Unlock instant, AI-driven research and patent intelligence for your innovation.

Kit for detecting human egfr gene mutation

A kit and gene technology, applied in the field of kits for detecting human EGFR gene mutation, can solve the problems of specificity and influence on analysis results, and achieve the effects of enhancing specificity, simple operation, and enhancing TM value and binding force.

Active Publication Date: 2019-09-10
FULONG MEDICAL MATERIALS CHINA
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still the following limitations and deficiencies in practical applications: ①Sometimes, even if mismatched bases are introduced at the 2nd and 3rd positions of the 3' end of the primer, the interference of wild type and other mutant types cannot be avoided; ②The samples When selecting the blood of tumor patients, due to the low content of mutated genes, more interference components in the blood will directly affect the analysis results; ③ If the mutated site is a weakly mismatched base sequence, the specificity of the method will be affected influences

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Kit for detecting human egfr gene mutation
  • Kit for detecting human egfr gene mutation
  • Kit for detecting human egfr gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The peripheral blood and tissue sections of 50 patients with clinical diagnosis of non-small cell lung cancer were collected. In order to ensure the accuracy of the experiment, the peripheral blood was detected by the kit of the present invention, and the tissue sections were verified by the traditional sequencing method.

[0041] The specific implementation steps are:

[0042] 1. Design and preparation of the kit of the present invention

[0043] The kit comprises primer pair A1 and probe B1 for detecting EGFR gene exon 18 G719S mutation, primer pair A2 and probe B2 for detecting EGFR gene exon 18 G719C mutation, and for detecting EGFR gene Primer pair A3 and probe B3 for G719A mutation in exon 18, primer pair A4 and probe B4 for detecting EGFR gene exon 20 T790M mutation, and primer pair A5 for detecting EGFR gene exon 20 S768I mutation and probe B5, primer pair A6 and probe B6 for detecting EGFR gene exon 21 L858R mutation, primer pair A7 and probe B7 for detecting ...

Embodiment 2

[0073] This example is mainly to prove the specificity of the kit of the present invention. Taking the L858R mutation site on exon 21 as an example, AMRS primers are specially designed and mismatches are introduced. Since kits using AMRS technology to detect EGFR gene mutations are also widely used in the market, a comparative analysis was performed using this method.

[0074] The peripheral blood and tissue sections of 50 cases of clinically diagnosed patients with non-small cell lung cancer were collected. In order to ensure the accuracy of the experiment, the peripheral blood was detected with the kit of the present invention, and the tissue sections were sequenced and verified by traditional sequencing methods.

[0075] The specific implementation steps are:

[0076] 1. The design of AMRS primer and kit of the present invention

[0077] Experimental group: the detection system for detecting the No. 21 L858R mutation site in the kit of the present invention was used for co...

Embodiment 3

[0091] This embodiment is mainly to prove the specificity of the kit of the present invention. Taking the G719S mutation on exon 18 as an example, the G719S mutant plasmid DNA is used for analysis, and a total of 3 concentration gradients are made, and in the background of 20ng / μL (not the Fluorescent detection is carried out under the clinical samples with mutations), which is used to illustrate the high sensitivity of the kit of the present invention.

[0092] 1. Extraction of sample genomic DNA

[0093] The whole blood DNA extraction kit of QIAGEN Company was used for the extraction of peripheral blood genomic DNA, and 200 UL of whole blood was extracted each time, and the OD value of the obtained DNA was measured to ensure that the concentration of the extracted DNA sample was 10 ng / μL, and 2 μL (i.e. 20ng / μL) for fluorescent PCR reaction, and the specific operation method was operated according to the routine operation steps of the kit.

[0094] 2. Dilution of Mutant Pla...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to the technical field of biological detection, in particular to a kit for detecting human EGFR (epidermal growth factor receptor) gene mutation. The kit comprises primers and a probe for detecting the EGFR gene. The primers comprise the forward primers, the competitive primers and the reverse primers. The competitive primers are further provided with GC tail ends on the original basis that the competitive primers are contrastive and complementary to the forward specific primers, and the TM value and the binding force are improved. The kit for detecting human EGFR gene mutation has high specificity and high background interference resistance and can accurately detect which single base has mutations, the kit can detect the mutant gene from a complex clinical sample, for example, detection of peripheral blood and the like, the kit is not limited by tumor cell tissues anymore, and the kit can still detect the target mutant gene under the high concentration background of 20 ng / ul.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a kit for detecting human EGFR gene mutation. Background technique [0002] Lung cancer is one of the most common malignant tumors and one of the most lethal cancers, among which 80-85% of patients are non-small cell lung cancers (NSCLC). The morbidity and mortality ranks first in my country, among which tyrosine kinase inhibitors targeting epidermal growth factor receptor (EGFR) are increasingly prominent in the treatment of non-small cell lung cancer (2,3). EGFR is the target of cancer treatment, and there are two main types of targeted drugs: one is small molecule tyrosine kinase inhibitor (TKI) that acts on receptor cells, mainly including gefitinib, erlotinib Ni, EKB-569, PKI-166, GW-2016 and CI-1033; another type of monoclonal antibody (MAb) acting on the extracellular domain of the receptor, including cetuximab (cetuximab), ABX-EGF and EMD72000. These drugs ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886
Inventor 汤世博
Owner FULONG MEDICAL MATERIALS CHINA