Methylated molecular marker, detection kit and application of methylated molecular marker in oral mucosa canceration screening

Abstract

The invention relates to the technical field of biology, and particularly discloses a methylated molecular marker for early warning of canceration of oral mucosa and a detection kit thereof. The methylation molecular markers are a ZNF582 gene and an SHOX2 gene. According to the application, important molecular information is provided for screening the oral cancer by detecting the methylation levels of the ZNF582 gene and the SHOX2 gene; the molecular marker provided by the invention has relatively high accuracy, sensitivity and specificity, and can provide important reference for early warning of oral mucosa canceration; and the detection method is simple and rapid, low in cost and convenient to sample, can help to realize canceration early warning of non-invasive oral potential malignant diseases, and has a good application prospect.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a methylation molecular marker, a detection kit and an application thereof in screening for oral mucosal carcinogenesis. Background Art

[0002] Oral potential malignant lesions (OPMDs) are a group of oral mucosal diseases that may have the potential to become cancerous, including oral leukoplakia (OLK), oral erythema (OE), oral lichen planus (OLP), oral submucous fibrosis (OSF), etc. It is reported that 5% to 18% of OPMDs may develop into oral cancer, among which the malignant transformation rate of OLK is 2% to 3%, the malignant transformation rate of OE is 14% to 50%, and the malignant transformation rate of OLP is 1.14%. Oral cancer is one of the most common malignant tumors in the head and neck, but the five-year survival rate of oral cancer diagnosed and treated in the early stage can reach more than 80%.

[0003] Histopathological examination results are the gold standard for the diagnosis of oral cancer. They provide the most reliable cytological and histological basis through microscopic examination of tissue sections. However, as a common examination method, biopsy is invasive, time-consuming, and difficult for patients to comply with for a long time, which limits its practicality as a means of cancer monitoring. At the same time, doctors are often influenced by subjective standards when judging whether patients need biopsies, which may lead to missing the opportunity for early treatment. Therefore, finding non-invasive screening technologies has become the focus of research. These technologies have the advantages of less trauma, simple and rapid, repeatable operation, and easy acceptance by patients, and have been applied in clinical practice.

[0004] Gene methylation plays an important role in the progression of OPMDs to oral squamous cell carcinoma (OSCC). Studies have shown that there are differences in DNA methylation patterns between patients with OPMDs and OSCC, and these differences may help diagnose and predict disease progression. Changes in methylation can affect the expression of key genes, thereby promoting cell malignancy and cancer development. Significant progress has been made in the study of methylated genes as molecular biomarkers to predict the risk of carcinogenesis. By discovering differential methylation patterns, researchers have identified methylation sites that appear during carcinogenesis, providing a basis for potential molecular markers. A large number of studies have verified the association between certain methylation markers and the risk of carcinogenesis. These markers can be detected through blood or tissue samples, providing a new means for early screening. Researchers are exploring the combined use of methylation markers to improve the accuracy and sensitivity of predictions. At the same time, specific methylation markers are associated with the clinical prognosis of cancer, providing important information for personalized treatment and management.

[0005] At present, the application of gene methylation technology in monitoring oral mucosal carcinogenesis needs further research. Summary of the invention

[0006] The purpose of this application is to overcome the shortcomings of the above-mentioned prior art and provide a methylation molecular marker for monitoring oral mucosal canceration, a detection kit and its application in oral mucosal canceration screening. The methylation molecular marker or detection kit of this application has the advantages of being simple, rapid, low-cost and highly accurate, and can be used to monitor oral mucosal canceration.

[0007] To achieve the above purpose, the technical solution adopted by this application is:

[0008] The present application provides a methylation molecular marker for monitoring oral mucosal carcinogenesis, wherein the methylation molecular markers are the ZNF582 gene and the SHOX2 gene.

