A methylation molecular marker, detection kit and its application in oral mucosal cancer screening

Abstract

This application relates to the field of biotechnology, specifically disclosing a methylation molecular marker and its detection kit for early warning of oral mucosal cancer. The methylation molecular markers are the ZNF582 and SHOX2 genes. This application provides important molecular information for oral cancer screening by detecting the methylation levels of the ZNF582 and SHOX2 genes. The molecular markers of this application have high accuracy, sensitivity, and specificity, providing an important reference for early warning of oral mucosal cancer. Furthermore, the detection method is simple, rapid, low-cost, and convenient to sample, which can help achieve non-invasive early warning of potential oral malignant diseases, showing promising application prospects.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a methylation molecular marker, a detection kit, and its application in the screening of oral mucosal cancer. Background Technology

[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 erythroplakia (OE), oral lichen planus (OLP), and oral submucosal fibrosis (OSF). It has been reported that 5%–18% of OPMDs may develop into oral cancer, with a malignancy rate of 2%–3% for OLK, 14%–50% for OE, and 1.14% for OLP. Oral cancer is one of the most common malignant tumors of the head and neck, but the five-year survival rate for oral cancer diagnosed and treated early can reach over 80%.

[0003] Histopathological examination is the gold standard for diagnosing oral cancer, providing the most reliable cytological and histological evidence through microscopic examination of tissue sections. However, biopsy, as a commonly used examination method, has drawbacks such as being invasive, time-consuming, and difficult for patients to adhere to long-term, limiting its practicality as a cancer monitoring tool. Furthermore, physicians' decisions regarding the need for a biopsy are often influenced by subjective criteria, potentially leading to missed opportunities for early treatment. Therefore, the search for non-invasive screening technologies has become a key research focus. These technologies offer advantages such as minimal invasiveness, simplicity, speed, repeatability, and patient acceptance, and have already been applied in clinical practice.

[0004] Gene methylation plays a crucial role in the development of oral squamous cell carcinoma (OSCC) from oral OPMDs. Studies have shown differences in DNA methylation patterns between OPMD and OSCC patients, which may aid in diagnosis and prediction of 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 research on methylated genes as molecular biomarkers for predicting cancer risk. By identifying differential methylation patterns, researchers have determined methylation sites that occur during carcinogenesis, providing a foundation for potential molecular markers. Numerous studies have validated the association between certain methylation markers and cancer risk; these markers can be detected through blood or tissue samples, providing new tools for early screening. Researchers are exploring the combined application of methylation markers to improve the accuracy and sensitivity of predictions. Furthermore, the association of specific methylation markers with the clinical prognosis of cancer provides important information for personalized treatment and management.

[0005] Currently, the application of gene methylation technology in monitoring oral mucosal carcinogenesis requires further research. Summary of the Invention

[0006] The purpose of this application is to overcome the shortcomings of the prior art and provide a methylation molecular marker, a detection kit, and its application in oral mucosal cancer screening for monitoring oral mucosal cancer. 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 cancer.

[0007] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0008] This application provides a methylation molecular marker for monitoring oral mucosal carcinogenesis, wherein the methylation molecular marker is the ZNF582 gene and the SHOX2 gene.

[0009] Through extensive research and experimentation, the inventors of this application discovered that the ZNF582 and SHOX2 genes are significantly associated with the carcinogenesis progression of oral mucosal malignancies (OPMDs). The degree of methylation at the methylation sites of these genes is strongly correlated with the carcinogenesis progression of OPMDs. This application is the first to use the ZNF582 gene, the SHOX2 gene, or a combination of both genes as methylation molecular markers for monitoring oral mucosal carcinogenesis. The combination of the ZNF582 and SHOX2 genes as an important parameter for early warning of oral mucosal carcinogenesis is more accurate.

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

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

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

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

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

[0015] The target region selected above is derived from the GRCh38 (hg38) version.

[0016] This application utilizes the ZNF582 and SHOX2 genes to monitor oral mucosal carcinogenesis by detecting methylation sites in these genes, demonstrating high accuracy, sensitivity, and specificity.

