 34 seconds, 72 °C extension for 10 seconds; and 35 cycles of fluorescence signal detection during annealing.

[0169] Step 4, detect the fluorescence signal, and take the Ct value as the standard for judging the result.

[0170] When the cycle number required for the fluorescence signal of the internal reference gene (actin) to reach the set threshold value is Ct≤36, it indicates that the DNA amount is within the allowable range, and the result is reliable; if the cycle number required for the fluorescence signal of the internal reference gene (actin) to reach the set threshold value is CT value > 36, it is considered as an invalid sample. In the case of valid detection, if the CT value of the GHR gene is ≤40 or the CT value of the ZNF569 gene is ≤40 or the CT value of the CNR1 gene is ≤40, the test result of the sample is "positive"; if the CT value of the GHR gene is > 40, the CT value of the ZNF569 gene is > 40, and the CT value of the CNR1 gene is > 40, the test result of the sample is "negative".

[0171] The results show (Table 5) that the sensitivity of the site of the tissue sample of the application is 100% for single detection, and the sensitivity of the combined detection result is 100%. The Ct values detected in cancer tissues (T) and paracancer tissues (N) were analyzed for differences, as shown in Figure 5 、 Figure 6 and Figure 7 ZNF569 site, GHR site, and CNR1 site all have significant differences in cancer tissues (T) and paracancer tissues (N).

[0172] Table 5 results of tissue sample detection

[0173]

[0174]

[0175] 2. Blood as sample

[0176] Select 166 blood samples, of which 83 are gastric cancer, 30 are precancerous lesions and 53 are normal, and quantitatively detect the methylation levels of GHR, ZNF569 and CNR1 genes. The preferred primer probe combination and qPCR reaction system of the application are used to detect 83 blood samples of clinical gastric cancer patients, 30 blood samples of precancerous lesion patients and 53 blood samples of healthy people, and the sample information is shown in Tables 6-8.

[0177] Table 6 clinical diagnosis information of gastric cancer blood samples

[0178]

[0179]

[0180]

[0181] Table 7 clinical diagnosis information of precancerous lesion blood samples

[0182]

[0183]

[0184] Table 8 clinical diagnosis information of normal blood samples

[0185]

[0186]

[0187] Sample detection experiment process:

[0188] Step 1, molecular hybridization capture.

[0189] 1. Nucleic acid extraction

[0190] 1. Take 500ul blood into a centrifuge tube and add lysis solution, proteinase K and magnetic beads (TIANGEN, DP709), shake and mix, then incubate at room temperature for 20min.

[0191] 2. Place the centrifuge tube on the magnetic stand for 2min, then carefully remove the liquid with a pipette after the magnetic beads are completely adsorbed, and take out the centrifuge tube.

[0192] 3. Add 750 μl of the deproteinization solution, mix well by inverting the tube, and suspend the magnetic beads. Centrifuge briefly to remove the droplets on the inner wall of the cap.

[0193] 5. Place the centrifuge tube on the magnetic stand for 1 min. After the magnetic beads are completely adsorbed, carefully remove the liquid with a pipette, and take out the centrifuge tube.

[0194] 6. Add 750 μl of the rinsing solution, mix well by inverting the tube, and suspend the magnetic beads. Centrifuge briefly to remove the droplets on the inner wall of the cap.

[0195] 7. Place the centrifuge tube on the magnetic stand for 1 min. After the magnetic beads are completely adsorbed, carefully remove the liquid with a pipette, and take out the centrifuge tube.

[0196] 8. Repeat steps 6 and 7 once.

[0197] 9. Place the centrifuge tube on the magnetic stand, and aspirate all the liquid and discard. Dry at room temperature for 10 min.

[0198] 10. Add 50 μl of the elution buffer, suspend the magnetic beads with a pipette, and incubate at 56 °C for 5 min. Shake gently every 2 min to elute the nucleic acid.

[0199] 11. Place the centrifuge tube on the magnetic stand for 2 min. After the magnetic beads are completely adsorbed, carefully transfer the nucleic acid solution to a new centrifuge tube.

