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Composition and kit for detecting EGFR gene deletion mutation, and detection method

A technology for mutation detection and gene deletion, applied in the field of biomedical nucleic acid detection, can solve the problems of unfavorable clinical application, high modification price, and high quantification of mutation abundance

Pending Publication Date: 2020-06-30
SICHUAN MACCURA BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when this method is applied to digital PCR, there is a possibility that the wild-type fluorescent signal will be overwhelmed, resulting in high quantification of mutation abundance.
Blocking wild-type amplification methods often use peptide nucleic acid (Peptide Nucleic Acid, PNA), locked nucleic acid (Locked Nucleic Acid, LNA) modification to block the amplification of wild-type templates, on the one hand, when this method is applied to digital PCR , there is also the possibility that the wild-type fluorescent signal will be overwhelmed. On the other hand, these modifications are expensive, which is not conducive to clinical application

Method used

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  • Composition and kit for detecting EGFR gene deletion mutation, and detection method
  • Composition and kit for detecting EGFR gene deletion mutation, and detection method
  • Composition and kit for detecting EGFR gene deletion mutation, and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0124] A composition for detection of EGFR gene exon 19 deletion mutation comprises:

[0125] Composition for mutation detection:

[0126] The upstream primer F1 has a nucleotide sequence as shown in SEQ ID NO.1;

[0127] The upstream primer F1-1 has a nucleotide sequence as shown in SEQ ID NO.3;

[0128] Hydrolysis probe P1 has the nucleotide sequence shown in SEQ ID NO.5;

[0129] Downstream primer R has a nucleotide sequence as shown in SEQ ID NO.7;'

[0130] Compositions for wild type detection:

[0131] The upstream primer F2 has a nucleotide sequence as shown in SEQ ID NO.2;

[0132] The upstream primer F2-1 has a nucleotide sequence as shown in SEQ ID NO.4;

[0133] Hydrolysis probe P2 has the nucleotide sequence shown in SEQ ID NO.6;

[0134]The downstream primer R has the nucleotide sequence shown in SEQ ID NO.7.

[0135] The 5' end of the hydrolysis probe P1 is labeled with a FAM fluorescent group, the 5' end of the hydrolysis probe P2 is labeled with a HEX fl...

Embodiment 2

[0142] A kit for detecting deletion mutation of exon 19 of EGFR gene. The kit includes primer-probe premix, reaction buffer, negative control, positive control and blank control. The main components of the components are shown in Table 1.

[0143] The primer probe premix in the kit is the mixture obtained by mixing the composition of Example 1 according to the ratio in Table 3;

[0144] The negative control is a fragmented healthy human genomic DNA sample, which has been sequenced to confirm that there is no deletion mutation in exon 19 of the EGFR gene. During preparation, it was quantified by digital PCR and prepared into a negative control substance of 20,000 copies / μL using Tris-EDTA buffer. Specific as Figure 4 shown.

[0145] The positive control is the DNA sample of the fragmented NCI-H1650 mutant cell line, which contains the EGFR gene exon 19 deletion mutation, specifically the mutation subtype is delE746-A750; the specific base level is c.2235_2249del15 (Cosmic ID:...

Embodiment 3

[0150] Perform assays on cell line samples.

[0151] Sample preparation:

[0152] Using QIAGEN Corporation The DNA Mini Kit was used to extract the nucleic acid of the NCI-H1650 cell line containing the mutation deletion of exon 19 of the EGFR gene according to the operation instructions of the kit, and the genomic DNA of the cell line with the mutation deletion of exon 19 of the EGFR gene was obtained.

[0153] Reaction preparation:

[0154] After the sample preparation is completed, the configuration of the reaction system is carried out, and the PCR reaction system is prepared according to the ratio described in Table 3.

[0155] At the same time, the EGFR WT&E746_A750del Assay Kit (Cat. No. 10041170) from Bio-Rad was used as a contrast reagent.

[0156] Add 20 μL of the prepared PCR reaction system to the sample well of the micro-droplet generator card, then add 70 μL of micro-droplet generator oil to the oil hole of the micro-droplet generator card, and finally seal t...

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Abstract

The invention discloses a composition and a kit for detecting EGFR gene deletion mutation, and a detection method. The composition comprises a mutant-type detection composition and / or a wild-type detection composition, wherein the mutant-type detection composition comprises an upstream primer F1, an upstream primer F1-1, a hydrolysis probe P1 and a downstream primer; the wild-type detection composition comprises an upstream primer F2, an upstream primer F2-1, a hydrolysis probe P2 and a downstream primer, wherein each of the upstream primer F1 and the upstream primer F2 comprises a non-matching region and a matching region; and the matching regions of the upstream primers F1 and F2 are specifically bound to the to-be-detected sequence regions respectively. The unmatched regions of the upstream primers F1 and F2 have the same sequences of the upstream primers and hydrolysis probes for corresponding detection. Compared with the prior art, the composition has the advantages that the sensitivity and the height specificity are remarkably improved, the primers and the test cost are greatly reduced, and the application value is extremely high.

Description

technical field [0001] The invention relates to the technical field of biomedical nucleic acid detection, in particular to a composition, a kit and a detection method for detecting EGFR gene deletion mutations. Background technique [0002] For a long time, histopathological diagnosis has been the gold standard of tumor diagnosis and the basis of clinical treatment. However, if the same treatment plan is adopted for tumor patients with the same histological type and stage, only a part of the tumor patients will respond. According to statistics, the inefficiency of traditional medicine in tumor treatment is as high as 75%. The poor curative effect of malignant tumors is that it is difficult to judge its malignant characteristics and drug efficacy characteristics from the level of tissue diagnosis. Studies have shown that the molecular characteristics of tumor lesions determine its malignant characteristics, metastasis characteristics, recurrence characteristics and drug res...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2563/107
Inventor 赵雨航葛志琪
Owner SICHUAN MACCURA BIOTECH CO LTD
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