[0017] This application also provides a detection reagent for detecting the above-mentioned methylation molecular markers, the detection reagent comprising a primer combination for detecting the methylation molecular markers;

[0018] The primer pair consists of a primer pair targeting the ZNF582 gene and a primer pair targeting the SHOX2 gene;

[0019] The nucleotide sequences of the primer pairs targeting the ZNF582 gene are shown in SEQ ID NO: 1-2;

[0020] The nucleotide sequences of the primer pairs targeting the SHOX2 gene are shown in SEQ ID NO: 3-4.

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

[0022] By designing primer combinations targeting methylated sequences and performing PCR amplification on nucleic acid sequences containing methylation sites, the presence of methylated fragments indicates the presence of methylation at those sites. Conversely, the absence of methylated fragments indicates the absence of methylation at those sites. In this application, the ΔCT value is used as an analytical indicator, which not only makes the determination but also eliminates errors caused by template concentrations between different samples, thus improving the practicality of this application.

[0023] This application also provides the application of the above-mentioned detection reagents in the preparation of detection kits, which are used to provide detection results of methylation markers for monitoring oral mucosal carcinogenesis.

[0024] This application also provides a test kit, which includes the above-described test reagents.

[0025] As a preferred embodiment of the detection kit described in this application, the detection kit further includes primer pairs for an internal reference gene and a real-time PCR detection reagent.

[0026] In a preferred embodiment of the detection kit described in this 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 NO: 5-6.

[0027] Preferably, the fluorescence quantitative PCR detection reagent includes commonly used reagents for fluorescence quantitative PCR detection such as PCR buffer, SYBR qPCR Master Mix, and ddH2O.

[0028] The kit also includes at least one of the following: oral swabs, sample collection containers, cell preservation solution, nucleic acid extraction reagents and consumables, and sulfite conversion reagent. All of the above reagents / devices can be purchased individually from the market or prepared in-house; therefore, they can be incorporated into the kit according to actual needs.

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

[0030] The kit described in this application enables accurate detection of small numbers of samples, thereby reducing the difficulty of sample collection and facilitating testing. There are no strict limitations on the material and shape of the sample collection container; it can be flexibly selected based on conventional practices in the field.

[0031] Nucleic acid extraction reagents are used to extract DNA; specifically, they can be conventional nucleic acid extraction reagents in this field. This application mainly uses magnetic bead extraction. Sulfite conversion reagents can convert cytosine (C) in nucleic acid fragments to uracil (U), while 5-methylcytosine (5mC) remains unchanged.

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

[0033] This application also provides instructions on how to use the above-mentioned reagent kit, including the following steps:

[0034] (1) Extract genomic DNA from the biological sample to be tested;

[0035] (2) The genomic DNA of the biological sample to be tested is subjected to sulfite conversion;

[0036] (3) Use the primer combination in the above kit to perform quantitative PCR detection of methylation of DNA after sulfite conversion;

[0037] (4) Analyze the test results.

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

[0039] This application also provides the application of the above-mentioned methylation molecular markers, or the above-mentioned detection reagents, or the above-mentioned detection kits in a detection platform for detecting and interpreting methylation markers for monitoring oral mucosal carcinogenesis.

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

[0041] 1) Detection unit, used to detect the methylation level of molecular markers in biological samples;

[0042] 2) Analysis unit for interpreting the detection results of methylation markers used in the monitoring of oral mucosal cancer;

[0043] The analysis unit interprets the results of methylation marker detection for oral mucosal cancer monitoring based on the following criteria:

[0044] If the amplification curve of the internal reference gene is S-shaped and the Ct value is ≤35, and the amplification curve of the target region of the ZNF582 gene is S-shaped, and the ΔCt of the ZNF582 gene is ≤12.547, then the ZNF582 gene is judged to be 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, then the ZNF582 gene is judged to be methylated negative.

[0045] If the amplification curve of the internal reference gene is S-shaped and Ct≤35, and the amplification curve of the target region of the SHOX2 gene is S-shaped and the ΔCt of the SHOX2 gene is ≤7.435, then the SHOX2 gene is judged to be 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, then the SHOX2 gene is judged to be methylated negative.

