Method for detecting multi-locus low-frequency mutation of free target DNA of lung cancer plasma and kit

A low-frequency mutation, multi-site technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of restricting wide application, time-consuming, unable to meet one-by-one screening, etc., to improve detection throughput, Effect of detection sensitivity improvement

Inactive Publication Date: 2018-04-06
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, ctDNA only accounts for less than 1% of the total free DNA in plasma, especially in patients with early cancer, which is even lower at about 0.01%. Therefore, it is not easy to accurately detect tumor-associated low-frequency mutation information from such a high background free DNA. thing
Existing ctDNA detection technologies such as droplet digital PCR technology (ddPCR), digital PCR and flow-based

Method used

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  • Method for detecting multi-locus low-frequency mutation of free target DNA of lung cancer plasma and kit
  • Method for detecting multi-locus low-frequency mutation of free target DNA of lung cancer plasma and kit
  • Method for detecting multi-locus low-frequency mutation of free target DNA of lung cancer plasma and kit

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Embodiment 1

[0061] This embodiment takes the following gene mutation as an example to verify the present invention: EGFR gene mutation c.2573T>G, p.L858R, c.2369C>T, T790M, del E746-A750; KRAS gene mutation p.G12C; the specific steps are as follows :

[0062] (1) Sample preparation: EGFR gene mutation c.2573T>G, p.L858R, c.2369C>T, T790M derived from cell line H1975, mutation del E746-A750 derived from cell line H1650, KRAS gene mutation p.G12C derived from In cell line A549, all mutation sites were identified by sanger sequencing. The above-mentioned mutant DNA was diluted with wild-type DNA to a ratio of 40%, and then the four diluted DNA were mixed in a ratio of 1:1:1:1, so that the ratio of each mutant DNA was 10%. Then the wild-type DNA was diluted to make the ratio of the final mutant type to the wild type 1:100, 1:1000, 1:10000, and the mixed DNA in three ratios was interrupted to 200-500bp by covaris S220;

[0063] (2) Prepare digital PCR mixture: Add 2x genotyping master mix, l...

Embodiment 2

[0073] Collect clinical lung cancer tissue samples and corresponding blood samples for one-time multiple lung cancer gene mutation detection

[0074] DNA extraction from test samples: DNA extraction from tissue samples was performed using DNeasy Blood & Tissue Kit from Qiagen Company, and the operation was performed according to the instructions of the extraction kit. Blood cell-free DNA was extracted using QIAamp Circulating Nucleic Acid Kit from Qiagen Company, and operated according to the kit instructions. The OD260 / OD280 of the extracted tissue DNA should be between 1.8 and 2.1 by Nanodrop, the free blood DNA is detected by 2100, and the fragments are 150 to 200bp, and both tissue DNA and blood free DNA are quantified by Qubit 2.0;

[0075] (1) Prepare digital PCR mixture: add 2x genotyping master mix, lung cancer-specific primers (final concentration 0.2-0.4uM), 10ng of the above DNA to 8 PCR tubes, and add water to a 20ul reaction system;

[0076] (2) Put the prepared ...

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Abstract

The invention relates to the technical field of biology and the field of nucleic acid detection and relates to method for detecting multi-locus low-frequency mutation of free target DNA of lung cancerplasma and a kit. The invention provides high-sensitivity and high-flux method for detecting multi-locus mutation of free DNA of lung cancer plasma once and a kit. According to the detection method,a ddPCR technique and a next generation sequencing technique are combined for detecting the mutation of lung cancer crDNA. The detection method comprises the following steps: (1) preparing a digital PCR mixed liquid including a to-be-tested sample DNA template, a primer and a PCR premixing liquid; (2) preparing a digital PCR micro-reaction drop, and carrying out PCR amplified reaction; (3) recycling and purifying a PCR product; (4) preparing and amplifying PCR premixing liquid for the second time; (5) carrying out purification and library quality control on the PCR product; and (6) carrying out computer sequencing and data analysis. According to the detection method, hotspot mutation information of 10 genes including EGFR/BRAF/KRAS/PIK3A/MET2/ERBB2/AKT1/NRAS/TP53/PTEN of lung cancer can bedetected for one time, the cost can be remarkably lowered, and the high-sensitivity and high-flue detection of mutation information of lung cancer ctDNA can be realized.

Description

technical field [0001] The invention relates to the fields of biotechnology and nucleic acid detection, in particular to a method for detecting low-frequency mutations at multiple sites in plasma free target DNA of lung cancer and a kit thereof. Background technique [0002] According to reports, lung cancer is the fastest growing malignant tumor in the world, and non-small cell lung cancer (NSCLC) accounts for about 80% of lung cancer. The latest research estimates that in 2015, there were about 733,000 new cases of lung cancer in my country (accounting for 17.1% of the overall new cancer cases), and 610,000 deaths (accounting for 21.1% of the overall cancer deaths), which seriously threatened the health of the Chinese population. life and health. About 75% of the patients were found in the middle and advanced stages, and surgical resection and chemotherapy are still the main treatment methods, but there are many complications, and the 5-year survival rate of patients is ve...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686C12Q1/6869
CPCC12Q1/686C12Q1/6869C12Q1/6886C12Q2600/156C12Q2600/118C12Q2563/159
Inventor 刘赟邱文青
Owner FUDAN UNIV
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