[0009] The inventors of this application have found through extensive research and experiments that ZNF582 gene and SHOX2 gene are genes significantly associated with the progression of OPMDs to cancer, and the methylation degree of the gene methylation site is strongly correlated with the progression of OPMDs to cancer. This application is the first to use ZNF582 gene or SHOX2 gene or both genes in combination as methylation molecular markers for monitoring oral mucosal cancer. The combination of ZNF582 gene and SHOX2 gene as an important parameter for early warning of oral mucosal cancer is more accurate.

[0010] Furthermore, the inventors found that the present application can effectively serve as an important parameter for the progression of potential oral malignant lesions to oral cancer by detecting the methylation levels of the ZNF582 gene and the SHOX2 gene; the molecular markers of the present application have high accuracy, sensitivity and specificity, and are of great significance for monitoring oral mucosal carcinogenesis; and the detection method is simple, fast, low-cost, and easy to sample, which can help achieve non-invasive early warning of cancer in potential oral malignant diseases, and has good application prospects.

[0011] As a preferred embodiment of the methylation molecular marker for oral mucosal canceration monitoring described in the present application, the methylation molecular marker is a nucleic acid methylated in at least one target region of the target gene ZNF582, or / and a nucleic acid methylated in at least one target region of the target gene SHOX2;

[0012] The target region is selected from the following fragments:

[0013] ZNF582 gene: negative strand chr19: 56,375,846-56,393,538;

[0014] SHOX2 gene: chr3: 158,095,905-158,106,420.

[0015] The fragments selected for the above target regions were derived from the GRCh38 (hg38) version.

[0016] The present application uses ZNF582 gene and SHOX2 gene for monitoring oral mucosal carcinogenesis by detecting the methylation sites of ZNF582 gene and SHOX2 gene, with high accuracy, sensitivity and specificity.

[0017] The present application also provides a detection reagent for detecting the above-mentioned methylation molecular marker, wherein the detection reagent comprises a primer combination for detecting the methylation molecular marker;

[0018] The primer combination is a primer pair for the ZNF582 gene and a primer pair for the SHOX2 gene;

[0019] The nucleotide sequences of the primer pairs for the ZNF582 gene are shown in SEQ ID NOs: 1-2;

[0020] The nucleotide sequences of the primer pairs targeting the SHOX2 gene are shown in SEQ ID NOs: 3-4.

[0021] The detection reagent of the present application can be used to accurately detect whether the ZNF582 gene and the SHOX2 gene are methylated, and is used to monitor oral mucosal carcinogenesis.

[0022] By designing a primer combination for the methylation sequence, a nucleic acid sequence containing a methylation site is amplified by PCR. If an amplified fragment can be obtained, it indicates that methylation exists at the site of the nucleic acid fragment. If no amplified fragment is obtained, it indicates that methylation does not exist at the site of the nucleic acid fragment. In this application, the ΔCT value is used as an analysis indicator, and while making judgments, the error caused by the template concentration between different samples can also be eliminated, thereby improving the practicality of the application.

[0023] The present application also provides the use of the above detection reagent in the preparation of a detection kit, wherein the detection kit is used to provide the detection results of methylation markers for monitoring oral mucosal carcinogenesis.

[0024] The present application also provides a detection kit, which includes the above-mentioned detection reagent.

[0025] As a preferred embodiment of the detection kit described in the present application, the detection kit also includes a primer pair of an internal reference gene and a fluorescent quantitative PCR detection reagent.

[0026] As a preferred embodiment of the detection kit described in the present application, the internal reference gene includes the ACTB gene, and the nucleotide sequence of the primer pair of the internal reference gene is shown in SEQ ID NOs: 5-6.

[0027] Preferably, the fluorescent quantitative PCR detection reagents include PCR buffer, SYBR qPCR Master Mix, ddH2O and other commonly used fluorescent quantitative PCR detection reagents.

[0028] The kit also includes at least one of an oral swab, a sample collection container, a cell preservation solution, a nucleic acid extraction reagent and consumables, and a sulfite conversion reagent. The above reagents / devices can be purchased separately through market channels or configured by oneself, so they can be configured into the kit according to actual needs.

