Bladder cancer diagnosis system and method of 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 result error, low sensitivity, high cost of patients, etc., and achieve the effects of improving sensitivity, strong primer specificity, and easy operation.

Inactive Publication Date: 2019-10-25
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

Benefits of technology

This patented technology allows for easy identification of bladdestones from other diseases that may also cause symptoms such as urothelial carcinoma. By accurately measuring changes caused during menstrual bleeding on an individual's own body over several hours it will help diagnose these conditions more efficiently than traditional methods like histopathology. Additionally, this new technique does away with any harmful procedures involved while still being effective at identifying individuals who are likely to develop them.

Problems solved by technology

This patents describes different methods or techniques for diagnosing bladdestones like bladish urologies caused by benign neoplasms called CISs during medical procedures. However current methods involve complicated steps involving multiple treatments followed by histopathologic assessment, making them time-consuming and costly. There remains room for improvement over existing approaches due to their poor specificity and potential harmful side effects on patients' health.

Method used

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  • Bladder cancer diagnosis system and method of detecting methylation level of target gene in urine
  • Bladder cancer diagnosis system and method of detecting methylation level of target gene in urine
  • Bladder cancer diagnosis system and method of detecting methylation level of target gene in urine

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Experimental program
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Effect test

Embodiment 1

[0059] Sample Handling (Urine Samples)

[0060] 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;

[0061] 2) Urine DNA was extracted using a Qiagen kit;

[0062] 3) Urine DNA was subjected to bisulfite conversion using the EZ Gold Methylation Kit.

[0063] 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

[0065] HRM-PCR detection of gene methylation level

[0066] High melting curve polymerase chain reaction (HRM-PCR) experiments were performed as follows:

[0067] 1) Prepare the reagents required for HRM-PCR (1 reaction):

[0068] a.5ul of 2X Zymo premix;

[0069] b. 0.5ul of primers, the primer sequence is shown in SEQ ID NO: 1-14;

[0070] c.0.5ul of LC Green Plus Saturated fluorescent dyes;

[0071] d.3ul of water;

[0072] The sample of e.1ul, this sample has been processed according to the method for embodiment 1;

[0073] 2) Perform HRM-PCR assay to obtain gene methylation level, the reaction steps and conditions are as follows:

[0074] a. Preactivation at 95.0°C for 10 minutes;

[0075] 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;

[0076] 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 ...

Embodiment 3

[0078] Construction of bladder cancer risk score model

[0079] 1. Sample collection

[0080] Eighty-one human urine samples were collected, including 44 bladder cancer patients and 37 normal controls.

[0081] 2. Model Construction

[0082] 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.

[0083] All statistical ana...

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Abstract

The invention discloses a method of noninvasively detecting bladder cancer. The method can provide early screening of bladder cancer, estimate the risk of a subject to experience bladder cancer, and provide typing of bladder cancer and/or prognostic prediction, so that diagnostic sensitivity, specificity and accuracy are improved; noninvasive, convenient, efficient and rapid diagnosis of bladder cancer is achieved. The invention also provides a bladder cancer diagnosis system, as well as a method of detecting methylation level of a target gene in urine.

Description

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Claims

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Application Information

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Owner 宽盈医疗科技(上海)有限公司
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