Detection reagent for methylation of myeloblastoma and preparation method thereof

Abstract

The invention provides an innovative method for methylation detection of medulloblastoma, and high-sensitivity and non-invasive methylation detection can be realized by combining a liquid biopsy technology and a novel methylation marker LINC00511 gene. According to the method, quantum dot labeling and surface enhanced Raman scattering technologies are utilized, so that the detection sensitivity and specificity of the methylation marker are remarkably improved, and the method is suitable for real-time monitoring of the low-abundance methylation marker. Compared with a traditional methylation detection method, the method provided by the invention can realize rapid and accurate detection in biological samples such as blood, urine and the like. The detection technology can be used for early diagnosis of medulloblastoma, can also effectively monitor the treatment reaction, has a wide clinical application prospect, and is especially suitable for the fields of non-invasive diagnosis and personalized treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, in particular to a methylation detection reagent for medulloblastoma and a preparation method thereof. BACKGROUND

[0002] Medulloblastoma is a common brain tumor in children. Traditional diagnostic methods rely on imaging examination and tissue biopsy, but have problems such as strong invasiveness, difficulty in early diagnosis, and false negatives caused by tumor heterogeneity. Therefore, there is an urgent need for a non-invasive, real-time, and sensitive detection method to monitor the progression and efficacy of medulloblastoma.

[0003] DNA methylation is a key regulatory mechanism of epigenetics and is closely related to the occurrence and development of tumors. Studies have found that specific methylation markers exist in medulloblastoma, such as MGMT, MLH1, CDKN2A, etc. These markers can be used for early diagnosis and prognosis evaluation. However, traditional methylation detection relies on tissue samples, has low sensitivity, and is highly invasive.

[0004] Liquid biopsy technology analyzes circulating tumor DNA (ctDNA) or circulating free DNA (cfDNA) in blood, urine, and other body fluid samples, providing non-invasive detection and dynamic information on tumors. Liquid biopsy technology has high sensitivity and real-time monitoring capabilities, making it suitable for early tumor screening, treatment response monitoring, and personalized treatment.

[0005] The present application combines liquid biopsy technology with less widely reported methylation markers such as LINC00511 and HOTAIR, providing a non-invasive, real-time, and highly sensitive detection platform that can effectively improve early diagnosis and treatment monitoring of medulloblastoma, providing support for personalized treatment and prognosis evaluation. SUMMARY

[0006] To address the above problems, the present application provides a methylation detection reagent for medulloblastoma and a preparation method thereof. The reagent combines liquid biopsy technology with novel medulloblastoma-specific methylation markers, enabling non-invasive detection through blood, urine, and other body fluid samples, with high sensitivity and specificity. It can monitor methylation marker changes in real time, provide early diagnosis, treatment response evaluation, and personalized treatment data support, and meet the needs of precision medicine for medulloblastoma.

[0007] To achieve the above-mentioned purposes, the present application adopts the following technical solutions: A methylation detection reagent for medulloblastoma, comprising the following components: Methylation probes, adaptive biomolecular recognition molecules, and liquid biopsy systems for one or more of blood, urine, saliva, thoracic and abdominal cavity fluid, and lymphatic fluid.

[0008] Optionally, in the methylation detection reagent for medulloblastoma, the adaptive biorecognition molecule is one or more of antibodies, monoclonal antibodies, single-chain antibodies, peptides, and nanobodies.

[0009] Optionally, in the methylation detection reagent for medulloblastoma, the methylation probe is a labeled probe based on fluorescence or surface-enhanced Raman scattering technology.

[0010] Optionally, in the methylation detection reagent for medulloblastoma, the in vivo biopsy system is used to separate exosomes, circulating cell-free DNA, or circulating tumor DNA from the sample.

[0011] Optionally, in the methylation detection reagent for medulloblastoma, the separation of exosomes, circulating cell-free DNA, or circulating tumor DNA is performed by ultracentrifugation or magnetic particle separation technology.

[0012] Optionally, the methylation detection reagent for medulloblastoma includes a methylation probe designed based on the CpG island region of genomic DNA, targeting medulloblastoma-specific methylation sites, including one or more of LINC00511, HOTAIR, MGMT, TP53, PTEN, and RB1.