[0029] In some specific embodiments, the oral swab and the sample collection container are used to collect exfoliated cell samples from the oral cavity with potential malignant diseases; the capacity of the sample collection container is 5 to 10 mL.

[0030] The kit of the present application can realize accurate detection of a small amount of samples, thereby reducing the difficulty of sample collection and facilitating detection. There is no strict limitation on the material and shape of the sample collection container, which can be flexibly selected according to conventional operations in the field.

[0031] The nucleic acid extraction reagent is used to extract DNA, and specifically can be a conventional nucleic acid extraction reagent in the art. The present application mainly uses the magnetic bead method for extraction. The sulfite conversion reagent can convert cytosine (C) in nucleic acid fragments into uracil (U), while 5-methylcytosine (5mC) does not undergo conversion.

[0032] The above primer pair for detecting ACTB gene was used to detect the expression level of ACTB control gene.

[0033] The present application also provides a method for using the above kit, comprising the following steps:

[0034] (1) Extracting genomic DNA from biological samples to be tested;

[0035] (2) performing sulfite conversion on the genomic DNA of the biological sample to be tested;

[0036] (3) using the primer combination in the above kit to perform methylation quantitative PCR detection on the sulfite-converted DNA;

[0037] (4) Analyze the test results.

[0038] The present application utilizes the above primer combination to detect the aforementioned molecular markers (ZNF582 and SHOX2 gene methylation sites) in biological samples (oral mucosal exfoliated cells), which can achieve accurate detection of ZNF582 and SHOX2 methylation levels and can be used to monitor oral mucosal carcinogenesis.

[0039] The present application also provides the use of the above-mentioned methylation molecular marker, or the above-mentioned detection reagent, or the above-mentioned detection kit in a detection platform for detecting and interpreting methylation markers for monitoring oral mucosal carcinogenesis.

[0040] The present application also provides a detection platform or device for detecting and interpreting methylation markers for monitoring oral mucosal canceration, the detection platform or device comprising:

[0041] 1) A detection unit, used to detect the methylation level of a molecular marker in a biological sample;

[0042] 2) Analysis unit, used for interpretation of test results of methylation markers for oral mucosal cancer monitoring;

[0043] The analysis unit is used for interpretation of the methylation marker detection results for oral mucosal canceration monitoring based on the following determination method:

[0044] Under the premise that the amplification curve of the internal reference gene is S-shaped and the Ct value is ≤35, if the amplification curve of the target region of the ZNF582 gene is S-shaped and the ΔCt of the ZNF582 gene is ≤12.547, it is judged that the ZNF582 gene is methylated positive; if the ΔCt of the ZNF582 gene is >12.547 or there is no amplification curve in the target region of the ZNF582 gene, it is judged that the ZNF582 gene is methylated negative;

[0045] Under the premise that the amplification curve of the internal reference gene is S-shaped and Ct≤35, if the amplification curve of the target region of the SHOX2 gene is S-shaped and the ΔCt of the SHOX2 gene is ≤7.435, it is judged that the SHOX2 gene is methylated positive; if the ΔCt of the SHOX2 gene is >7.435 or there is no amplification curve in the target region of the SHOX2 gene, it is judged that the SHOX2 gene is methylated negative;

[0046] When ZNF582 and / or SHOX2 genes showed methylation-positive expression, the sample was regarded as a methylation-positive sample.

[0047] ΔCt = Ct value of target gene - Ct value of reference gene;

[0048] The target gene includes ZNF582 gene or SHOX2 gene.

[0049] Preferably, the detection unit comprises a fluorescence quantitative PCR instrument. In some specific embodiments, the detection unit further comprises a DNA extraction device for extracting DNA from a biological sample, including a magnetic rack and the like.

