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Method and kit for identifying state of prostate cancer

A state-of-the-art technology for prostate cancer, applied in the direction of biochemical equipment and methods, microbial determination/testing, DNA/RNA fragments, etc.

Pending Publication Date: 2021-08-10
BEIJING EXELLON MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of means to effectively detect the methylation status of these cancer-related genes and process the detection results

Method used

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  • Method and kit for identifying state of prostate cancer
  • Method and kit for identifying state of prostate cancer
  • Method and kit for identifying state of prostate cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: DNA Extraction

[0076] DNA Extraction Reagent consists of Lysis Buffer, Binding Buffer, Washing Buffer and Elution Buffer. Lysis buffer consists of protein denaturants, detergents, pH buffers and nuclease inhibitors. Binding buffers consist of protein denaturants and pH buffers. The cleaning buffer is divided into cleaning buffer A and cleaning buffer B, and cleaning buffer A is composed of protein denaturant, nuclease inhibitor, detergent, pH buffer and ethanol; cleaning buffer B is composed of nuclease inhibitor, pH buffer and ethanol composition. The elution buffer consists of nuclease inhibitors and pH buffers. The protein denaturant is: guanidine hydrochloride; the detergent is: Tween20; the pH buffer is: Tris-HCl; the nuclease inhibitor is: EDTA.

[0077] In this embodiment, the plasma sample of a prostate cancer patient is taken as an example to extract plasma DNA. The extraction method comprises the following steps:

[0078] (1) Take 1 mL of pl...

Embodiment 2

[0090] Example 2: Bisulfite Treatment of DNA

[0091] Bisulfite treatment of DNA is treated with bisulfite reagent, which consists of bisulfite buffer and protection buffer; bisulfite buffer is a mixed liquid of sodium bisulfite and water ; The protection buffer is a mixed liquid of oxygen free radical scavenger hydroquinone and water.

[0092] In this embodiment, the DNA extracted in Example 1 is used as the processing object, and the DNA is treated with bisulfite, and the specific steps include:

[0093] (1) Preparation of bisulfite buffer: weigh 1g of sodium bisulfite powder, add water to prepare 3M buffer;

[0094] (2) Preparation of protection buffer: weigh 1g of hydroquinone reagent, add water to prepare 0.5M protection buffer;

[0095] (3) Mix 100 μL DNA solution, 200 μL bisulfite buffer solution and 50 μL protection solution, and shake to mix evenly;

[0096] (4) Heat treatment: 95°C for 5min, 80°C for 60min, 4°C for 10min;

[0097] (5) Add 1 mL of DNA binding buff...

Embodiment 3

[0107] Example 3: Detection of DNA methylation by real-time fluorescent PCR and verification of primer sets

[0108] In this embodiment, real-time fluorescent PCR is used as an example to measure the methylation level of biomarker genes. The detected genes were APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB and RASSF1A genes, and the internal reference gene was ACTB. In this example, the DNA treated with bisulfite in Example 2 was used as a template for real-time fluorescent PCR amplification. The DNA samples to be tested, the negative quality control, the positive quality control, and the no-template control were all tested in triplicate holes. The negative quality control and the positive quality control were prepared in the following manner: take 400 μL of human leukocyte DNA with a concentration of 10 ng / μL and add it to TE buffer solution containing 1% BSA, mix well, and dilute the solution to 200 mL to obtain the concentration Negative quality control substanc...

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Abstract

Provided herein is a method of identifying the state of prostate cancer in a subject, and the method comprises the steps: 1) detecting the methylation level of a biomarker gene in a biological sample from the subject wherein the biomarker gene is selected from one or more of the following genes: APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB, and RASSF1A; 2) comparing the methylation level detected in the step 1) with the normal methylation level of the corresponding biomarker gene in the population to determine the state of prostate cancer in the subject. Also provided herein are kits for identifying the state of prostate cancer in a subject. The method and the kit provided by the invention provide a rapid, reliable and accurate new way for prediction, diagnosis and evaluation of prostate cancer.

Description

technical field [0001] The present invention relates to methods and kits for identifying prostate cancer status in a subject. Background technique [0002] Prostate cancer is the most common malignant tumor of the male reproductive system, and it is the second leading cause of cancer death in men in the United States. In recent years, the incidence of prostate cancer in my country has increased significantly. Prostate cancer has leapt to the sixth most common tumor in Chinese men, and its growth rate is the fastest among all male tumors. The cure rate of prostate cancer detected in the early stage is as high as 95%, while the 5-year survival rate in the late stage is less than 30%. The prevention and treatment situation of prostate cancer in China is very severe today. [0003] At present, the diagnosis of prostate cancer mainly includes PSA blood test, digital rectal examination and ultrasound. However, in recent years, studies have reported that the accuracy of prostate ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/154
Inventor 徐春叶李明明蒲珏
Owner BEIJING EXELLON MEDICAL TECH CO LTD
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