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Reagent combination and kit for detecting liver cancer and application of reagent combination and kit

A technology for detecting reagents and kits, which is applied in the field of detection of methylation levels, can solve the problems of low sensitivity and low specificity, and achieve the effects of high sensitivity, good specificity, and less harm to patients

Active Publication Date: 2021-01-29
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This project has been certified by the FDA as a breakthrough medical device, but its clinical research is mainly based on European and American populations and has the defect of low sensitivity; the patent CN107164508A detects liver cancer by detecting the content of 5-hydroxymethylcytosine in 9 genes, which has a relatively good performance The detection performance is high, with a sensitivity of 90% and a specificity of 91.3%, but the included samples do not include liver cirrhosis, hepatitis and other high-risk groups of liver cancer, so there may be a deficiency of low specificity

Method used

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  • Reagent combination and kit for detecting liver cancer and application of reagent combination and kit
  • Reagent combination and kit for detecting liver cancer and application of reagent combination and kit
  • Reagent combination and kit for detecting liver cancer and application of reagent combination and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Embodiment 1, the screening of methylation gene

[0063] The present invention collected 785 cases of cancer tissues, 461 cases of paracancerous tissues or normal controls from the TCGA data set (https: / / tcga.xenahubs.net) of the UCSC Xena website and the GEO database of the National Center for Biotechnology Information (NCBI). Tissue and 656 healthy whole blood methylation data. Differential analysis was performed with liver cancer and control data, and the physical location and genetic information annotation of the differential sites were performed. In order to ensure that the screened fragments have a consistent methylation level, the screening of methylated gene fragments must meet the following requirements at the same time: 1) It is required that the selected gene fragments have no less than 2 adjacent sites with consistent methylation levels; 2) Perform differential analysis on liver cancer and paracancerous tissues or normal control tissues, and select liver can...

Embodiment 2

[0064] Example 2. Detection of gene methylation level in clinical samples

[0065] Collect 10ml of peripheral blood from each sample for detection and analysis of the methylation level of DNA methylation markers in the sample. The experimental procedure is as follows:

[0066] 1. Sample preparation

[0067] The sample preparation of the present invention is by MagMAX TM Cell-Free DNA Isolation Kit extracts 4ml of plasma, and 45μL of eluent is eluted. The extracted free nucleic acid needs to meet the following quality control conditions: the total amount of extracted nucleic acid is greater than 20ng.

[0068] 2. Library preparation

[0069] In the present invention, all free nucleic acids qualified for quality control are treated with bisulfite using EZ DNA Methylation-Lightning TM Kit (Zymo Research, Irvine, CA, USA). Subsequently, the sample DNA treated with bisulfite was used to construct a pre-library using the single-strand library construction method. After the pre-...

Embodiment 3

[0077] Example 3, the reagent combination of the present invention detects the methylation level of clinical samples

[0078] Samples from 63 patients with primary liver cancer, 25 patients with liver cirrhosis, 15 patients with hepatitis and 7 healthy people were collected, and the methylation level of the cg16657538 methylation site of the ZNF397OS gene was detected and analyzed according to the method described in Example 2 , and the methylation level of the combination of the ZNF397OS gene and other genes in the sample to verify its effect on detecting liver cancer. The test results are shown in Table 1, figure 1 and figure 2 shown.

[0079] Table 1 The prediction performance of the combination of ZNF397OS gene and other genes in the Logistic liver cancer classification model

[0080]

[0081] Among them, 1 represents the GRASP gene (to detect the average methylation level of the fragment shown in SEQ ID NO: 3), 2 represents the PAK1 gene (to detect the average meth...

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Abstract

The invention provides a reagent combination for detecting liver cancer. The reagent combination comprises a detection reagent for detecting the methylation level of a cg16657538 methylation site of aZNF397OS gene. Meanwhile, the invention also provides application of the reagent combination and a kit comprising the reagent combination. By using the reagent combination provided by the invention,the liver cancer can be detected with 0.910 sensitivity, 0.950 specificity and 0.941 under-curve area in a clinical tissue sample and with 0.7619 sensitivity, 0.9574 specificity and 0.8948 under-curvearea in a free DNA sample by using fewer markers; the liver cancer can be sensitively and specifically detected by detecting one methylation site of the gene, so cost and the time are saved; therefore, the kit can be used for sensitively and specifically detecting the liver malignant change in the early stage of the liver malignant change clinically.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, specifically to the field of liver cancer detection, and more specifically to the detection of the methylation level of liver cancer gene markers. Background technique [0002] Liver cancer is one of the most common and deadly diseases in the world, and it is a malignant tumor with great harm. Up to 50% of liver cancer patients in the world are in China, and it is the fourth common malignant tumor and the second leading cause of cancer death in my country. According to the "2015 Analysis of the Epidemic Situation of Malignant Tumors in China" released in 2019, about 370,000 people were diagnosed with liver cancer in my country in 2015; about 326,000 people died of liver cancer, which is seriously threatening the lives and health of our people. The onset of liver cancer is insidious, and there are no specific symptoms in the early stage. Most liver cancer patients are already in the mid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/154
Inventor 郭鑫武陈明洪梅刘让蛟戴立忠
Owner SANSURE BIOTECH INC