[0200] 1.2 Hybridization capture

[0201] 1. Take the DNA solution dissolved in the previous step into a PCR tube, and add the probe, hybridization buffer, and water.

[0202] 2. Set the PCR program according to the following parameters:

[0203] ① 98 °C for 5 min;

[0204] ② 65 °C for 1 hour;

[0205] ③ 65 °C Hold.

[0206] 1.3 Purification and recovery

[0207] 1. Take out the streptavidin magnetic beads, and place them on the magnetic stand. Aspirate the supernatant.

[0208] 2. Add the washing solution, vortex, and place on the magnetic stand. Carefully aspirate the supernatant.

[0209] 3. Add the binding solution, and add the solution in step 1.2 to the magnetic beads containing the binding solution. Place on a rotating mixer for 30 min at room temperature.

[0210] 4. After the rotation incubation is complete, add the washing solution WB1, place it on a magnetic rack, and carefully aspirate the supernatant.

[0211] 5. Add rinsing solution WB2, place on a magnetic rack, and carefully aspirate the supernatant.

[0212] 6. Add 80% ethanol, place on a magnetic rack, and carefully aspirate the supernatant.

[0213] 7. Open the tube cap and let it stand at room temperature for 5 minutes to allow the remaining alcohol to evaporate completely. Remove the centrifuge tube from the magnetic rack, add 45 μL of purified water to the centrifuge tube, and vortex to mix thoroughly to completely disperse the magnetic beads. The resulting solution can be used for subsequent sulfite conversion.

[0214] Step 2: Perform sulfite conversion on the extracted DNA sample (EZ DNA Methylation Kit, purchased from ZYMO RESEARCH).

[0215] Step 3: Perform qPCR amplification according to the following amplification system (total volume 40 μL).

[0216] 2×Premix Ex Taq TM (Probe qPCR) 20 μL, Rox II (50×) 0.8 μL, final concentration of each primer 0.2–0.3 μM, final concentration of probe 0.2 μM, DNA template >10 ng, and water to bring the volume to 40 μL. Use the sulfite-modified DNA obtained in step 2 as a template.

[0217] 2×Premix Ex Taq in qPCR amplification TM (Probe qPCR) and Rox II (50×) were from TAKARA.

[0218] The qPCR reaction conditions are as follows:

[0219] Pre-denaturation at 96℃ for 3 minutes; 15 cycles: denaturation at 95℃ for 15 seconds, annealing and extension at 70℃ for 20 seconds, annealing and extension at 64℃ for 20 seconds, and extension at 72℃ for 10 seconds; 35 cycles: denaturation at 95℃ for 15 seconds, annealing and extension at 70℃ for 20 seconds, annealing and extension at 60℃ for 34 seconds, and extension at 72℃ for 10 seconds; and fluorescence signal was detected during annealing in all 35 cycles.

[0220] Step 4: Detect the fluorescence signal and use the Ct value as the standard for judging the result.

[0221] If the cycle number Ct required for the fluorescence signal of the internal reference gene (actin) to reach the set threshold value is ≤36, it indicates that the amount of DNA loaded is within the allowable range, and the result is reliable; if the cycle number CT value required for the fluorescence signal of the internal reference gene (actin) to reach the set threshold value is >36, it is considered as an invalid sample. Under the condition of valid detection, if the CT value of the GHR gene is ≤40 or the CT value of the ZNF569 gene is ≤40 or the CT value of the CNR1 gene is ≤40, the test result of the sample is "positive"; if the CT value of the GHR gene is >40, the CT value of the ZNF569 gene is >40, and the CT value of the CNR1 gene is >40, the test result of the sample is "negative".

[0222] In 166 blood samples (83 gastric cancer, 30 precancerous lesions, 53 normal), the methylation levels of GHR, ZNF569 and CNR1 genes were detected, and the results showed (Table 9) that the sensitivity of the ZNF569 gene alone was 73.33% (precancerous lesions), 83.13% (gastric cancer), and the specificity was 100%; the sensitivity of the GHR gene alone was 63.33% (precancerous lesions), 81.93% (gastric cancer), and the specificity was 100%; the sensitivity of the CNR1 gene alone was 70.00% (precancerous lesions), 80.72% (gastric cancer), and the specificity was 100%; the sensitivity of the combined detection of the three genes was 80.00% (precancerous lesions), 91.57% (gastric cancer), and the specificity was 100%.

