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Composition for detecting free DNA methylation of plasma based on digital PCR and application of composition

A technology of methylation and composition, which is applied in the field of detection of plasma free DNA methylation composition, can solve the problems of inaccurate clinical detection of gastric cancer, difficulty in realizing high-precision and absolute quantitative detection of plasma DNA methylation, etc. , to achieve the effect of solving tissue sample dependence, broad application prospects and industrialization prospects, and saving time

Pending Publication Date: 2021-08-20
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, at present, fluorescent quantitative PCR is still used to detect the free DNA methylation of the above-mentioned genes. The basic principle is to use specific primers and probes to distinguish methylated and non-methylated gene sequences after being treated with bisulfite. This method is widely used as a relatively quantitative detection technique for highly sensitive DNA methylation. However, the detection result of the fluorescence quantitative method is relatively quantitative, and it is difficult to achieve high-precision and absolute quantitative detection of plasma DNA methylation, which leads to the detection of gastric cancer. clinical testing is imprecise

Method used

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  • Composition for detecting free DNA methylation of plasma based on digital PCR and application of composition
  • Composition for detecting free DNA methylation of plasma based on digital PCR and application of composition
  • Composition for detecting free DNA methylation of plasma based on digital PCR and application of composition

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] (1) Extraction of plasma cfDNA

[0036] 1) Add 30 μL of magnetic bead suspension, 55 μL of proteinase K, and 150 μL of Bead BindingBuffer to 1 mL of plasma sample in sequence, and vortex to mix;

[0037] 2) Centrifuge at 200×g for 30s to remove the liquid on the lid, place the Ep tube on a 2mL magnetic stand, incubate for 1min until the solution is clear, and remove the upper layer solution with a pipette;

[0038] 3) Remove the Ep tube, add 200 μL Bead Elution Buffer to it, vortex to mix, transfer the mixture to the Bead Elution tube, put it in the metal bath, set the program at 20°C, 300 rpm, and incubate for 5 minutes;

[0039] 4) Remove it and place it on a magnetic stand for 1 min until the solution is clear. Transfer the supernatant to a new elution tube, discard the magnetic beads, add 300 μL ACB buffer to the supernatant, vortex to mix, and briefly centrifuge;

[0040] 5) Add the mixture from the previous step to the QIAamp UCP Elution Column, centrifuge at 60...

Embodiment 2

[0056] Design of primers for digital PCR amplification: The methylated sequence and non-methylated sequence of the gene Reprimo bisulfite conversion were used as the target gene to design primers. The primer sequences are listed in Table 1.

[0057] Table 1 Primer sequence list

[0058]

[0059]

[0060] Among them, the fluorophore labeled at the 5' end of M-Probe is FAM, the quencher group labeled at the 3' end is BHQ1; the fluorophore labeled at the 5' end of U-Probe is VIC, and the quencher group labeled at the 3' end is The killing group is BHQ1.

[0061] 1) Assuming that the sample to be tested is N, the configuration of the reaction system is shown in Table 2:

[0062] Table 2 ddPCR reaction system

[0063]

[0064] 2) After fully mixing the reaction system, dispense it into 17 μL of each reaction tube;

[0065] 3) Take 3 μL of bisulfite-converted DNA and add them to corresponding reaction tubes, with a final volume of 20 μL;

[0066] 4) Put a new droplet g...

Embodiment 3

[0079] Design of primers for digital PCR amplification: The methylated sequence and unmethylated sequence of the gene RNF180 after bisulfite conversion were used as target genes to design primers. The primer sequences are listed in Table 4.

[0080] Table 4 Primer sequence list

[0081]

[0082] Among them, the fluorophore labeled at the 5' end of M-Probe is FAM, the quencher group labeled at the 3' end is BHQ1; the fluorophore labeled at the 5' end of U-Probe is VIC, and the quencher group labeled at the 3' end is The killing group is BHQ1.

[0083] 1) Assuming that the sample to be tested is N, the configuration of the reaction system is shown in Table 5:

[0084] Table 5 ddPCR reaction system

[0085]

[0086] 2) After fully mixing the reaction system, dispense it into 17 μL of each reaction tube;

[0087] 3) Take 3 μL of bisulfite-converted DNA and add them to corresponding reaction tubes, with a final volume of 20 μL;

[0088] 4) Put a new droplet generation card...

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Abstract

The invention discloses a composition, a kit and a detection method for detecting methylation of a gene Reprimo and / or a gene RNF180 in gastric cancer plasma cfDNA based on a digital PCR technology. The method for absolutely and quantitatively detecting the methylation of the Reprimo gene or the RNF180 gene in the gastric cancer plasma cfDNA based on the micro-droplet digital PCR technology is high in detection sensitivity, accurate in result and capable of absolutely quantifying, and has wide application prospects and industrialization prospects. More importantly, methylation of a gastric cancer marker Reprimo gene or RNF180 gene is subjected to joint detection, experiments prove that the diagnostic value of multi-parameter joint diagnosis analysis for distinguishing a healthy control group and a GC group as well as a benign group and the GC group is superior to that of single parameter detection, so that early discovery and early prevention of gastric cancer diagnosis can be realized, the important significance is achieved for treatment and prognosis of the gastric cancer.

Description

[0001] Cross References to Related Applications [0002] This application claims the priority of the patent application No. 202110608386.4 submitted to the State Intellectual Property Office of China on June 1, 2021, the entire content of which is incorporated herein by reference. technical field [0003] The invention belongs to the technical field of methylation detection, and relates to a composition for detecting plasma free DNA methylation based on digital PCR and an application thereof. Background technique [0004] According to the latest global cancer data for 2020 released by the International Agency for Research on Cancer (IARC), there were 1,089,103 new cases of gastric cancer compared with the previous year, and it has been ranked fifth in the number of new cancer cases worldwide. Because it is usually diagnosed at an advanced stage, gastric cancer has a high mortality rate, accounting for 7.7% of the total global cancer deaths in 2020, ranking fourth. Early det...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851
CPCC12Q1/6886C12Q1/6851C12Q2600/154C12Q2523/125C12Q2531/113C12Q2563/159C12Q2563/107
Inventor 田亚平张朋军隋亚鑫
Owner GENERAL HOSPITAL OF PLA
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