DNA methylation biomarker composition, detection method and kit

A methylation marker and kit technology, which can be used in biochemical equipment and methods, recombinant DNA technology, and microbial determination/examination, etc., can solve the problem of low sensitivity, invasiveness, and limited diagnostic ability of small lesions in urine exfoliative cytology. And other issues

Active Publication Date: 2020-03-10
ANCHORDX MEDICAL CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, cystoscopy combined with biopsy pathology is the gold standard for cystoscopy diagnosis, but this inspection method is invasive, prone to complications, and patient compliance is low
However, imaging examinations have limited diagnostic ability for small lesions, urine exfoliation cytology ex...

Method used

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  • DNA methylation biomarker composition, detection method and kit
  • DNA methylation biomarker composition, detection method and kit
  • DNA methylation biomarker composition, detection method and kit

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Experimental program
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Effect test

Embodiment 1

[0172] Co-methylation of methylated regions for bladder cancer detection, diagnosis, classification or prediction, treatment monitoring, prognosis or other evaluation of bladder cancer, including multiple methyl groups indicated by [CG] in the nucleic acid sequences listed in Table 1 Comethylation of methylation sites and comethylation of multiple methylation sites of nucleic acids that are completely complementary in sequence to the nucleic acids indicated by [CG] in Table 1.

[0173] Table 1 Co-methylation composition of DNA methylated regions

[0174]

[0175]

[0176]

Embodiment 2

[0178]A test kit for co-methylation of multiple methylated regions of bladder cancer for detection, diagnosis, classification or prediction, treatment monitoring, prognosis or other assessment of bladder cancer, comprising co-methylation of multiple methylated regions Specific primer pairs and probes, as shown in Table 2:

[0179] Table 2-1 Primer and probe sequence combinations for detection of co-methylation in 22 methylated regions 1

[0180]

[0181]

[0182]

[0183] Table 2-2 Primer and probe sequence combinations for detection of co-methylation in 22 methylated regions 2

[0184]

[0185]

[0186] Table 2-3 Primer and probe sequence combinations for detection of co-methylation in 22 methylated regions 3

[0187]

[0188]

[0189]

[0190] In practical applications, the corresponding primers and probes will be selected according to the combination of specific methylated regions.

[0191] Internal reference primers and probes:

[0192]

Embodiment 3

[0196] Co-methylation detection of 2-3 methylated regions by multiplex fluorescent quantitative PCR

[0197] Use commercial complete methylation (positive control) and non-methylation (negative control) standard (purchased from QIAGEN company) to carry out every 2-3 Co-methylation detection of methylated regions.

[0198] The specific process is as follows:

[0199] 1. DNA extraction

[0200] The extraction kit was purchased from QIAGEN, and was carried out according to the instructions of the kit.

[0201] 2. DNA bisulfite conversion

[0202] The DNA bisulfite conversion kit was purchased from Zymo, and was carried out according to the instructions of the kit.

[0203] 3. Multiplex PCR amplification

[0204] Using primer pairs for 22 methylated regions (SEQ ID NO.1-8), multiplex PCR was carried out in one reaction well (see Table 2 for the primer sequence), and the target sequence containing the target region was amplified, and the product size was in About 70-130bp.

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Abstract

The invention relates to a DNA methylation biomarker composition for bladder cancer detection. The biomarker composition comprises any two or more of sequences shown in the formulas of SEQ ID NO.1 toSEQ ID NO.22, or any two or more of completely complementary sequences shown in the formulas of SEQ ID NO.1 to SEQ ID NO.22. The invention also relates to a detection kit for the methylation biomarkercomposition. The bladder cancer occurrence is discriminated and analyzed by using the composition in the co-methylation state of a plurality of specific methylation regions, the specific methylationcomposition has high sensitivity for discriminating the bladder cancer occurrence, and the detection method is simple, convenient and feasible. The primer pair composition of the kit overcomes the defect of false positive results caused by single methylation site detection mismatch in primer sequence design, and considers the interaction between multiple methylation biomarker primer and probe paircomposition.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a DNA methylation biomarker combination, a detection kit and a detection method. Background technique [0002] Bladder cancer is one of the most common malignant tumors of the urinary system. In 2014, the incidence of bladder cancer in China was 78,000 people per year, and the incidence rate is increasing year by year. Bladder cancer has the characteristics of high incidence and easy recurrence. Hematuria is a common clinical symptom of bladder cancer, and bladder cancer is detected in about 17% of patients with hematuria. Currently, the main diagnostic methods for bladder cancer include shadow cystoscopy, urine exfoliation cytology, urine FISH and tumor markers. Among them, cystoscopy combined with biopsy histopathology is the gold standard for cystoscopic diagnosis, but this examination method is invasive, prone to complications, and patient compliance is low. Howeve...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/112C12Q2600/118C12Q2600/154C12Q2600/16C12Q2600/166C12Q2523/125C12Q2537/143C12Q2563/107C12Q2531/113C12Q2545/101
Inventor 阮微媚蒋泽宇李霞陈志伟范建兵
Owner ANCHORDX MEDICAL CO LTD
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