[0223] Table 9 Detection results of blood samples

[0224]

[0225]

[0226] Comparative Example 1

[0227] According to the descending order of the β difference (Δβ) ≥0.3 (Table 1), primers and probes were designed for the corresponding sites of combinations 2, 3 and 4, and the sequences were as follows:

[0228] ZNF569-2713-FP: 5'-GTATAAAGAGTTCGGCGTTTAGAG-3' (SEQ ID NO. 44)

[0229] ZNF569-2713-RP: 5'-TATCCGCGAACTTAATA-3' (SEQ ID NO. 45)

[0230] ZNF569-2713-P: (SEQ ID NO. 46)

[0231] CNR1-6641-FP: 5'-CGAGTCGTTCGTTATTTGGTTGCGG-3' (SEQ ID NO. 47)

[0232] CNR1-6641-RP: 5'-CATCGAAAACCGACGAACGAAACG-3' (SEQ ID NO. 48)

[0233] CNR1-6641-P: (SEQ ID NO. 49)

[0234] The blood samples of the gastric cancer patients are detected by using the preferred primer probe combination and real-time fluorescent qPCR reaction system.

[0235] The steps are as follows:

[0236] Select 28 blood samples (Tables 6-8), of which 10 are gastric cancer, 9 are precancerous lesions, and 9 are normal, and quantitatively detect the methylation levels of the related genes.

[0237] Step 1, molecular hybridization capture.

[0238] 1.1 Nucleic acid extraction

[0239] 3. Take 500ul blood into a centrifuge tube and add lysis solution, proteinase K and magnetic beads (TIANGEN, DP709), shake and mix, then incubate at room temperature for 20 min.

[0240] 4. Place the centrifuge tube on the magnetic stand for 2 min, then carefully remove the liquid with a pipette after the magnetic beads are completely adsorbed, and take out the centrifuge tube.

[0241] 3. Add 750ul deproteinization solution, mix up and down for 30 sec to fully suspend the magnetic beads, and briefly centrifuge to remove the droplets on the inner wall of the tube cap.

[0242] 5. Place the centrifuge tube on the magnetic stand for 1 min, then carefully remove the liquid with a pipette after the magnetic beads are completely adsorbed, and take out the centrifuge tube.

[0243] 6. Add 750ul rinsing solution, mix up and down for 30 sec to fully suspend the magnetic beads, and briefly centrifuge to remove the droplets on the inner wall of the tube cap.

[0244] 12. Place the centrifuge tube on the magnetic stand for 1 min, then carefully remove the liquid with a pipette after the magnetic beads are completely adsorbed, and take out the centrifuge tube.

[0245] 13. Repeat steps 6 and 7 once.

[0246] 14. Place the centrifuge tube on the magnetic stand, aspirate all the liquid and discard, and air dry at room temperature for 10 min.

[0247] 15. Add 50 μΐ elution buffer, resuspend the magnetic beads by pipetting, incubate at 56°C for 5 min, shake gently every 2 min to ensure complete elution of nucleic acid.

[0248] 16. Place the centrifuge tube on the magnetic stand for 2 min, and when the magnetic beads are completely absorbed, carefully transfer the nucleic acid solution to a new centrifuge tube.

[0249] 1.2 Hybridization capture

[0250] 1. Take the DNA solution dissolved in the previous step into a PCR tube, and add probes, hybridization buffer and water.

[0251] 2. Set the PCR program according to the following parameters:

[0252] (IV) 98°C for 5 min;

[0253] (V) 65°C for 1 hour;

[0254] (VI) 65°C Hold.

[0255] 1.3 Purification and recovery

[0256] 1. Take the streptavidin magnetic beads and place them on the magnetic stand, and discard the supernatant.