[0050] The present application is carried out by QMSP (quantitative methylation-specific PCR) to quantitatively analyze the methylation levels of ZNF582 gene and SHOX2 gene; the detection step includes extracting DNA from oral mucosal exfoliated cells, treating with bisulfite to convert unmethylated cytosine into uracil, and then amplifying the methylation sites of ZNF582 gene and SHOX2 gene using methylation-specific primers (SEQ ID NO: 1-4); the detection results are read by a fluorescence quantitative analyzer and quantitatively analyzed by a standard curve, and finally the methylation levels of ZNF582 gene and SHOX2 gene are determined for monitoring oral mucosal carcinogenesis.

[0051] Preferably, the biological sample comprises oral mucosal exfoliated cells.

[0052] The present application uses exfoliated cells from the affected area as test samples. The sampling is simple and easy to obtain, and will not cause any pain or impact to the patient, and the patient acceptance is high.

[0053] Compared with the prior art, this application has the following beneficial effects:

[0054] The present application provides a methylation molecular marker, a detection kit and the use thereof in screening for oral mucosal carcinogenesis. The molecular markers (ZNF582 gene and SHOX2 gene) of the present application have high accuracy, sensitivity and specificity, and are of great significance for the monitoring of oral mucosal carcinogenesis. In addition, the detection method is simple and rapid, low in cost, and convenient in sampling. It can be used as an important parameter to achieve non-invasive early warning of carcinogenesis of potential oral malignant diseases, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is the frequency diagram of methylated genes in OPMDs cells;

[0056] Figure 2 is a relationship diagram between abnormal methylation genes and pathological grades in Example 1;

[0057] Figure 3 This is the ROC curve of the ΔCT critical value of ZNF582 gene methylation;

[0058] Figure 4 This is the ROC curve of the SHOX2 gene methylation ΔCT critical value. DETAILED DESCRIPTION

[0059] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0060] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0061] Example 1. Screening of molecular markers for monitoring oral mucosal carcinogenesis

[0062] 1. Materials and Methods

[0063] 1. Case selection:

[0064] A total of 59 patients with suspected mucosal diseases who visited the Oral Mucosa Department and the Oral and Maxillofacial Surgery Department of the Affiliated Hospital of Stomatology, Sun Yat-sen University from July 2021 to July 2022 were selected.

[0065] Case inclusion criteria:

[0066] (1) Patients with clinical and pathological diagnosis of oral erythema, oral leukoplakia, oral lichen planus, oral submucosal fibrosis, and other potential oral malignant lesions without dysplasia or with mild, moderate, or severe dysplasia of the mucosal epithelium, as well as squamous cell carcinoma;

[0067] (2) Not received hormone, laser, radiation or chemotherapy treatment;

[0068] (3) Able to sign the informed consent form.

[0069] Case exclusion criteria:

[0070] (1) Pregnant or lactating women;

[0071] (2) Those with serious systemic diseases such as heart, lung, liver, and kidney;

[0072] (3) Patients with other tumors or mental illnesses.

[0073] 2. Detection method:

[0074] The mucosal doctors of the Affiliated Stomatological Hospital of Sun Yat-sen University collected exfoliated cell and tissue specimens from the lesions of the patients, and the tissue specimens were sent to the Pathology Department of our hospital. The exfoliated cell specimens were sent to Changsha Jianlu Medical Laboratory for TCT, DNA ploidy analysis, and gene methylation testing.

[0075] 2.1 Collection method:

[0076] The patient rinses his mouth with water for 30 seconds and uses a low-temperature sterilized oral brush (Cervibrush) to brush 10-15 times in the same direction with medium force. After the exfoliated cells are collected, the tissue is cut at the sampling site for histopathological examination.

[0077] 2.2 Gene methylation:

[0078] Detect 10 oral cancer-related genes, including SOX1, ZNF582, SHOX2, PAX1, TIMP3, DAPK1, MGMT, TMEFF2, CDKN2A, and TFPI2. Record the types and numbers of methylated genes in the patients.

[0079] 2.3 Histopathological examination

[0080] After HE staining, two experienced oral pathologists evaluated the samples. The diagnosis was classified into no epithelial dysplasia, mild to moderate dysplasia, severe dysplasia, squamous cell carcinoma, etc. If there were more than two pathological diagnoses, the more severe diagnosis was used for judgment.

