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Detection method and kit for diagnosing bladder urothelium carcinoma

A technology of urothelial carcinoma and a kit, which is applied in the fields of biotechnology and medical diagnosis, can solve the problems of low sensitivity and specificity, unobvious early symptoms of bladder urothelial carcinoma, and difficulty in detection, and achieve excellent sensitivity and specificity sexual effect

Active Publication Date: 2022-03-01
安康优乐众成医疗器械有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The early symptoms of bladder urothelial carcinoma are not obvious, which makes it difficult to detect. Excellent detection methods such as bladder endoscopy are invasive and easily lead to complications. The existing non-invasive detection methods have problems of low sensitivity and specificity. , the present invention provides a detection method and kit for diagnosis of bladder urothelial carcinoma

Method used

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  • Detection method and kit for diagnosing bladder urothelium carcinoma
  • Detection method and kit for diagnosing bladder urothelium carcinoma
  • Detection method and kit for diagnosing bladder urothelium carcinoma

Examples

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Embodiment 1

[0046] The present invention is based on performing single-cell transcriptome, single-cell chromatin accessibility sequencing and Whole-genome DNA methylation sequencing, analysis found that cells of different origin types have different DNA methylation characteristics, causing differences in the degree of chromatin opening, which in turn affects differences in gene expression, and ultimately leads to different fates of cells. Some are normal cells, while some develop into muscle-invasive cancer cells, or non-muscle-invasive cancer cells. Through statistical analysis of these DNA methylation features, regions (DMRs) with significant differences in methylation levels in the three source cells were screened out, as shown in Table 1. The present invention is based on the method of methylation multiplex PCR, amplifies these DMR regions, and detects the methylation level on these DNA regions by high-throughput sequencing, and the specific primer combinations are shown in Table 2: ...

Embodiment 2

[0058] Seventeen methylated regions were detected using commercial fully methylated and unmethylated pattern standards (purchased from Zymo).

[0059] The specific process is as follows:

[0060] 1. DNA bisulfite conversion (BS treatment)

[0061] The DNA bisulfite conversion kit was purchased from Zymo, and was carried out according to the instructions of the kit, and the conversion product was obtained with NF-H 2 O was eluted for later use.

[0062] 2. Model Standard Gradient Incorporation

[0063] Single-strand quantification was performed after BS-treated fully methylated and non-methylated standard samples were disrupted by ultrasound. Gradient incorporation was carried out according to the DNA concentration, and 13 model standard gradients were set up in this application (methylation levels were 100%, 99%, 98%, 95%, 90%, 80%, 50%, 20%, 10% %, 5%, 2%, 1%, 0%).

[0064] 3. Preparation of Methylated Multiplex PCR Primer Sets

[0065] 19 pairs of primers were mixed fo...

Embodiment 3

[0105] Use bladder urothelial cancer cell lines RT4 and 5637 to detect biological standards to clarify the minimum detection limit of this application for bladder urothelial cancer and its typing

[0106] The specific process is as follows:

[0107] 1. gDNA extraction

[0108] Bladder urothelial carcinoma cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences. The extraction kit was purchased from QIAGEN (69506), and was carried out according to the instructions of the kit.

[0109] 2. DNA bisulfite conversion (reference example 2)

[0110] 3. Biological Standard Gradient Incorporation

[0111] The RT4 / 5637 gDNA treated with BS was ultrasonically fragmented to simulate cfDNA, and normal urine cfDNA did not need ultrasonic fragmentation to perform single-strand quantification. Gradient incorporation is carried out according to the DNA concentration. In this application, a total of 8 model standard gradients are set up for each group (the gDNA conten...

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Abstract

The invention relates to the field of biotechnology and medical diagnosis, in particular to a detection method and a kit for diagnosing bladder urothelium carcinoma. The invention provides application of a primer combination for detecting a DNA methylation difference region in a to-be-detected sample in preparation of a reagent or a kit for detecting or predicting whether a subject suffers from bladder urinary tract epithelial carcinoma or not or whether a bladder urinary tract epithelial carcinoma patient typing is muscular layer wetting or non-muscular layer wetting or not. The invention also relates to a kit for detecting or predicting whether a subject suffers from the bladder urothelial carcinoma or whether a bladder urothelial carcinoma patient typing is muscular layer infiltration or non-muscular layer infiltration. The detection method and the kit for diagnosing the bladder urinary tract epithelium cancer, provided by the invention, have excellent sensitivity and specificity, have great positive significance on clinical diagnosis and treatment of the bladder urinary tract epithelium cancer and later monitoring and prognosis evaluation, and provide a theoretical basis for further early diagnosis.

Description

technical field [0001] The invention relates to the fields of biotechnology and medical diagnosis, in particular to a detection method and a kit for diagnosing bladder urothelial carcinoma. Background technique [0002] Bladder cancer (BCa) is the most common malignant tumor of the urinary system, and its incidence is on the rise worldwide. BCa is mainly divided into three pathological types: bladder urothelial carcinoma (Bladder Urothelial Carcinoma, BLCA), squamous cell carcinoma and adenocarcinoma, of which BLCA accounts for 90%, and there will be 212,500 new deaths worldwide in 2020. [0003] BLCA can be further divided into muscle-invasive BCa (muscle-invasive) and non-muscle-invasive BCa (non-muscle-invasive), with non-muscle-invasive BCa accounting for approximately 75% of all cases. The biological behaviors of the two types of BLCA are different, and the prognosis is very different, so they need to be treated differently. Since non-muscle invasion does not invade t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/154C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2535/122C12Q2545/113
Inventor 张翼吴凯单柳颖金婉
Owner 安康优乐众成医疗器械有限责任公司
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