[0046] When the ZNF582 and / or SHOX2 genes show methylation positivity, the sample is considered a methylation positive sample.

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

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

[0049] Preferably, the detection unit includes a real-time PCR instrument. In some specific embodiments, the detection unit further includes a DNA extraction device for extracting DNA from biological samples, including a magnetic rack, etc.

[0050] This application utilizes QMSP (quantitative methylation-specific PCR) to quantitatively analyze the methylation levels of the ZNF582 and SHOX2 genes. The detection steps include extracting DNA from exfoliated cells of the oral mucosa, treating with bisulfite to convert unmethylated cytosine to uracil, and then amplifying the methylation sites of the ZNF582 and SHOX2 genes using methylation-specific primers (SEQ ID NO: 1-4). The detection results are read using a quantitative fluorescence analyzer and quantitatively analyzed using a standard curve to ultimately determine the methylation levels of the ZNF582 and SHOX2 genes for monitoring oral mucosal carcinogenesis.

[0051] Preferably, the biological sample includes exfoliated cells from the oral mucosa.

[0052] This application uses exfoliated cells from the affected area as the test sample. The sampling is simple and easy to obtain, and will not cause any pain or impact on the patient, so it is highly accepted by patients.

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

[0054] This application provides a methylation molecular marker, a detection kit, and its application in oral mucosal cancer screening. The molecular markers (ZNF582 gene and SHOX2 gene) of this application have high accuracy, sensitivity, and specificity, which are of great significance for monitoring oral mucosal cancer. Moreover, the detection method is simple, rapid, low-cost, and convenient to sample, and can be used as an important parameter to achieve non-invasive early warning of potential oral malignant diseases, showing good application prospects. Attached Figure Description

[0055] Figure 1 A frequency diagram of methylated genes in OPMDs cells;

[0056] Figure 2 This is a diagram showing the relationship between abnormal methylation genes and pathological grading in Example 1;

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

[0058] Figure 4 ROC curve of ΔCT threshold for SHOX2 gene methylation. Detailed Implementation

[0059] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0060] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified, and the raw materials used in each parallel experiment are the same.

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

[0062] I. Materials and Methods:

[0063] 1. Case selection:

[0064] Fifty-nine patients with suspected mucosal diseases who visited the Department of Oral Mucosa and the Department of Oral and Maxillofacial Surgery at the Affiliated Stomatological Hospital of Sun Yat-sen University between July 2021 and July 2022 were selected.

[0065] Case inclusion criteria:

[0066] (1) Patients with oral erythema, oral leukoplakia, oral lichen planus, oral submucosal fibrosis and other potential oral malignant lesions with mild, moderate and severe abnormal proliferation of mucosal epithelium without abnormal proliferation, as well as squamous cell carcinoma.

[0067] (2) Not treated with hormones, lasers, radiation or chemical drugs;

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

[0069] Exclusion criteria for cases:

[0070] (1) Pregnant or lactating women;

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

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

[0073] 2. Detection method:

[0074] Doctors at the Stomatological Hospital Affiliated to Sun Yat-sen University collected exfoliated cells and tissue specimens from the patient's lesion site. The tissue specimens were sent to the hospital's pathology department. The exfoliated cell specimens were sent to Changsha Jianlu Medical Laboratory for TCT, DNA ploidy analysis, and gene methylation detection.

[0075] 2.1 Material Collection Method:

[0076] The patient rinsed their mouth with water for 30 seconds, then used a low-temperature sterilized oral brush (Cervibrush) to brush 10-15 times in the same direction with moderate pressure. After collecting exfoliated cells, tissue was excised from the sampling site for histopathological examination.

[0077] 2.2 Gene methylation:

[0078] Ten oral cancer-related genes were detected: SOX1, ZNF582, SHOX2, PAX1, TIMP3, DAPK1, MGMT, TMEFF2, CDKN2A, and TFPI2. The types and numbers of methylated genes in patients were recorded.