[0081] 2.4 Statistical methods

[0082] In the statistical analysis, all data were processed by SPSS software. Continuous variables were expressed as x±s, and count data were expressed as composition ratio.

[0083] In addition, the analysis found that SOX1, ZNF582 and SHOX2 genes appeared more frequently in methylation abnormalities in OPMDs cells (e.g. Figure 1 ). Among them, there is a significant correlation between the methylation status of ZNF582 and SHOX2 genes and pathological grade, indicating that the methylation levels of these two genes may change with different pathological grades, suggesting that they are related to the severity or progression of the disease. However, the methylation status of SOX1 gene is not significantly correlated with pathological grade (as shown in Figure 2). Figure 2 Based on this result, ZNF582 and SHOX2 were finally selected as biomarkers for assessing the risk of pathological grading.

[0084] Example 2: Detection of molecular markers for monitoring oral mucosal canceration

[0085] 1. Materials and Methods

[0086] 1. Case selection:

[0087] Twenty-six patients with suspected mucosal diseases admitted to the Affiliated Hospital of Stomatology of Sun Yat-sen University, Foshan Stomatological Hospital, Zhongshan Stomatological Hospital, Stomatological Hospital of Southern Medical University and Shenzhen Maternal and Child Health Hospital from June to September 2024 were selected.

[0088] This study had been reviewed and approved by the Ethics Committee before it began, and all subjects signed informed consent.

[0089] Case inclusion criteria:

[0090] (1) Patients with clinical and pathological diagnosis of oral erythema, oral leukoplakia, oral lichen planus, oral submucosal fibrosis, and other potential oral malignant lesions without dysplasia or with mild, moderate, or severe dysplasia of the mucosal epithelium, as well as squamous cell carcinoma;

[0091] (2) Not received hormone, laser, radiation or chemotherapy treatment;

[0092] (3) Able to sign the informed consent form.

[0093] Case exclusion criteria:

[0094] (1) Pregnant or lactating women;

[0095] (2) Those with serious systemic diseases such as heart, lung, liver, and kidney;

[0096] (3) Patients with other tumors or mental illnesses.

[0097] 2. Detection method:

[0098] 2.1 Collection of oral exfoliated cells and histopathological examination:

[0099] Explain the purpose of collecting exfoliated cells to the patient; after the patient rinses his mouth, the medical staff gently collects cells from the lesion and the contralateral normal area with an oral brush, puts the brush in the preservation solution and stores it with a label. Pathological examination can then be performed.

[0100] Oral exfoliated cell samples were collected as follows:

[0101] (1) Use a dedicated oral exfoliated cell collector for sampling;

[0102] (2) For sample collection methods, refer to the instructions of the manufacturer of the collector and preservation solution used;

[0103] (3) To ensure the accuracy of the test results, the sampling operation must comply with the relevant requirements for the collection of oral exfoliated cells for clinical testing.

[0104] 2.2 DNA extraction from exfoliated cell samples:

[0105] Take the oral swab sample after wiping the cheek into a 2mL centrifuge tube, and use the magnetic bead oral swab genomic DNA extraction kit (DP703) of Tiangen Biochemical Technology (Beijing) Co., Ltd. with the plug-in magnetic stand (OSE-MF-01) to extract DNA. The specific steps are as follows:

[0106] (1) Add 500 μL tissue digestion solution GHA and 10 μL Proteinase K, vortex to mix, and incubate at 65°C for 15-30 minutes. Vortex to mix several times every 10 minutes, and take 300 μL for subsequent experiments;

[0107] (2) Add 600 μL of buffer GHC (with isopropanol added) and mix well;

[0108] (3) Add 10 μL of magnetic bead suspension G and mix for 12 minutes;

[0109] (4) Place on the magnetic stand for 30 seconds and carefully remove the liquid after the magnetic beads are adsorbed;