[0013] Optionally, the methylation detection reagent for medulloblastoma is quantified by a standard curve method or an internal reference method, and the detection limit range is 0.1 ng / mL to 1 ng / mL.

[0014] Optionally, the detection method for the methylation detection reagent in medulloblastoma includes the following specific steps: S1. Using the methylation detection reagent according to claim 1, non-invasive detection is performed on biological samples; S2. Screen for methylation markers associated with medulloblastoma and combine the methylation probes with adaptive biorecognition molecules to form highly specific detection probes; S3. By monitoring changes in methylation markers in real time, analyze the methylation characteristics of medulloblastoma and provide data support for the evaluation of treatment response; S4. Extract circulating cell-free DNA or circulating tumor DNA from blood or other body fluid samples using liquid biopsy techniques.

[0015] Optionally, the methylation detection reagent for medulloblastoma has a sensitivity of 90%-98% or higher and a specificity of 95%-98% for the methylation marker.

[0016] The beneficial effects of this invention are: The methylation detection reagent prepared by this invention has excellent sensitivity and specificity, enabling non-invasive detection using bodily fluid samples such as blood and urine, with a sensitivity of over 90% and a specificity of over 95%. This reagent can monitor changes in methylation markers in medulloblastoma in real time, providing data support for early diagnosis, treatment response assessment, and personalized treatment. Through liquid biopsy technology, this reagent significantly improves the non-invasiveness and accuracy of detection. The combination of methylation probes and adaptive biorecognition molecules significantly enhances the reagent's detection capability in various biological samples, showing broad application prospects. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0018] Figure 1 This is a line graph showing the sensitivity changes of different experimental groups at different times in this invention; Figure 2 This is a bar chart showing the sensitivity changes of different sample types in different experimental groups according to the present invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0020] Example 1: S1. Peripheral blood samples were collected from 10 patients diagnosed with medulloblastoma, with 10 mL of each sample. S2. Select the promoter region of the LINC00511 gene as the target methylation site; S3. Select CdSe quantum dots and use polyvinyl alcohol to modify their surface to improve their water solubility and stability. Design oligonucleotide primers that bind to the promoter region of the MGMT gene and label the fluorophore at the 3' end with fluorescent isothiocyanate fluorescein (FITC).

[0021] S4. The methylated probe was crosslinked with quantum dots using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) crosslinking reagents at 20°C for 4 h. After crosslinking, the probe was purified by ultrafiltration and gel permeation chromatography, and the purity and fluorescence intensity of the probe were finally verified by UV-Vis spectroscopy and fluorescence spectroscopy.

[0022] S5. Use fluorescence microscopy to monitor changes in MGMT methylation markers in blood samples in real time.

[0023] Example 2: S1. Peripheral blood samples were collected from 10 patients diagnosed with medulloblastoma, with 10 mL of each sample. S2. Select the promoter region of the LINC00511 gene as the target methylation site; S3. Design a methylation probe based on SERS technology and combine it with gold nanoparticles (AuNPs) to enhance the signal. AuNPs were selected as the SERS substrate, and the methylation probe was covalently bound to the surface of gold nanoparticles using a silanization method to ensure that the probe could specifically bind to the methylation site in the promoter region of the MGMT gene.

[0024] S4. Using EDC / NHS crosslinking reagent, the probe was crosslinked with gold nanoparticles at 20°C for 4 h. After crosslinking, the unbound material was removed by ultrafiltration and gel permeation chromatography. Finally, the purity and fluorescence intensity of the probe were verified by UV-Vis spectroscopy and fluorescence spectroscopy.

[0025] S5. Use the SERS system to monitor MGMT methylation markers in blood samples in real time.

[0026] Comparative Example 1: S1. Peripheral blood samples were collected from 10 patients diagnosed with medulloblastoma, with 10 mL of each sample. S2. Methylation of the LINC00511 gene was detected using conventional PCR methods. Primers targeting methylated (M) and unmethylated (U) CpG sites were designed, and HotStart Taq DNA polymerase was used. The reaction conditions were 95°C denaturation for 5 min, 40 cycles (95°C for 30 s, 60°C annealing for 30 s, 72°C extension for 45 s). The products were analyzed by 1% agarose gel electrophoresis.

