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Primer composition, reagent and kit for detecting human MET gene amplification and application of primer composition, reagent and kit

A technology for gene amplification and detection of human beings, applied in the field of primer compositions for detection of human MET gene amplification, can solve the problems of difficult clinical application, complex data analysis, high price, etc., and achieve non-invasive detection of MET gene amplification and expansion The range of use and the effect of high-sensitivity detection

Inactive Publication Date: 2021-03-12
杭州求臻医学检验实验室有限公司
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Problems solved by technology

Next-generation sequencing (NGS) technology can detect multiple samples at the same time, and can detect multiple genetic loci, but the cost is high, the experimental period is long, and the data analysis is complicated; fluorescence in situ hybridization (FISH) technology detection MET gene amplification has high specificity, but the sample processing cycle is long, the cost is high (probes are expensive), the result interpretation is s

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  • Primer composition, reagent and kit for detecting human MET gene amplification and application of primer composition, reagent and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0034] 1) Design and synthesis of primers and probes for detection of MET gene amplification based on micro-droplet digital PCR technology

[0035] Based on the genome analysis data, analyze the MET gene, use Oligo software to analyze the sites of TaqMan primers and probes, and select the best combination as follows:

[0036] MET gene upstream and downstream primers:

[0037] MET upstream primer SEQ ID NO1: 5'-AGTTCGCTACGATGCAAGAGT-3'

[0038] MET downstream primer SEQ ID NO2: 5'-AGTTGGGCTTACACTTCGGG-3'

[0039] 2) When using the above-mentioned primers for PCR amplification, the amplified fragment is 73bp, which is especially suitable for the amplification of small fragment DNA samples such as plasma free DNA.

[0040] 3), MET gene detection probe: SEQ ID NO3: 5'-TCCTCATTTGGATAGGCTTGTA

[0041] A gene that can be stably amplified and whose amplification effect is consistent with MET was selected as the internal reference gene.

[0042] 4), the internal reference gene sele...

Embodiment 2

[0049]Extract cfDNA from the sample to be tested:

[0050] The samples to be tested in this example are plasma samples, and the sample numbers are A, B, C, D, and E respectively; the cfDNA in the plasma samples is extracted using a kit, and the specific operation can be found in the QIAamp Circulating Nuleacid Kit kit manual of QIAGEN. , and the extracted cfDNA was used as a template for ddPCR detection, where A, B, and C were MET-positive samples, and D and E were MET-negative samples.

Embodiment 3

[0052] Prepare the PCR reaction solution in the PCR plate according to the ratio shown in Table 1: the concentrations of the cfDNA template and the internal reference gene are both 2.5 ng / μL.

[0053] Table 1 PCR reaction solution configuration

[0054] Reagent Dosage 2×ddPCR Master Mix 10μL cfDNA template 2μL Various primers 0.8μM Each probe 0.45μM Add DEPC water to 20 μL

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Abstract

The invention relates to the technical field of human MET gene detection, in particular to a primer composition, a reagent and a kit for detecting human MET gene amplification and application of the primer composition, the reagent and the kit, the application is a method for detecting human MET gene amplification, and the method comprises the following steps: S1, extracting cfDNA in a whole bloodsample of a subject; S2, taking the cfDNA extracted in the step S1 as a template, and carrying out quantitative digital PCR reaction on the cfDNA template of the sample to be detected, a primer composition for detecting human MET gene amplification, a probe or a kit to prepare a ddPCR mixed solution; S3, performing signal collection on a product after the PCR amplification reaction in the step S2;and S4, judging whether the sample to be detected contains MET gene amplification or not according to the type of the fluorescence signal collected in the step S3. The primer composition, the reagentand the kit are high in sensitivity, high in accuracy, convenient to interpret and easy to carry out.

Description

technical field [0001] The invention relates to the technical field of human MET gene detection, in particular to a primer composition, reagent and kit for detecting human MET gene amplification and applications thereof. Background technique [0002] The MET gene is located on the long arm of human chromosome 7 (7q31) and encodes a transmembrane tyrosine kinase receptor. After MET binds to the ligand hepatocyte growth factor HGF, it dimerizes and causes a variety of tyrosine residues in the cell. Phosphorylation of the base, and then activate a series of downstream signaling pathways to promote tumor cell proliferation, growth, migration, and angiogenesis. The cMET gene can cause tumors through three forms: MET exon 14 mutation, MET gene amplification and MET overexpression. Studies have shown that MET gene amplification can activate the ErbB3 / PI3K / AKT signaling pathway, leading to drug resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI), and the high expression of MET...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/106C12Q2600/158C12Q2600/166C12Q2531/113C12Q2561/101C12Q2563/159C12Q2545/101
Inventor 段小红杨春燕王冬冬丁蕊张腾龙周启明
Owner 杭州求臻医学检验实验室有限公司
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