[0110] (5) Add 900 μL of deproteinized solution PD, mix for 3 minutes, place on the magnetic stand again for 30 seconds, and then remove the liquid;

[0111] (6) Add 900 μL of PWD (with anhydrous ethanol added), mix for 3 minutes, place on the magnetic stand for 30 seconds, and then remove the liquid;

[0112] (7) Dry at room temperature for 10 to 15 minutes to ensure that the ethanol evaporates;

[0113] (8) Add 50-100 μL of elution buffer TB, mix well and incubate at 56°C for 10 minutes. After magnetic bead adsorption, transfer the DNA solution to a collection tube for storage.

[0114] 2.3 DNA sulfite conversion:

[0115] (1) Set aside the genomic DNA solution to be processed.

[0116] (2) Prepare Bisulfite Mix according to your own experimental requirements. When preparing, add 850 μl of buffer BM to each tube of dry powder and shake until the dry powder is completely dissolved. The whole process takes about 5 minutes.

[0117] (3) Prepare a bisulfite reaction system in a 200 μl centrifuge tube. The specific preparation system is shown in Table 1:

[0118] Table 1 Bisulfite reaction system

[0119]

[0120] (4) After the reaction system is prepared, bisulfite conversion is performed on an instrument such as a PCR instrument that can set a temperature change program. The entire process takes about 1 hour. The specific procedure is shown in Table 2:

[0121] Table 2 Bisulfite treatment thermal cycle conditions

[0122]

[0123] (5) Column equilibration step: Add 500 μl of equilibration solution BL to the adsorption column CB1 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm (~13,400 × g) for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube (please use the column that has been processed on the same day).

[0124] (6) After the bisulfite treatment procedure is completed, transfer the reaction system in the tube to a clean 1.5 ml centrifuge tube after a brief centrifugation.

[0125] (7) After the transfer, add 5 times the volume (600 μl) of binding buffer PB and mix thoroughly.

[0126] (8) Add the solution obtained in the previous step to the adsorption column CB1 (the adsorption column is placed in the collection tube), leave it at room temperature for 2 minutes, centrifuge it at 12,000 rpm (~13,400×g) for 30-60 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CB1 in the collection tube. Note: The volume of the adsorption column is 800 μl. If the sample volume is larger than 800 μl, it can be added in batches.

[0127] (9) Add 600 μl of rinse solution PW to the adsorption column CB1 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400 × g) for 30-60 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CB1 in the collection tube.

[0128] (10) Add 600 μl of solution DB to the adsorption column CB1, place at room temperature (15-25°C) for 15 min, centrifuge at 12,000 rpm (~13,400 × g) for 30-60 sec, discard the waste liquid in the collection tube, and place the adsorption column CB1 in the collection tube.

[0129] (11) Add 600 μl of rinse solution PW to the adsorption column CB1, centrifuge at 12,000 rpm (~13,400 × g) for 30-60 sec, and discard the waste liquid in the collection tube.

[0130] (12) Repeat step 11.

[0131] (13) Place the adsorption column CB1 in a collection tube and centrifuge at 12,000 rpm (~13,400 × g) for 2 min to remove as much rinsing solution as possible. Leave the adsorption column at room temperature for several minutes and dry it thoroughly to prevent residual rinsing solution from affecting the next step of the experiment. Note: Residual ethanol in the rinsing solution will affect subsequent PCR experiments.

[0132] (14) Take out the adsorption column CB1 and place it in a clean centrifuge tube. Add 20 μl of elution buffer EB to the middle of the adsorption membrane and leave it at room temperature for 2 min. Centrifuge at 12,000 rpm (~13,400 × g) for 2 min to collect the DNA solution.

[0133] 2.4QPCR detection:

[0134] (1) The sequences of ZNF582 gene detection primers, SHOX2 gene detection primers, and internal reference gene ACTB detection primers are shown in Table 3 below.

