Bladder cancer maker set and application thereof

A technology of bladder cancer and markers, applied in the field of molecular biology, can solve the problems of low sensitivity, error in results, high cost of patients, etc., and achieve the effect of improving sensitivity, easy operation and high acceptance

Inactive Publication Date: 2019-10-18
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 maker set and application thereof
  • Bladder cancer maker set and application thereof
  • Bladder cancer maker set and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Sample Handling (Urine Samples)

[0038] 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;

[0039] 2) Urine DNA was extracted using a Qiagen kit;

[0040]3) Urine DNA was subjected to bisulfite conversion using the EZ Gold Methylation Kit.

[0041] 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

[0043] HRM-PCR determination of gene methylation level

[0044] High melting curve polymerase chain reaction (HRM-PCR) experiments were performed as follows:

[0045] 1) Prepare the reagents required for HRM-PCR (1 reaction):

[0046] a.5ul of 2X Zymo premix;

[0047] b. 0.5ul of primers, the primer sequence is shown in SEQ ID NO: 1-14;

[0048] c.0.5ul of LC Green Plus Saturated fluorescent dyes;

[0049] d.3ul of water;

[0050] The sample of e.1ul, this sample has been processed according to the method for embodiment 1;

[0051] 2) Perform HRM-PCR assay to obtain gene methylation level, the reaction steps and conditions are as follows:

[0052] a. Preactivation at 95.0°C for 10 minutes;

[0053] 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;

[0054] 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 / ...

Embodiment 3

[0056] Construction of bladder cancer risk score model

[0057] 1. Sample collection

[0058] Eighty-one human urine samples were collected, including 44 bladder cancer patients and 37 normal controls.

[0059] 2. Model Construction

[0060] Univariate and multivariate logistic regression analyzes were performed on the methylation levels of 7 selected genes (HOXA9, ONECUT2, PCDH17, PENK, TWIST1, VIM, and ZNF154), and a detection model including 7 genes was constructed. Using the method of Example 2, the methylation levels of 7 genes in each sample were obtained respectively, and the operation process of the detection experiment was as follows figure 1 shown. First, a univariate logistic regression analysis was performed. Such as figure 2 As shown, the 7 selected genes were all significantly associated with bladder cancer. Multivariate logistic regression analysis was then performed to obtain the regression coefficients of the seven genes.

[0061] All statistical ana...

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Abstract

The invention discloses a bladder cancer maker set. The bladder cancer maker set comprises the following genes of HOXA9, ONECUT2, PCDH17, PENK, TWIST1, VIM and ZNF154. The invention further provides an application of the bladder cancer maker set to preparation of a reagent or a reagent kit for diagnosis of bladder cancer, and further provides a reagent composition for detecting the methylated level of the bladder cancer maker set. The bladder cancer maker set can be used for early screening of the bladder cancer, and subtype and / or prognosis forecast of the bladder cancer, so that the diagnosis and detection sensitivity, specificity and accuracy are improved. Through accurate risk assessment and forecast, the bladder cancer maker set disclosed by the invention can reduce unnecessary invasive inspection of low-risk (low-danger) patients.

Description

technical field [0001] The present invention belongs to the field of molecular biology. In particular, the present invention relates to bladder cancer marker panels and applications thereof. More specifically, the present invention relates to DNA methylation biomarkers for bladder cancer and their applications. Background technique [0002] With the deepening of people's understanding of tumors and the advancement of science and technology, many new tumor markers have been discovered and used for clinical diagnosis. Before 1980, tumor markers were mainly cell secretions such as hormones, enzymes, and proteins. For example, carcinoembryonic antigen and alpha-fetoantigen could be used as markers for various tumors such as liver cancer and gastric cancer. Carbohydrate antigen 125 could be used as markers for ovarian cancer. The marker of prostate specific antigen can be used as a marker of prostate cancer. However, the sensitivity and specificity of this class of tumor marke...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/112C12Q2600/154C12Q2600/118
Inventor 韩君
Owner 宽盈医疗科技(上海)有限公司
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