[0079] 2.3 Histopathological examination

[0080] Following HE staining, the diagnosis was evaluated by two experienced oral pathologists. The diagnosis was classified as aplastic angioepithelial neoplasia, mild to moderate dysplasia, severe dysplasia, squamous cell carcinoma, etc. If more than two pathological diagnoses were present, the more severe diagnosis was used.

[0081] 2.4 Statistical Methods

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

[0083] Furthermore, analysis revealed that the SOX1, ZNF582, and SHOX2 genes in OPMDs cells exhibited higher frequencies in cases of abnormal methylation (e.g., Figure 1 As shown in the figure). Among them, the methylation status of the ZNF582 and SHOX2 genes showed a significant association with pathological grade, indicating that the methylation levels of these two genes may change with different pathological grades, suggesting a correlation with disease severity or progression. However, the methylation status of the SOX1 gene did not show a significant correlation with pathological grade (e.g., Figure 2 (As shown). Based on these results, ZNF582 and SHOX2 were ultimately selected as biomarkers for assessing the risk of pathological grading.

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

[0085] I. Materials and Methods:

[0086] 1. Case selection:

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

[0088] This study was reviewed and approved by the ethics committee before it began, and all participants signed informed consent forms.

[0089] Case inclusion criteria:

[0090] (1) Patients with oral erythema, oral leukoplakia, oral lichen planus, oral submucosal fibrosis and other potential oral malignant lesions with mild, moderate and severe abnormal proliferation of mucosal epithelium without abnormal proliferation, as well as squamous cell carcinoma.

[0091] (2) Not treated with hormones, lasers, radiation or chemical drugs;

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

[0093] Exclusion criteria for cases:

[0094] (1) Pregnant or lactating women;

[0095] (2) Individuals with severe 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 exfoliated cells from the oral cavity and histopathological examination:

[0099] Explain the purpose of collecting exfoliated cells to the patient; after the patient rinses their mouth, medical staff gently collect cells from the lesion and the opposite normal area using an oral brush. The brush is then placed in preservation solution and labeled for storage. Pathological examination can be performed subsequently.

[0100] Oral exfoliated cell samples were collected according to the following method:

[0101] (1) Samples were collected using a dedicated oral exfoliative cell collector;

[0102] (2) For sample collection methods, please refer to the instructions of the sampler and preservation solution manufacturer.

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

[0104] 2.2 DNA extraction from exfoliated cell samples:

[0105] Collect oral swab samples from the inside of the cheek into 2mL centrifuge tubes. Extract DNA using the Magnetic Bead Method Oral Swab Genomic DNA Extraction Kit (DP703) from Tiangen Biotech (Beijing) Co., Ltd., along with the interlocking magnetic rack (OSE-MF-01). The specific steps are as follows:

[0106] (1) Add 500 μL of tissue digestion solution GHA and 10 μL of proteinase K, vortex mix, and incubate at 65°C for 15-30 minutes, vortex 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 it on the magnetic rack for 30 seconds, and carefully remove the liquid after the magnetic beads are attracted;

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

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

[0112] (7) Allow to air dry at room temperature for 10-15 minutes to ensure ethanol evaporates;

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

[0114] 2.3 DNA sulfite transformation:

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

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

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

[0118] Table 1. Bisulfite Reaction System

[0119]

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

[0121] Table 2 Thermal cycling conditions for bisulfite treatment

[0122]

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

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

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

[0126] (8) Add the solution obtained in the previous step to the adsorption column CB1 (place the adsorption column in the collection tube), let it stand at room temperature for 2 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 into the collection tube. Note: The volume of the adsorption column is 800 μl. If the sample volume is greater than 800 μl, it can be added in batches.

[0127] (9) Add 600 μl of washing 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, discard the waste liquid in the collection tube, and put the adsorption column CB1 into the collection tube.

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

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

[0130] (12) Repeat step 11.