[0135] ZNF582 and SHOX2 gene detection primers can be used to specifically amplify the methylation sites of the target regions of the ZNF582 and SHOX2 genes, wherein the target region of the ZNF582 gene is selected from the negative chain chr19: 56,375,846-56,393,538 (GRCh38 (hg38) version); the target region of the SHOX2 gene is selected from chr3: 158,095,905-158,106,420 (GRCh38 (hg38) version).

[0136] Table 3 Primer sequence information

[0137]

[0138] (2) Using the above primer combination, perform methylation quantitative PCR on the sulfite-converted DNA obtained in step 2.3 above.

[0139] In this embodiment, the ZNF582 gene, SHOX2 gene and ACTB gene of the same sample were retested in a single tube, that is, in addition to the necessary reaction components and templates, primers for all target genes and internal reference genes were added to one PCR tube each time, and the PCR reaction configuration system was shown in Table 4. PCR amplification was performed according to the amplification program shown in Table 5.

[0140] The PCR primer probe premix of three genes can be prepared first, and the PCR primer premix includes: ZNF582 gene forward primer, ZNF582 gene reverse primer, SHOX2 gene forward primer, SHOX2 gene reverse primer, ACTB gene forward primer, ACTB gene reverse primer.

[0141] The PCR reaction system is shown in Table 4.

[0142] Table 4 PCR reaction system

[0143]

[0144] The PCR amplification program is shown in Table 5:

[0145] Table 5 PCR amplification program

[0146]

[0147] (3) QPCR result analysis:

[0148] a. Baseline and threshold line adjustment:

[0149] After PCR is completed, the baseline is adjusted for each gene individually. The fluorescence value 1-2 cycles before the minimum Ct value of the sample in a PCR is set as the baseline value, and the threshold is set at the inflection point of the S-shaped amplification curve to obtain the Ct value of each gene in the sample.

[0150] b. Sample validity confirmation:

[0151] The VIC channel amplification curve of the internal reference gene ACTB must be S-shaped and the Ct value must be ≤35, and the sample is valid;

[0152] The VIC channel amplification curve of the internal reference gene ACTB is S-shaped, and the Ct value is greater than 35 or there is no amplification curve, which means the sample is invalid.

[0153] c. Confirmation of target gene methylation:

[0154] When the amplification curve of the ZNF582 gene FAM channel is S-shaped and the ΔCt value is ≤12.547, the sample is considered to be positive for ZNF582 gene methylation; when the ΔCt value is greater than 12.547 or there is no amplification curve, the sample is considered to be negative for ZNF582 gene methylation;

[0155] When the amplification curve of the SHOX2 gene FAM channel is S-shaped and the ΔCt value is ≤7.435, the sample is considered to be positive for SHOX2 gene methylation; when the ΔCt value is greater than 7.435 or there is no amplification curve, the sample is considered to be negative for SHOX2 gene methylation.

[0156] When ZNF582 and / or SHOX2 genes showed methylation-positive expression, the sample was regarded as a methylation-positive sample.

[0157] ΔCt=Ct value of target gene (ZNF582 gene or SHOX2 gene)-Ct value of internal reference gene.

[0158] The results are shown in Tables 6 and 7.

[0159] The following data are the critical values ​​(thresholds) calculated by the ROC curve;

[0160] Different from general markers, the results of this application are obtained by judging the critical value obtained by the gold standard of pathological examination, which is a well-founded proposal from clinical evidence to general standards.

[0161] Table 6 Test results of 26 samples

[0162]

[0163]

[0164] Table 7 Contingency table of methylation combined test results and pathological grades

[0165]

[0166] As shown in Table 7, based on the contingency table of methylation combined test results and pathological grading, the sensitivity was calculated to be 93.75% and the specificity was 80%. This indicates that the test has a high sensitivity in detecting severe dysplasia or oral cancer, and also shows good specificity in excluding no dysplasia or mild to moderate dysplasia, and has good clinical application prospects.

[0167] ROC curve of ZNF582 gene methylation ΔCT critical value Figure 3 As shown, the ROC curve of SHOX2 gene methylation ΔCT critical value is as follows Figure 4 The area under the curve (AUC), sensitivity and specificity were calculated from the ROC curve.