[0257] 2. Add the washing solution, vortex, and place it on the magnetic stand, and carefully discard the supernatant.

[0258] 3. Add the binding solution, and add the solution from step 1.2 to the magnetic beads containing the binding solution, and place it on a rotary mixer at room temperature for 30 min.

[0259] 4. After the rotary incubation, add the rinse solution WB1, place it on the magnetic stand, and carefully discard the supernatant.

[0260] 5. Add the rinse solution WB2, place it on the magnetic stand, and carefully discard the supernatant.

[0261] 6. Add 80% ethanol, place it on the magnetic stand, and carefully discard the supernatant.

[0262] 7. Open the tube cap, let it stand at room temperature for 5 min to completely evaporate the remaining alcohol, remove the centrifuge tube from the magnetic stand, add 45 μΐ of purified water to the centrifuge tube, vortex to completely disperse the magnetic beads, and the resulting solution can be used for subsequent bisulfite conversion.

[0263] Step 2: Bisulfite conversion of the extracted DNA sample (EZ DNA Methylation Kit, purchased from ZYMO RESEARCH).

[0264] Step 3, qPCR amplification was performed according to the following amplification system (total volume 40 μL).

[0265] 2x Premix Ex Taq TM (Probe qPCR) 20 μL, Rox II (50x) 0.8 μL, final concentration of each primer 0.2-0.3 μM, final concentration of probe 0.2 μM, DNA template > 10 ng, and water to 40 μL. The DNA modified by sulfite in step 2 was used as a template.

[0266] 2x Premix Ex Taq in qPCR amplification TM (Probe qPCR) and Rox II (50x) from TAKARA company.

[0267] The qPCR reaction conditions are as follows:

[0268] 96 °C pre-denaturation for 3 minutes; 15 cycles: 95 °C denaturation for 15 seconds, 70 °C annealing and extension for 20 seconds, 64 °C annealing and extension for 20 seconds, 72 °C extension for 10 seconds; 35 cycles, 95 °C denaturation for 15 seconds, 70 °C annealing and extension for 20 seconds, 60 °C annealing and extension for 34 seconds, 72 °C extension for 10 seconds; and 35 cycles of fluorescence signal detection during annealing.

[0269] Step 4, detect the fluorescence signal, and take the Ct value as the standard for judging the result.

[0270] When the cycle number required for the fluorescence signal of the internal reference gene (actin) to reach the set threshold value is Ct≤36, it indicates that the DNA amount is within the allowable range, and the result is reliable; if the cycle number required for the fluorescence signal of the internal reference gene (actin) to reach the set threshold value is CT value > 36, it is considered as an invalid sample. In the case of valid detection, if the CT value of the GHR gene is ≤40 or the CT value of the ZNF569 gene is ≤40 or the CT value of the CNR1 gene is ≤40, the test result of the sample is "positive"; if the CT value of the GHR gene is > 40, the CT value of the ZNF569 gene is > 40, and the CT value of the CNR1 gene is > 40, the test result of the sample is "negative".

[0271] The detection results (Table 10) show that among the four combinations, combination 1 (cg08630279 (ZNF569), cg24773720 (GHR) and cg20622089 (CNR1)) used in the present application and combinations 2 and 3 have the lowest false positive rate in healthy people, and combination 4 has a false positive rate that is too high, so it is not a preferred site; in addition, combinations 1, 2 and 3 have higher detection effects in gastric cancer and precancerous lesions, and the positive detection result of combination 4 in precancerous lesions is too low, so it is not a preferred site.

[0272] Table 10 Comparative detection results of four combined detection combinations in blood samples

[0273]

[0274] The preferred embodiments of the present application have been described in detail above. It should be understood that modifications and variations can be made by those skilled in the art in light of the teachings of the present application without departing from the spirit of the present application. It is therefore to be understood that within the scope of the claims and their equivalents, the present application can be practiced otherwise than as specifically described.

