Digital PCR detection kit and digital PCR detection method for mutation detection of gene mutation high-incidence region

A detection kit and mutation detection technology, which is applied in the field of digital PCR, can solve the problem that it is impossible to realize the quantitative detection of gene mutation high-incidence areas with one detection.

Inactive Publication Date: 2020-03-06
TARGETINGONE CORP +1
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Mutation detection in high-incidence areas of gene mutations has always been a difficulty in mutation detection. Existing various detection technologies cannot realize the detection of various mutation types in high-incidence areas of gene mutations and the quantitative detection of various mutation types through one detection

Method used

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  • Digital PCR detection kit and digital PCR detection method for mutation detection of gene mutation high-incidence region
  • Digital PCR detection kit and digital PCR detection method for mutation detection of gene mutation high-incidence region
  • Digital PCR detection kit and digital PCR detection method for mutation detection of gene mutation high-incidence region

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Embodiment 1

[0022] Embodiment one: basic principle of the present invention

[0023] Such as figure 1 shown, where figure 1 In A, when the template is wild-type, after the template is dispersed in the droplet, the wild-type (WT) probes and internal control (IC) probes labeled with different fluorescent dyes are detected in the same droplet. The fluorescent signal of the positive droplet is the combination of the fluorescent signals of the two probes; figure 1 In B, when the template is a mutant type, after the template is dispersed in the droplet, the WT probe has no signal or the signal decreases because it cannot bind to the mutant template or the binding efficiency decreases, and there is only the fluorescent signal of the IC probe in the positive droplet Or the IC probe signal has different positions according to the different binding efficiencies of the wild-type probe and different mutant templates, so that mutant 1 and mutant 2 can be detected and distinguished; figure 1 In C, w...

Embodiment 2

[0024] Example 2: Detection of EGFR gene exon 19 deletion mutation

[0025] The micro-droplet digital PCR reaction conditions for detection of EGFR gene exon 19 deletion mutations are as follows: PCR amplification reaction mixture includes: 2X MasterMix (with UNG) (Xinyi Manufacturing Technology (Beijing) Co., Ltd.), 200-1000nM primers (sequence is SEQ ID NO: 1-2), 100-800nM detection probe (sequence is SEQ ID NO: 3-4), template DNA (extracted by extraction reagent) 2 ul, water up to 30 ul, and mix the reagents evenly.

[0026] Table 1. Primer and probe sequences for detection of EGFR gene exon 19 deletion mutation detection

[0027]

[0028]

[0029] Note: "+" in Table 1 indicates LNA bases, that is, locked nucleic acid bases.

[0030] Use a sample preparation instrument (Xinyi Manufacturing Technology (Beijing) Co., Ltd.) to prepare micro-droplets according to the instruction manual. Then put the 8-strip tubes containing micro-droplets on the PCR instrument for ampli...

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Abstract

The invention discloses a digital polymerase chain reaction (PCR) detection kit and a digital PCR detection method for mutation detection of a gene mutation high-incidence region. The kit comprises apair of amplification primers, a wild-type specificity detection probe and a internal control probe; the amplification primers are used as a universal PCR amplification upstream primer and a universalPCR amplification downstream primer for detecting a plurality of mutation sites in a gene mutation high-incidence region, and an amplification region comprises a gene mutation high-incidence region to be detected; the wild-type specificity detection probe aims at the gene mutation high-incidence region; and the internal control probe aims at a gene conservative sequence in the amplification region of the gene mutation high-incidence region, wherein different fluorescent dyes are marked by the wild-type specificity detection probe and the internal control probe. The kit and the method can be used to distinguish the wild type and the mutant type of the gene mutation high-incidence region, can further determine the specific mutant type according to a positive signal position of the probes, and can realize quantification of various different mutant types to determine proportions of various mutations.

Description

technical field [0001] The invention relates to the field of digital PCR, in particular to a digital PCR detection kit and a method for detecting mutations in high-incidence areas of gene mutations. Background technique [0002] Gene mutation refers to the change of the hereditary gene (usually deoxyribonucleic acid, DNA) in the cell. Gene mutations include point mutations caused by single base changes, or deletions, duplications, and insertions of multiple bases. Gene mutations are caused by errors in the replication of genes during cell division, or by the effects of chemicals, genotoxicity, radiation or viruses. There can be mutations at multiple sites on a gene, and there are multiple similar or adjacent mutation sites at certain positions, and this area is called a high-incidence mutation area. For example, such mutation-prone regions include: multiple deletion mutations of codons 746-752 in exon 19 of the epidermal growth factor receptor EGFR gene. Usually, mutation...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/106C12Q2600/156
Inventor 祝令香彭志勇郭永杨文军高娜
Owner TARGETINGONE CORP
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