[0168] The area under the ROC curve results for the ZNF582 gene are shown in Table 8.

[0169] Table 8

[0170]

[0171] The area under the ROC curve results for the SHOX2 gene are shown in Table 9.

[0172] Table 9

[0173]

[0174] The detection efficiency of the reagent or kit of the present application can be determined based on the above data.

[0175] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A methylation molecular marker for monitoring oral mucosal carcinogenesis, characterized in that: The methylation molecular markers are ZNF582 gene and SHOX2 gene.

2. The methylation molecular marker according to claim 1, characterized in that The methylation molecular marker is a nucleic acid methylated in at least one target region of the target gene ZNF582, or / and a nucleic acid methylated in at least one target region of the target gene SHOX2; The target region is selected from the following fragments: ZNF582 gene: negative strand chr19: 56,375,846-56,393,538; SHOX2 gene: chr3: 158,095,905-158,106,420.

3. A detection reagent for detecting the methylation molecular marker according to claim 1, characterized in that: The detection reagent includes a primer combination for detecting the methylation molecular marker; The primer combination is a primer pair for the ZNF582 gene and a primer pair for the SHOX2 gene; The nucleotide sequences of the primer pairs for the ZNF582 gene are shown in SEQ ID NOs: 1-2; The nucleotide sequences of the primer pairs targeting the SHOX2 gene are shown in SEQ ID NOs: 3-4.

4. Use of the detection reagent according to claim 3 in preparing a detection kit, characterized in that: The detection kit is used to provide detection results of methylation markers for monitoring oral mucosal carcinogenesis.

5. A detection kit, characterized in that: The detection kit comprises the detection reagent as claimed in claim 3.

6. The detection kit according to claim 5, characterized in that The detection kit also includes a primer pair of an internal reference gene and a fluorescent quantitative PCR detection reagent.

7. The detection kit according to claim 6, characterized in that: The internal reference gene includes the ACTB gene, and the nucleotide sequence of the primer pair of the internal reference gene is shown in SEQ ID NOs: 5-6.

8. Use of the methylation molecular marker according to claim 1 or 2, or the detection reagent according to claim 3, or the detection kit according to any one of claims 5 to 7 in a detection platform for detecting and interpreting methylation markers for monitoring oral mucosal carcinogenesis.

9. A detection platform or device for detecting and interpreting methylation markers for monitoring oral mucosal canceration, characterized in that: The detection platform or device comprises: 1) A detection unit, used to detect the methylation level of a molecular marker in a biological sample; 2) Analysis unit, used for interpretation of test results of methylation markers for oral mucosal cancer monitoring; The analysis unit is used for interpretation of the methylation marker detection results for oral mucosal canceration monitoring based on the following determination method: Under the premise that the amplification curve of the internal reference gene is S-shaped and the Ct value is ≤35, if the amplification curve of the target region of the ZNF582 gene is S-shaped and the ΔCt of the ZNF582 gene is ≤12.547, it is judged that the ZNF582 gene is methylated positive; if the ΔCt of the ZNF582 gene is >12.547 or there is no amplification curve in the target region of the ZNF582 gene, it is judged that the ZNF582 gene is methylated negative; Under the premise that the amplification curve of the internal reference gene is S-shaped and Ct≤35, if the amplification curve of the target region of the SHOX2 gene is S-shaped and the ΔCt of the SHOX2 gene is ≤7.435, it is judged that the SHOX2 gene is methylated positive; if the ΔCt of the SHOX2 gene is >7.435 or there is no amplification curve in the target region of the SHOX2 gene, it is judged that the SHOX2 gene is methylated negative; When ZNF582 and / or SHOX2 genes showed methylation-positive expression, the sample was considered as a methylation-positive sample; ΔCt=Ct value of target gene-Ct value of reference gene; the target gene includes ZNF582 gene or SHOX2 gene.

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