[0027] Comparative Example 2: S1. Peripheral blood samples were collected from 10 patients diagnosed with medulloblastoma, with 10 mL of each sample. S2. The promoter region of the LINC00511 gene was selected as the target methylation site. Specific methylation primers were designed, and PCR amplification was performed using fluorescently labeled methylation probes. The PCR reaction system used HotStart Taq DNA polymerase. The reaction conditions were: 95°C for 5 minutes denaturation, 40 cycles of 95°C for 30 seconds denaturation, 60°C for 30 seconds annealing, 72°C for 45 seconds extension, and a final extension at 72°C for 10 minutes. Fluorescence signals were monitored using a real-time PCR instrument to quantitatively analyze the methylation status, and methylation changes were calculated using a standard curve.

[0028] Performance testing 1. Sensitivity Test First, standardized methylated DNA samples were used to simulate five concentration points (0.01 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, and 5 ng / mL). A target methylation marker, LINC00511, was selected, and real-time monitoring was performed using a quantum dot-labeled methylation probe and a SERS probe method. At least three replicates were set for each concentration to ensure the reliability of the test results. The reaction system consisted of a 20 μL PCR system, with cfDNA concentrations set according to the five concentration points mentioned above. The reaction conditions were: initial denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 45 seconds, and final extension at 72°C for 10 minutes. The sensitivity was calculated using a standard curve method by comparing fluorescence intensity with standard concentrations. The sensitivity was required to be at least 90% to ensure that the method could effectively detect low-abundance methylation markers and was suitable for clinical applications.

[0029] Table 1. Sensitivity data of different samples at different concentrations.

[0030] Example 1 demonstrated the highest sensitivity in the detection of low-concentration methylation biomarkers, reaching 92% at 0.01 ng / mL, significantly outperforming conventional PCR and fluorescent probe methods. Compared to Example 2, Example 1 exhibited higher detection sensitivity, particularly demonstrating stronger detection capability in the detection of low-concentration biomarkers.

[0031] 2. Specificity test First, the promoter region of the target methylation biomarker LINC00511 gene was selected as the test target, and methylation-specific probes targeting these targets were designed. Then, the non-target gene MGMT was selected as a reference for cross-testing, and probes targeting the methylation sites of this gene were designed and used simultaneously with the target methylation probes. Methylation biomarkers in the samples were detected using quantum dot labeling or SERS probe methods at five different concentrations: 0.01 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, and 5 ng / mL. The signals of the methylation biomarkers were quantitatively analyzed using the standard curve method. Finally, the specificity was calculated and ensured to be at least 95% to ensure that the detection method could specifically identify the target methylation biomarker.

[0032] Table 2. Specificity data of different samples at different concentrations

[0033] Example 1 demonstrated the best specificity at all concentrations, maintaining a high specificity of 95% even at 0.01 ng / mL. In contrast, although Example 2 also showed high specificity, it was slightly lower than Example 1 at low concentrations. Comparative Examples 1 and 2 showed poor specificity in low-concentration samples and failed to achieve the detection results of Examples 1 and 2.

[0034] 3. Real-time detection capability test The methylation marker LINC00511 in blood samples was monitored in real time using a real-time PCR instrument and a SERS system. First, cfDNA was extracted from peripheral blood samples of 10 medulloblastoma patients to ensure sample quality. A methylation probe based on quantum dot labeling and SERS technology was designed and detected at concentrations of 0.1 ng / mL and 5 ng / mL. Changes in the methylation marker were monitored at five time points: 30 min, 1 h, 2 h, 4 h, and 6 h. Reaction conditions: HotStart Taq DNA polymerase was used in the PCR system, which included dNTPs, MgCl2, and buffer. Amplification conditions were: denaturation at 95°C for 5 min, followed by 40 cycles: 95°C for 30 s, 60°C for 30 s, 72°C for 45 s, and a final extension at 72°C for 10 min.

[0035] Table 3. Sensitivity and specificity data of different samples at different concentrations and times.

