Primer probe combination for methylation detection of RASSF1A gene and P16 gene and application of primer probe combination

A primer-probe and methylation technology, applied in the field of molecular biology, can solve the problems of difficult early detection and early qualitative diagnosis, unrealistic tumor tissue detection, and difficulty in early diagnosis of liver cancer, achieving high specificity and detection operation. Simple, convenient and highly accurate results

Inactive Publication Date: 2018-12-28
天津知因生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the heterogeneity of tumors, for patients whose cancer cells have metastasized, only detecting tumor tissue in a certain part cannot reflect the overall situation of the patient, and it is also impractical to detect all tumor tissues. also has a certain hysteresis
Early liver cancer is hardly noticed by doctors and p

Method used

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  • Primer probe combination for methylation detection of RASSF1A gene and P16 gene and application of primer probe combination
  • Primer probe combination for methylation detection of RASSF1A gene and P16 gene and application of primer probe combination
  • Primer probe combination for methylation detection of RASSF1A gene and P16 gene and application of primer probe combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1 Assembly of the kit

[0115] 1. The design of primer probe combination, the specific sequence is shown in Table 1 below:

[0116] Table 1 Sequence of primer probe

[0117]

[0118]

[0119] 2. The reagent composition in the kit is shown in Table 2.

[0120] Table 2 Reagent composition in the kit

[0121]

[0122] Among them, the nucleotide sequences of positive control and negative control are shown in Table 3:

[0123] Table 3 Nucleotide sequence of positive control and negative control

[0124]

[0125]

[0126] Put the above components, instructions and droplet generation card into the kit body.

Embodiment 2

[0127] Example 2 RASSF1A gene and P16 gene methylation detection

[0128] Using the kit in Example 1 to perform methylation detection of RASSF1A gene and P16 gene includes the following steps:

[0129] (1) Extraction of template DNA

[0130] ①Plasma separation: Collect 5mL-10mL human peripheral blood with EDTA anticoagulation tube; centrifuge the blood sample at 1600g for 10 minutes; carefully transfer the collected plasma sample to a 2mL centrifuge tube; centrifuge the plasma sample at 4℃ , 16000g, 10 minutes; carefully transfer the collected plasma sample to a new 2mL centrifuge tube; collect the completed plasma sample to save at -20℃;

[0131] ② Extraction of free DNA: Use DNA extraction reagents to extract free DNA from peripheral blood, monitor DNA quality, and select free DNA with OD260 / OD280 between 1.6 and 2.0;

[0132] ③DNA modification: The free DNA obtained in step ② is modified with sulfite according to the instructions of the kit EZ DNAMethylation-DirectTM KIT (D5020) of ...

Embodiment 3

[0151] Compared with Example 2, the PCR reaction conditions are the same as in Example 2 except that the PCR reaction conditions are pre-denaturation at 98°C for 3 minutes, 93°C for 40s; annealing at 55°C for 45s; 45 cycles, and finally at 98°C for 8 minutes.

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Abstract

The invention belongs to the technical field of molecular biology and relates to a detection product for genomic methylation, in particular to a primer probe combination for methylation detection of an RASSF1A gene and a P16 gene and application of the primer probe combination. The primer probe combination for the methylation detection of the RASSF1A gene and the P16 gene comprises detection primer probes; and the detection primer probes comprise an RASSF1A gene methylation detection specific primer pair, an RASSF1A gene specific probe, a P16 gene methylation detection specific primer pair anda P16 gene specific probe. According to the primer probe combination, methylation sites of the RASSF1A gene and the P16 gene are accurately detected, detection precision of ctDNA is remarkably improved, and absolute quantification is realized; and in addition, the primer probe combination has high specificity and accuracy, and is suitable for early screening of liver cancer.

Description

Technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a gene methylation detection product, in particular to a primer probe combination for detection of RASSF1A gene and P16 gene methylation and its application. Background technique [0002] Primary hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. In the world, the incidence of liver cancer ranks sixth among tumors, and the mortality rate ranks third, with more than 60 new cases every year. Million. In China, the incidence of liver cancer ranks third among all tumors, and its mortality rate is the second. Approximately 230,000 people die from liver cancer each year, accounting for 55% of all liver cancer deaths worldwide. The World Health Organization pointed out: If cancer patients can be detected early, the cure rate can reach 80%. However, liver cancer has no obvious clinical symptoms in the early stage, develops quickly and easily meta...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6858C12Q1/6886C12Q2600/136C12Q2600/154C12Q2600/16C12Q2600/166C12Q2545/101C12Q2531/113C12Q2563/159C12Q2537/143
Inventor 罗敏赵勇娟韩玥斌刘焕
Owner 天津知因生物科技有限公司
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