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DNA methylation biomarker combination and application thereof

A methylation marker, methylation technology, applied in recombinant DNA technology, DNA/RNA fragment, determination/inspection of microorganisms, etc., to achieve the effect of speeding up diagnosis and treatment

Pending Publication Date: 2021-12-07
ANCHORDX MEDICAL CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To date, there are no commercially available non-invasive urine tests for the preoperative stratification and identification of patients with HR-NMIBC or MIBC

Method used

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  • DNA methylation biomarker combination and application thereof
  • DNA methylation biomarker combination and application thereof
  • DNA methylation biomarker combination and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] The sequence compositions including the 5 markers used for bladder cancer risk stratification correspond to the following table: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 . In addition to the composition of the above-mentioned 5 markers, Table 1 also includes nucleic acid sequences of 22 other listed biomarkers. Each biomarker is indicated by the degree of co-methylation at multiple methylation sites indicated by the sequence [CG].

[0103] Table 1 Co-methylation composition of DNA methylated regions

[0104]

[0105]

[0106] For the above methylated sequences, specific primer pairs and probes for co-methylation of multiple methylated regions are shown in Table 2.

[0107] Table 2-1 Primer and probe sequence combination 1 for co-methylation detection of 22 methylated regions Table 2-1 Primer and probe sequence combination 1 for co-methylation detection of 22 methylated regions

[0108]

[0109]

[0110]

[0111] Table 2-2 Primer ...

Embodiment 2

[0123] Example 2 Multiplex fluorescent quantitative PCR for co-methylation detection of 5 methylated regions in 22 methylated regions

[0124] The 22 methylated regions comprising the 5 methylated biomarkers (SEQ ID NO.1-22) for co-methylation detection of every 2-3 methylated regions.

[0125] The specific process is as follows:

[0126] 1. DNA extraction

[0127] The extraction kit was purchased from QIAGEN, and was carried out according to the instructions of the kit.

[0128] 2. DNA bisulfite conversion

[0129] The DNA bisulfite conversion kit was purchased from Zymo, and was carried out according to the instructions of the kit.

[0130] 3. Multiplex PCR amplification

[0131] Using primer pairs for 22 methylated regions (SEQ ID NO.1-22), multiplex PCR was carried out in one reaction well (see Table 2 for the primer sequence, and the sequence in Table 2-2 was used in this embodiment) to amplify The target sequence containing the target region is produced, and the prod...

Embodiment 3

[0154] Parallel detection of co-methylation of any 1-3 methylated regions among 22 methylated regions

[0155] When the co-methylation of the target methylated region is detected in parallel as any 1-3 of the 22 methylated regions, using the combination scheme in Table 4 in Example 2, the following detection methods can be used. The specific detection process is as follows:

[0156] 1. DNA extraction

[0157] The extraction kit was purchased from QIAGEN, and was carried out according to the instructions of the kit.

[0158] 2. DNA bisulfite conversion

[0159] The DNA bisulfite conversion kit was purchased from Zymo, and was carried out according to the instructions of the kit.

[0160] 3. Fluorescent quantitative PCR assay

[0161] Select the primers and probes of 1-3 methylated regions and the primers and probes of the internal reference, and measure them in one reaction well (see Table 2 for the sequences of primers and probes, and see Example 2 for the combination sche...

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Abstract

The invention relates to a DNA methylation marker combination for bladder cancer risk stratification. The DNA methylation marker combination comprises a combination of SEQ ID NO.3 or a complete complementary sequence thereof, SEQ ID NO.5 or a complete complementary sequence thereof, and SEQ ID NO.7 or a complete complementary sequence thereof. Clinical application of a preoperative three-level risk stratification model is provided according to the screened proper molecular marker combination, so that reasonable utilization of a current diagnosis and treatment mode is promoted, excessive invasive cystoscopy can be avoided for BC negative patients, HR-NMIBC or MIBC can accelerate and comprise a more thorough operation plan, and a judged LMR-NMIBC patient can follow a standard diagnosis mode.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a combination of DNA methylation biomarkers and an application thereof. Background technique [0002] Bladder cancer (BC) is the tenth most common cancer worldwide and the ninth leading cause of cancer death in men. According to histological grade, TNM classification, tumor size and lesion, low-intermediate risk non-muscle-invasive bladder cancer (LMR-NMIBC), high-risk non-muscle-invasive bladder cancer (HR-NMIBC) and muscle-invasive bladder cancer (MIBC) The three risk stratifications of bladder cancer predict different prognosis and risk of recurrence of patients, and thus determine different monitoring methods and treatment methods. Although 70-80% of patients diagnosed with non-muscle-invasive bladder cancer (NMIBC) and up to 50% of LMR-NMIBC showed a good prognosis, the 5-year The recurrence rate is as high as 80%, and up to 50% develop MIBC, and once MIBC develops...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/154C12Q2600/118C12Q2600/112
Inventor 阮微媚林天歆黄健陈旭蒋泽宇李霞陈志伟范建兵
Owner ANCHORDX MEDICAL CO LTD
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