[0131] (13) Place the adsorption column CB1 into the collection tube and centrifuge at 12,000 rpm (~13,400 × g) for 2 min to remove as much of the wash solution as possible. Allow the adsorption column to air dry completely at room temperature for several minutes to prevent residual wash solution from affecting subsequent experiments. Note: Residual ethanol in the wash solution may affect subsequent PCR experiments.

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

[0133] 2.4 qPCR detection:

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

[0135] The ZNF582 and SHOX2 gene detection primers can specifically amplify the methylation sites in the target regions of the ZNF582 and SHOX2 genes. The target region of the ZNF582 gene is selected from negative strand 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) Use the above primer combination to perform methylation quantitative PCR on the DNA obtained after sulfite conversion in step 2.3.

[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 template, primers for all the target genes and internal reference genes were added to each PCR tube. The PCR reaction configuration system is shown in Table 4, and PCR amplification was performed according to the amplification program shown in Table 5.

[0140] Three types of PCR primer and probe premixes can be prepared first. The PCR primer premixes include: ZNF582 gene forward primer, ZNF582 gene reverse primer, SHOX2 gene forward primer, SHOX2 gene reverse primer, ACTB gene forward primer, and 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 procedure is shown in Table 5:

[0145] Table 5 PCR amplification program

[0146]

[0147] (3) Analysis of qPCR results:

[0148] a. Baseline and threshold line adjustment:

[0149] After PCR is completed, the baseline is adjusted for each gene individually. The minimum Ct value of the sample in one PCR is set as the fluorescence value 1-2 cycles in advance, 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 verification:

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

[0152] If the VIC channel amplification curve of the internal reference gene ACTB is S-shaped, the Ct value is >35, or there is no amplification curve, then the sample is invalid.

[0153] c. Confirmation of methylation of the target gene:

[0154] When the amplification curve of the FAM channel of the ZNF582 gene 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 >12.547 or there is no amplification curve, the sample is considered to be negative for ZNF582 gene methylation.

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

[0156] When the ZNF582 and / or SHOX2 genes show methylation positivity, the sample is considered 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 critical values ​​(thresholds) calculated using ROC curves;

[0160] Unlike general biomarkers, the results in this application are obtained through threshold determination based on the gold standard of pathological examination, which is an evidence-based proposal from clinical evidence to general standards.

[0161] Table 6. Detection results of 26 samples

[0162]

[0163]

[0164] Table 7. Contingency Table of Methylation Combined Test Results and Pathological Grading

[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 to be 80%. This indicates that the test has high sensitivity in detecting severe dysplasia or oral cancer, and also shows good specificity when excluding dysplasia without abnormalities or mild to moderate dysplasia, thus demonstrating good prospects for clinical application.

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

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

[0169] Table 8

[0170]

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

[0172] Table 9

[0173]

[0174] The detection efficacy of the reagents or kits in this 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 solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. Use of a primer combination of methylation molecular markers for monitoring oral mucosa carcinogenesis in the preparation of a detection platform for detecting and interpreting potential malignant lesions of the oral cavity, characterized in that, The methylation molecular markers are ZNF582 gene and SHOX2 gene; 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 pair for the ZNF582 gene are shown in SEQ ID NO: 1-2; The nucleotide sequences of the primer pair for the SHOX2 gene are shown in SEQ ID NO: 3-4; The methylation marker detection results for the monitoring of oral mucosa carcinogenesis are interpreted 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 determined that the ZNF582 gene is methylation 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 determined that the ZNF582 gene is methylation negative; 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 SHOX2 gene is S-shaped, and the ΔCt of the SHOX2 gene is ≤7.435, it is determined that the SHOX2 gene is methylation 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 determined that the SHOX2 gene is methylation negative; When the ZNF582 gene and / or the SHOX2 gene is determined to be methylation positive, it is considered as a potential malignant lesion in the oral cavity; ΔCt=Ct value of target gene-Ct value of internal reference gene; the target gene includes the ZNF582 gene or the SHOX2 gene; the internal reference gene is the internal reference gene ACTB.

2. Use according to claim 1, wherein The nucleotide sequences of the primer pair of the internal reference gene are shown in SEQ ID NO: 5-6.

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