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Kit for detecting imatinib targeted drug use related gene mutation

An imatinib target and kit technology, applied in the field of biomedicine, can solve the problems of high detection cost, low detection method sensitivity, less than 5% effective rate of chemotherapeutic drugs, etc., and achieves convenient and fast operation, good specificity and purification. Efficient effect

Pending Publication Date: 2020-04-07
WUHAN BIOTECH GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

GIST is extremely insensitive to traditional chemotherapy, the effective rate of chemotherapy drugs is less than 5%, and the median survival rate in advanced stage is only about 18 months
However, the existing commercially available kits for gene C-kit or PDGFRA mutations related to imatinib targeted drug can only detect the mutation of one of the genes, and some detection methods have low sensitivity and cannot accurately detect the C-kit gene or PDGFRA mutation. Whether the PDGFRA gene is mutated, and the detection cost is high

Method used

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  • Kit for detecting imatinib targeted drug use related gene mutation
  • Kit for detecting imatinib targeted drug use related gene mutation
  • Kit for detecting imatinib targeted drug use related gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The embodiment of the present invention provides a primer probe composition for detecting mutations of genes C-kit and PDGFRA related to imatinib-targeted drug use. The primer probe composition for detecting mutations of C-kit and PDGFRA genes includes Forward and reverse primers and fluorescent probes for detecting mutations in exon 9 of the C-kit gene, for detecting forward and reverse primers and fluorescent probes for exon 11 of the C-kit gene, for detecting C- Forward and reverse primers and fluorescent probes of the No. 13 exon of the kit gene are used to detect the No. 17 exon of the C-kit gene. Forward and reverse primers and fluorescent probes are used to detect the No. 12 exon of the PDGFRA gene Forward and reverse primers and fluorescent probes of the exon, and forward and reverse primers and fluorescent probes for detecting the No. 18 exon of the PDGFRA gene, wherein:

[0057] The forward primer of exon 9 of the C-kit gene is shown in SEQ ID NO.1 in the sequ...

Embodiment 2

[0077] The embodiment of the present invention provides a kit for detecting mutations of imatinib-targeted drug-related genes C-kit and PDGFRA. The kit includes the primer-probe composition provided in Example 1 of the present invention, each primer and fluorescent The probe concentration is 10 μmol / L, which can detect the mutations of exons 9, 11, 13 and 17 of C-kit gene and the mutations of exons 12 and 18 of PDGFRA gene .

[0078] The kit also includes PCR reaction solution, sequencing reaction solution, digestive enzyme system, PCR product purification reagent, negative quality control substance and positive quality control substance. The PCR reaction solution includes Mg-containing 2+ 5×PCR amplification buffer, 2.5mmol / L dNTPs and 5U / μL Taq DNA polymerase, the sequencing reaction solution includes 2.5×BigDye and 5×BigDye sequencing buffer.

[0079] The digestive enzyme system includes 1-4U / μL shrimp alkaline enzyme and 4-7U / μL exonuclease I.

[0080] On the basis of t...

Embodiment 3

[0083] Example 3, Detection of genes related to imatinib targeted drug

[0084] Patients with gastrointestinal stromal tumors who had a history of imatinib were selected as subjects for a retrospective study.

[0085] S1. Extracting sample genomic DNA

[0086]Applicable sample type: 10% neutral formalin-fixed paraffin-embedded gastrointestinal stromal tumor tissue section.

[0087] Sample requirements to be tested:

[0088] (1) Paraffin-embedded pathological section samples should be confirmed to contain tumor lesion cells. In order to ensure that the section tissue contains a sufficient proportion of tumor cells, one HE-stained slide of the same tissue should be added;

[0089] (2) Cut 5 pieces of 8-10 μm thick white slices from each sample, and the tumor cell area is larger than 5mm×5mm, slice serially, and seal them in a 1.5mL centrifuge tube;

[0090] (3) Paraffin-embedded pathological sections should not be stored at room temperature for more than 12 months. In order t...

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Abstract

The invention discloses a kit for detecting imatinib targeted drug use related gene mutation. The imatinib targeted drug use related gene comprises a C-kit gene and a PDGFRA gene. The kit comprises primers and fluorescent probes which are respectively used for detecting the ninth, eleventh, thirteenth and seventeenth exons of the C-kit gene and the twelfth and eighteenth exons of the PDGFRA gene,a target gene segment is amplified through fluorescent quantitative PCR, and then a sequencing reaction is carried out, the kit further comprises a digestive enzyme system composed of shrimp alkalinephosphatase and exonuclease I, the PCR product purification effect is better, and the sample loss is less. The kit is high in detection accuracy and sensitivity, good in repeatability and convenient and rapid to operate, and provides a rapid and reliable experimental basis for test, analysis and evaluation of imatinib targeted medication.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a kit for detecting gene mutations related to imatinib targeted drug. Background technique [0002] Targeted medicine (targeted medicine) is currently the most advanced medicine used to treat cancer. The biggest difference between it and traditional medicine lies in its mechanism of action. Conventional chemotherapeutic drugs play a role by poisoning cells. Since they cannot accurately identify tumor cells, they will also harm normal cells while killing tumor cells, thus producing relatively large toxic and side effects. Targeted drugs are developed for tumor genes, which can recognize the characteristic sites on tumor cells determined by tumor cell-specific genes, and block the control cells in tumor cells by binding to them (or other similar mechanisms). Growth and proliferation signal transduction pathways, thereby killing tumor cells and preventing their proliferation. Due to suc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6869C12N15/11
CPCC12Q1/6886C12Q1/6869C12Q2600/106C12Q2600/156C12Q2531/113C12Q2521/319C12Q2521/525C12Q2545/113
Inventor 熊慧柳诚刚曾海肖樊高峰英
Owner WUHAN BIOTECH GENE ENG
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