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Bladder cancer diagnosis system, reagent combination and method for detecting methylation level of target gene in urine

A diagnostic system and methylation technology, applied in the field of biomedical detection, can solve the problems of low sensitivity, high cost of patients, and error in results, and achieve the effects of improving sensitivity, easy operation, and high reliability

Inactive Publication Date: 2019-10-29
宽盈医疗科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional bladder cancer examinations are cystoscopy and cytological biopsy, but cystoscopy is an invasive examination, which is expensive and will bring some pain to the patient, and the results may be subject to subjective judgments by the operator. ; while cytology biopsy has the defect of low sensitivity (about 34%)

Method used

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  • Bladder cancer diagnosis system, reagent combination and method for detecting methylation level of target gene in urine
  • Bladder cancer diagnosis system, reagent combination and method for detecting methylation level of target gene in urine
  • Bladder cancer diagnosis system, reagent combination and method for detecting methylation level of target gene in urine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Sample Handling (Urine Samples)

[0057] 1) Collect 30ml of urine in a urine collection tube pre-stored with 600ul EDTA solution, mix well and centrifuge at 3000g for 10 minutes;

[0058] 2) Urine DNA was extracted using a Qiagen kit;

[0059] 3) Urine DNA was subjected to bisulfite conversion using the EZ Gold Methylation Kit.

[0060] Urine storage conditions: if the storage time is less than 48 hours, store at 4°C; if the storage time exceeds 48 hours, store at -80°C.

Embodiment 2

[0062] HRM-PCR detection of gene methylation level

[0063] High melting curve polymerase chain reaction (HRM-PCR) experiments were performed as follows:

[0064] 1) Prepare the reagents required for HRM-PCR (1 reaction):

[0065] a.5ul of 2X Zymo premix;

[0066] b. 0.5ul of primers, the primer sequence is shown in SEQ ID NO: 1-8;

[0067] c.0.5ul of LC Green Plus Saturated fluorescent dyes;

[0068] d.3ul of water;

[0069] The sample of e.1ul, this sample has been processed according to the method for embodiment 1;

[0070] 2) Perform HRM-PCR assay to obtain gene methylation level, the reaction steps and conditions are as follows:

[0071] a. Preactivate at 95.0°C for 10 minutes;

[0072] b. Denaturation at 95.0°C for 15 seconds, annealing at 60.0°C for 30 seconds, extension at 72.0°C for 15 seconds, and 50 cycles;

[0073] c. Obtain the HRM curve according to the following reaction conditions: raise the temperature from 72.0°C to 95.0°C at a rate of 1.6°C / sec and...

Embodiment 3

[0075] Construction of bladder cancer risk score model

[0076] 1. Sample collection

[0077] Eighty-one human urine samples were collected, including 44 bladder cancer patients and 37 normal controls.

[0078] 2. Model Construction

[0079] Univariate and multivariate logistic regression analyzes were performed on the methylation levels of 4 selected genes (HOXA9, PCDH17, POU4F2, and ONECUT2), and a detection model including 4 genes was constructed. Utilize the method of embodiment 2, respectively obtain the methylation level of 4 genes in each sample, the operating procedure of detection experiment is as follows figure 1 shown. First, a univariate logistic regression analysis was performed. The 4 selected genes were all significantly associated with bladder cancer. Multivariate logistic regression analysis was then performed to obtain the regression coefficients of the four genes.

[0080] All statistical analyzes were performed using SPSS 19.0 (Statistical Product a...

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Abstract

The invention discloses a noninvasive detection method of bladder cancer. The noninvasive detection method of bladder cancer can realize early screening of bladder cancer, risk assessment about the fact that a subject suffers from bladder cancer, and parting and / or prognostic prediction of bladder cancer, the sensitivity, specificity and accuracy of diagnosis are improved, and the effect of noninvasive, convenient and rapid, efficient and rapid diagnosis of bladder cancer is realized. The invention further provides a bladder cancer diagnosis system, a reagent combination for detecting the methylation level of a target gene in urine, and a method for detecting the methylation level of the target gene in the urine.

Description

technical field [0001] The invention belongs to the field of biomedical detection. In particular, the present invention relates to methods for the non-invasive detection of bladder cancer. More specifically, the present invention relates to a method for non-invasive detection of bladder cancer using DNA methylation biomarkers of bladder cancer. Background technique [0002] Bladder cancer is a common malignant tumor with high lethality. According to statistics, there are approximately 429,800 new cases and 165,100 deaths worldwide each year. The traditional bladder cancer examinations are cystoscopy and cytological biopsy, but cystoscopy is an invasive examination, which is expensive and will bring some pain to the patient, and the results may be subject to subjective judgments by the operator. ; And cytology biopsy has the defect of low sensitivity (about 34%). Therefore, non-invasive detection has become one of the important development directions of bladder cancer det...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11G16B20/20G16B40/10G16H50/20
CPCC12Q1/6886C12Q1/6858G16B20/20G16B40/10G16H50/20C12Q2600/154C12Q2600/118C12Q2600/112C12Q2523/125C12Q2531/113C12Q2527/107C12Q2537/165
Inventor 韩君
Owner 宽盈医疗科技(上海)有限公司
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