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Primers, probes and kit for screening liver cancer and application of primers, probes and kit

A kit and probe technology, which is applied in the field of primers, probes and kits for screening liver cancer, and can solve the problems of no liver cancer screening products being approved for marketing.

Active Publication Date: 2021-12-31
GUANGZHOU DRIPNANO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

DNA methylation is recognized as the most valuable nucleic acid marker for early tumor screening. At present, there are colorectal cancer, lung cancer, gastric cancer and other tumor screening diagnostic products based on DNA methylation detection that have been certified by FDA or CFDA. Liver cancer-free screening product approved for marketing

Method used

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  • Primers, probes and kit for screening liver cancer and application of primers, probes and kit
  • Primers, probes and kit for screening liver cancer and application of primers, probes and kit
  • Primers, probes and kit for screening liver cancer and application of primers, probes and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The screening of embodiment 1 methylation gene and the design of primer probe

[0050] The present invention constructs large methylation data sets related to liver cancer by mining data in the TCGA database and high-throughput methylation sequencing of clinical samples, uses bioinformatics methods to construct analysis models of different methylation sites, and screens out Valuable plasma cell-free DNA methylation biomarkers, further screened out methylation markers with high specificity and high sensitivity through verification of clinical samples.

[0051] Specifically, the present invention screens candidate targets suitable for plasma free DNA methylation through the following steps: Based on the TCGA database and high-throughput methylation sequencing data of clinical samples, differential analysis is performed on the methylation data of liver cancer patients and healthy people, and screening Liver cancer patients showed high methylation and healthy people showed ...

Embodiment 2

[0074] Example 2 Kit for Screening and Diagnosing Liver Cancer

[0075] This example is a kit for screening and diagnosing liver cancer containing the primer pair and probe described in Example 1, specifically including nucleic acid extraction reagents, methylation conversion reagents, PCR reaction solutions, positive quality controls, and negative quality control substances. Control product; wherein, the PCR reaction solution includes primers and probes shown in SEQ ID NO: 1~SEQID NO: 3, primers and probes shown in SEQ ID NO: 4~SEQID NO: 6, and primers and probes shown in SEQ ID NO: 4~SEQID NO: 6, Primers and probes shown in NO: 7 to SEQ ID NO: 9, primers and probes shown in SEQ ID NO: 10 to SEQ ID NO: 12, PCR buffer, dNTP, MgCl 2 The corresponding relationship with TaqDNA polymerase, primers and probes is shown in Table 1; the positive quality control product uses human methylated genomic DNA, and the negative quality control product uses human unmethylated genomic DNA.

Embodiment 3

[0076] Example 3 Application of the kit of the present invention in the screening and diagnosis of liver cancer

[0077] This embodiment includes the following detection steps:

[0078] 1. Materials, reagents, instruments

[0079] The nucleic acid extraction reagents and methylation conversion kits used in this example are all self-developed reagents of our company; PCR buffer, dNTP, and TaqDNA polymerase were purchased from Takara Company; MgCl 2 Purchased from Sigma; primers and probes were synthesized by Shanghai Sangon Co., Ltd.; fluorescent quantitative PCR instrument was ABI7500.

[0080] 2. Sample preparation

[0081] The positive reference product uses human methylated genomic DNA, the negative reference product uses human unmethylated genomic DNA, and the sensitivity reference product is 10ng / mL human unmethylated genomic DNA contains 0.1% human methylated genomic DNA; to be processed The samples were a plasma sample from a patient with confirmed liver cancer and a...

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Abstract

The invention relates to primers, probes and kit for screening liver cancer and application of the primers, probes and kit. The primers and probes comprise a first amplification primer pair and a corresponding first detection probe which are used for detecting CELF6 gene methylation, a second amplification primer pair and a corresponding second detection probe which are used for detecting SLMO1 gene methylation, and a third amplification primer pair and a corresponding third detection probe which are used for detecting SPINT2 gene methylation. According to the invention, methylation data related to the liver cancer in TCGA and GEO databases are collected and analyzed, methylation genes highly related to the liver cancer are screened out through a large amount of researches, a gene methylation detection system is constructed, and primers and probes with excellent detection effects are obtained through further design and optimization, so that high-sensitivity and high-specificity screening diagnosis on the liver cancer is realized.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a primer, a probe, a kit and an application thereof for screening liver cancer. Background technique [0002] Liver cancer is one of the most common malignant tumors, which seriously threatens human life and health. Traditional clinical diagnosis mainly relies on liver ultrasonography and serum alpha-fetoprotein (AFP) for early screening of liver cancer. It is recommended that high-risk groups undergo an examination at least every 6 months. Based on domestic and foreign reports and clinical practice, the diagnostic sensitivity of AFP is about 60%, the misdiagnosis rate is 40%, the specificity is about 80%, and the misdiagnosis rate is 20%. 84%, but for intrahepatic nodules of 5-20mm, the sensitivity of ultrasound is only 33%. Moreover, ultrasound detection is very dependent on instruments and manual operations, and is affected by the limitation of medical resource dis...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/154C12Q2600/166C12Q2531/113C12Q2563/107C12Q2545/101C12Q2545/113
Inventor 阳卫超
Owner GUANGZHOU DRIPNANO BIOTECH CO LTD
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