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A method for early warning of liver cancer

A technology for liver cancer and target region, applied in the field of single-gene or multi-gene CpG island methylation status on liver, can solve the problems of low sensitivity and specificity, high rate of missed diagnosis and misdiagnosis, and poor effect.

Active Publication Date: 2020-04-28
江苏吉睿生物技术研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity and specificity of these inspection methods are low, the missed diagnosis rate and misdiagnosis rate are high, and the effect in the prediction and early diagnosis of liver cancer is poor.

Method used

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  • A method for early warning of liver cancer
  • A method for early warning of liver cancer
  • A method for early warning of liver cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0190] Example 1 Plasma cfDNA Extraction

[0191] 1.1 Separation and storage of plasma samples

[0192] Take 8mL of peripheral blood from the subject and centrifuge at 2500rpm at 4°C for 15min to separate the sample into three layers: plasma, white blood cells and red blood cells. Use a pipette gun to take out the uppermost layer of plasma in the anticoagulant tube and divide it into several 1.5mL centrifuge tubes After labeling, store at -80°C.

[0193] 1.2 cfDNA extraction

[0194] A commercial plasma cell-free DNA extraction kit (Guangzhou Meiji Biotechnology Co., Ltd., D3182-03A) was used, and all operations were strictly performed in accordance with the kit instructions.

[0195] 1.3 cfDNA quality detection

[0196] BioAnalyzer 2100 (Agilent) was used to detect the quality of the extracted cfDNA. When the test results showed that the main peak of cfDNA was distributed around 160bp, it indicated that the quality of the extracted cfDNA met the requirements.

[0197] 1.4...

Embodiment 2

[0209] The design of embodiment 2 primers and probes

[0210] According to the sequence of the determined target gene, a series of specific primers and detection probe sequences as shown in Table 2 were designed. The sequences of the primers and probes used in this experiment are respectively: SEQ ID No: 21 as the upstream primer of the P16 gene, SEQ ID No: 22 as the downstream primer of the P16 gene, and SEQ ID No: 23 as the probe of the P16 gene; SEQ ID No:25 is used as the upstream primer of SFRP1 gene, and SEQ ID No:26 is used as the downstream primer of SFRP1 gene, and SEQ ID No:27 is used as the probe of SFRP1 gene; SEQ ID No:36 is used as the upstream primer of RASSF1A gene, SEQ ID No:37 is used as the downstream primer of RASSF1A gene, and SEQ ID No:38 is used as the probe of RASSF1A gene; SEQ ID No:44 is used as the probe of GSTP1 gene, and SEQ ID No:42 is used as the upstream primer of GSTP1 gene, SEQ ID No: 43 is used as the downstream primer of GSTP1 gene; SEQ ID No...

Embodiment 3

[0215] Embodiment 3 detection system research

[0216] 3.1 Determine the PCR reaction conditions

[0217] The test results showed that the P16, SFRP1, RASSF1A, and β-actin quadruple gene detection system had high sensitivity and specificity under the PCR reaction conditions shown in Table 3, and the GSTP1, APC, β-actin triple gene detection system was in The PCR reaction conditions shown in Table 4 have higher sensitivity and specificity, and the PCR amplification program is shown in Table 5.

[0218] table 3

[0219]

[0220]

[0221] Table 4

[0222] Reagent Final concentration EPI HS taq 0.75-1.5U / 20μL Buffer 1.0～1.5X MgCl 2

2.0-3.5mM dNTP 200-300μM GSTP1-F 0.2±0.1μM GSTP1-R 0.2±0.1μM GSTP1-P 0.2±0.1μM APC-F 0.2±0.1μM APC-R 0.2±0.1μM APC-P 0.2±0.1μM β-actin-F 0.3±0.1μM β-actin-R 0.3±0.1μM β-actin-P 0.2±0.1μM template DNA 10~100ng dd H 2 o

Make ...

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Abstract

The invention relates to a kit for judging whether a subject suffers from liver cancer or predicting the risk of liver cancer of the subject. The kit includes a reagent capable of identifying the methylation status of characteristic genes and / or a regulatory region thereof. The characteristic genes include P16, SFRP1 and RASSF1A. The invention also provides a method for judging whether the subjectsuffers from liver cancer or predicting the risk of liver cancer of the subject.

Description

technical field [0001] This application relates to the field of biomedicine, in particular to a method and application for detecting abnormal methylation of cfDNA genes in peripheral blood, specifically by detecting CpG islands of single or multiple genes in P16, SFRP1, RASSF1A, GSTP1 and APC Method and application of methylation status in risk assessment of liver cirrhosis patients progressing to liver cancer. Background technique [0002] Hepatocellular carcinoma (HCC) is currently the most common malignant tumor of the liver. Existing studies have shown that long-term chronic hepatitis virus infection (HBV / HCV) often easily leads to the occurrence of liver cancer. Studies have found that about 12%-20% of hepatitis B patients will develop liver cirrhosis within 5 years, and 2%-3% of patients with liver cirrhosis will deteriorate into liver cancer every year, while the 5-year survival rate of liver cirrhosis is 55%. The survival rate is only 5%. Therefore, early warning o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6827
CPCC12Q1/6827C12Q1/6886C12Q2600/154C12Q2523/125
Inventor 尚小云邓小军朱成
Owner 江苏吉睿生物技术研究院有限公司