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A digital pcr detection kit and detection method for t790m gene mutation

A technology for detection kits and detection methods, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problems of early, non-invasive, quantitative detection of unfavorable T790M gene mutations

Active Publication Date: 2021-08-20
SIMCERE DIAGNOSTICS CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection of T790M is a detection of low-abundance target sequences, and the existing detection methods are not conducive to the early, non-invasive and quantitative detection of T790M gene mutations

Method used

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  • A digital pcr detection kit and detection method for t790m gene mutation
  • A digital pcr detection kit and detection method for t790m gene mutation
  • A digital pcr detection kit and detection method for t790m gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1 Design T790M amplification primers and detection probes for digital PCR

[0061] 1. Design of primers and probes

[0062] 1. According to the nucleotide sequence of the 20th exon of human EGFR, design the amplification product including the upstream and downstream primers of the T790M site. The specific primer sequences are shown in Table 1.

[0063] 2. Design detection probes according to the nucleotide information at the T790M gene mutation: mutant nucleic acid probe (T790M-Probe-MU) and wild-type nucleic acid probe (T790M-Probe-WT), wherein the mutant probe is Taqman-MGB probe, the 5' end of its nucleic acid sequence is marked with FAM, and the 3' end is marked with MGB; the wild-type probe is Taqman-MGB probe, the 5' end of its nucleic acid sequence is marked with VIC, and the 3' end is marked with MGB, specifically See Table 2 for probe sequences.

[0064] Table 1

[0065]

[0066]

[0067]

[0068] Table 2

[0069] Primer probe na...

Embodiment 2

[0097] The optimization of embodiment 2 primer concentration and probe concentration

[0098] 1. Primer concentration optimization

[0099] The upstream and downstream primers (the 13th pair) described in Example 1 were serially diluted so that the final concentrations of the upstream and downstream primers in the reaction system were 700, 800, 900, 1000, 1100 or 1200nM respectively, and passed the numbers PCR detection of the detection effect at different concentrations, specific detection results see Figure 4 . according to Figure 4 From the results shown, it can be seen that the primer concentration has little effect on the fluorescence value and the differentiation of droplets, and 900nM was finally selected as the optimized primer concentration. Wherein the specific detection sample, the configuration of the reaction system, the microdroplet generation method and the reaction conditions are as shown in Example 1, and the gradient dilution method is as follows: take t...

Embodiment 3

[0106] The optimization of embodiment 3 reaction system annealing temperature

[0107] Optimize the annealing temperature of digital PCR according to the following method: set the temperature gradient on the PCR instrument from 55.0°C to 64.0°C, use the genomic DNA of the H1975 cell line as the detection template to perform digital PCR amplification detection, and determine the optimal annealing according to the droplet differentiation The temperature was 55.6°C. For specific test results, see Figure 6~7 . Among them, the specific detection sample, reaction system, droplet generation and sealing operation are shown in Example 2, and the specific PCR reaction program is shown in Table 7.

[0108] Table 7

[0109]

[0110]

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Abstract

The invention relates to a digital PCR detection kit for T790M gene mutation and a detection method thereof. The kit of the present invention includes amplification primers and detection probes, wherein the amplification primers include upstream and downstream primers, and the nucleotide sequences of the upstream and downstream primers are respectively shown in SEQ ID NO: 25-26 , the detection probes include mutant probes and wild-type probes, the nucleotide sequences of the mutant probes and wild-type probes are respectively shown in SEQ ID NO: 65-66. The kit and detection method of the present invention have the advantages of high accuracy and sensitivity, short detection time, and can realize the qualitative and quantitative detection of samples with low abundance and low mutation rate, and help to realize the early, non-invasive, and rapid detection of T790M gene mutation. Quantitative detection.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a digital PCR detection kit for T790M gene mutation and a detection method thereof. Background technique [0002] T790M gene mutation is a type of gene mutation found in tumor patients, specifically refers to the mutation of threonine (T) at the 790th amino acid site of exon 20 of the epidermal growth factor receptor (EGFR) It is methionine (G), and the gene level shows that ACG is mutated to ATG. Since methionine occupies a larger space than threonine, it forms steric hindrance and changes the affinity of ATP in the kinase region of EGFR, leading to the development of small molecule drugs such as EGFR-tyrosine kinase inhibitors (TKIs). It cannot effectively block the EGFR activation signal, thus losing its killing effect on tumor cells. The current research results show that the T790M gene mutation is related to the drug resistance, curative effect prediction and prognosis of tum...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6858C12Q2561/101C12Q2563/107C12Q2563/159
Inventor 张利清黄霖霆张琴曹乾升李杜衡张林任用
Owner SIMCERE DIAGNOSTICS CO LTD