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Liver cancer early warning method

A liver cancer and genetic technology, applied in the field of early warning of liver cancer, can solve the problems of high missed diagnosis rate and misdiagnosis rate, poor effect, low sensitivity and specificity

Active Publication Date: 2019-11-22
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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0273] Example 1 Plasma cfDNA Extraction

[0274] 1.1 Separation and storage of plasma samples

[0275] 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.

[0276] 1.2 cfDNA extraction

[0277] 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.

[0278] 1.3 cfDNA quality detection

[0279] 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.

[0280] 1.4...

Embodiment 2

[0293] The design of embodiment 2 primers and probes

[0294] 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 ...

Embodiment 3

[0299] Embodiment 3 detection system research

[0300] 3.1 Determine the PCR reaction conditions

[0301] 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.

[0302] table 3

[0303]

[0304]

[0305] Table 4

[0306]

[0307]

[0308] table 5

[0309]

[0310] 3.2 Sensitivity Verification

[0311] Generally, the content of cfDNA in the human body is 10-15ng / mL plasma, and the half-life is 0.5-2h, so it is necessary to establish a highly sensitive detection method.

[0312] First, gene fragments were synthesized according to the positive quality control sequences of P16, SFRP1, RASSF1A, GSTP1, and APC genes in Table 1 and ...

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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 Applications(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
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