Claims

1. A methylation marker combination for detecting gastric cancer, characterized by, The methylation marker combination consists of target gene sequences or fragments thereof containing the following CpG methylation sites respectively: Methylation marker 1: CpG methylation site cg08630279 of ZNF569 gene; Methylation marker 2: CpG methylation site cg24773720 of GHR gene; Methylation marker 3: CpG methylation site cg20622089 of CNR1 gene.

2. A primer probe combination for detecting the methylation marker combination according to claim 1, characterized in that The primer probe combination includes an upstream primer, a downstream primer and a probe for each methylation marker methylation site, wherein: The upstream primer for the methylation marker 1 methylation site is ZNF569-FP as shown in SEQ ID NO. 1, the downstream primer is ZNF569-RP as shown in SEQ ID NO. 2, and the probe is ZNF569-P as shown in SEQ ID NO. 3; The upstream primer for the methylation marker 2 methylation site is GHR-FP as shown in SEQ ID NO. 4, the downstream primer is GHR-RP as shown in SEQ ID NO. 5, and the probe is GHR-P as shown in SEQ ID NO. 6; The upstream primer for the methylation marker 3 methylation site is CNR1-FP as shown in SEQ ID NO. 7, the downstream primer is CNR1-RP as shown in SEQ ID NO. 8, and the probe is CNR1-P as shown in SEQ ID NO.

9.

3. The primer probe combination of claim 2, wherein, The nucleotide sequences of the primers and the probes are designed according to the nucleotide sequences of the corresponding methylation sites of each target gene after bisulfite modification, and the bisulfite modified sequences are as follows: The bisulfite modified sequence of ZNF569 gene is as shown in SEQ ID NO. 10; The bisulfite modified sequence of GHR gene is as shown in SEQ ID NO. 11; The bisulfite modified sequence of CNR1 gene is as shown in SEQ ID NO.

12.

4. A kit for detecting gastric cancer, characterized by comprising the polynucleotide of claim 1 or 2. The primer probe combination of claim 2 or 3 is included.

5. The kit of claim 4, wherein A positive control is also included; the positive control includes a plasmid containing the methylation sequence of the target gene after bisulfite modification and / or a gastric cancer cell line; the vector plasmid is selected from pUC57, pUC57-Kan, pUC57-Simple, pUC57-mini, pUC18 or pUC19; the gastric cancer cell line is selected from MKN-45, AGS, SGC-7901, BGC-823 or HGC-27; and the methylation sequence of the target gene after bisulfite modification is as follows: The bisulfite modified sequence of ZNF569 gene is as shown in SEQ ID NO. 10; The bisulfite modified sequence of GHR gene is as shown in SEQ ID NO. 11; The bisulfite modified sequence of CNR1 gene is as shown in SEQ ID NO.

12.

6. The kit of claim 5, wherein A quality control gene primer pair and a quality control gene probe are also included, the quality control gene primer pair includes a quality control gene forward primer and a quality control gene reverse primer; the quality control gene is beta actin, and the primer and probe sequences are as follows: The quality control gene forward primer is as shown in SEQ ID NO. 13; The reverse primer of the quality control gene is shown as SEQ ID NO.

14. The probe of the quality control gene is shown as SEQ ID NO.

15.

7. The kit of claim 6, wherein A negative control is also included, which is normal human genomic DNA or DNA of the negative cell line 293T.

8. The kit of claim 7, wherein The detection result is determined by qPCR analysis result of the primer probe combination: Quality control effectiveness: Ct of beta-actin ≤ 36, the result is reliable; Ct > 36, the result is unreliable; Site positive determination: Ct ≤ 40 of ZNF569, GHR and CNR1 single site is determined as methylation positive, and Ct > 40 of each site is negative; Combined determination: any site is methylation positive, which is determined as gastric cancer positive, and three sites are all negative, which is determined as gastric cancer negative.

9. Use of the methylation marker combination of claim 1 in the preparation of primer probes or methylation detection reagents for gastric cancer detection.

10. Use of the kit of claim 4 in the preparation of molecular analysis products for detecting gastric cancer screening, detection, diagnosis or prognosis evaluation.

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