[0036] Example 1 demonstrated the best sensitivity and specificity at all concentrations and time points, especially at 5 ng / mL, where both sensitivity and specificity reached 99%. In contrast, Comparative Example 2 was slightly lower in sensitivity and specificity than Example 1, while Comparative Example 1 showed lower sensitivity and specificity than both Example 1 and Example 2 at all time points, indicating that the conventional method performs poorly in low-abundance methylated samples.

[0037] 4. Sample type comparison test Blood, urine, and saliva samples (10 mL each) were collected from 10 patients diagnosed with medulloblastoma. cfDNA was extracted from each sample using magnetic microparticle separation, and quantification using Qubit was performed to ensure the cfDNA concentration and quality met experimental requirements. A methylation probe based on quantum dot labeling and SERS technology was designed, targeting the promoter region of the LINC00511 gene, a methylation marker, and amplified by PCR using HotStart Taq DNA polymerase. Amplification conditions included denaturation at 95°C for 5 minutes, 40 cycles (95°C for 30 seconds, 60°C for 30 seconds, 72°C for 45 seconds), and a final extension at 72°C for 10 minutes. Fluorescence signals were monitored using a real-time PCR instrument, or changes in methylation markers in the samples were detected using a SERS system. The sensitivity and specificity of different blood, urine, and saliva samples were recorded.

[0038] Table 4. Sensitivity and specificity data for different sample types.

[0039] According to the data in the table, Example 1 exhibited the highest sensitivity and specificity across all sample types, particularly in blood samples, where the sensitivity and specificity reached 95% and 96%, respectively. In contrast, Example 2 and Comparative Example 2 performed poorly across different sample types, especially in saliva samples, where the sensitivity and specificity were lower than in Example 1, demonstrating that the quantum dot-labeled probe + SERS probe method has greater reliability in the application of multiple sample types.

[0040] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A reagent for detecting methylation in medulloblastoma, characterized in that, The reagent comprises the following components: Methylation probes, adaptive biometric molecules, and liquid biopsy systems for use in one or more of blood, urine, saliva, pleural and peritoneal fluid, and lymph.

2. The methylation detection reagent for medulloblastoma according to claim 1, characterized in that, The adaptive biometric molecule is one or more of antibodies, monoclonal antibodies, single-chain antibodies, peptides, and nanobodies.

3. The methylation detection reagent for medulloblastoma according to claim 1, characterized in that, The methylation probe is a labeling probe based on fluorescence or surface-enhanced Raman scattering technology.

4. The methylation detection reagent for medulloblastoma according to claim 1, characterized in that, The biopsy system is used to separate exosomes, circulating cell-free DNA, or circulating tumor DNA from samples.

5. The methylation detection reagent for medulloblastoma according to claim 4, characterized in that, The separation of exosomes, circulating cell-free DNA, or circulating tumor DNA is performed by ultracentrifugation or magnetic particle separation technology.

6. The methylation detection reagent for medulloblastoma according to claim 1, characterized in that, The methylation probes are designed based on CpG island regions of genomic DNA and target medulloblastoma-specific methylation sites, including one or more of LINC00511, HOTAIR, MGMT, TP53, PTEN, and RB1.

7. The methylation detection reagent for medulloblastoma according to claim 1, characterized in that, The detection reagent is quantified using the standard curve method or the internal reference method, and the detection limit range is 0.1 ng / mL to 1 ng / mL.

8. A detection method for a methylation detection reagent in medulloblastoma, the detection method as described in any one of claims 1-7, characterized in that, The steps are as follows: S1. Using the methylation detection reagent according to claim 1, non-invasive detection is performed on biological samples; S2. Screen for methylation markers associated with medulloblastoma and combine the methylation probes with adaptive biorecognition molecules to form highly specific detection probes; S3. By monitoring changes in methylation markers in real time, analyze the methylation characteristics of medulloblastoma and provide data support for the evaluation of treatment response; S4. Extract circulating cell-free DNA or circulating tumor DNA from blood or other body fluid samples using liquid biopsy techniques.

9. The methylation detection reagent for medulloblastoma according to claim 1, characterized in that, The methylation marker has a sensitivity of 90%-98% or higher and a specificity of 95%-98%.

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