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Primer probe, kit and application for detection of egfr gene l858r mutation

A primer-probe and mutation detection technology, which is applied in the field of primer-probes for EGFR gene L858R mutation detection, can solve the problems of long detection cycle, difficulty in meeting actual clinical needs, and high operational requirements of second-generation sequencing technology, so as to shorten detection time, The effect of short detection time and complex operation procedures

Active Publication Date: 2022-06-07
ENFIN BIOTECH (JIANGSU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although next-generation sequencing technology and PCR-based methods are the "gold standard" for detecting ctDNA mutations, the shortcomings of these two types of technologies are also significant
The next-generation sequencing technology has a long detection period (2-3 weeks) and high cost, which is difficult to meet the actual clinical needs
The PCR-based method has high operational requirements and is prone to introduce sol contamination, resulting in false negative or false positive results
In addition, both methods require well-trained professionals as well as expensive equipment

Method used

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  • Primer probe, kit and application for detection of egfr gene l858r mutation
  • Primer probe, kit and application for detection of egfr gene l858r mutation
  • Primer probe, kit and application for detection of egfr gene l858r mutation

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Experimental program
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Effect test

Embodiment 1

[0053] 1. Detection of EGFR gene L858R primer and probe

[0054] Different from conventional RPA amplification primers, the present embodiment is based on the RPA amplification of tail primers, due to the modification of the C3 arm between the tail primer specific extension sequence and the detection sequence, it is ensured that the detection sequence will not be extended by the primers extended to form a double strand, and only two rounds of amplification need to be formed when the RPA amplification is carried out (see ). Figure 1 Subsequently, the amplification product of the two-tailed structure will be amplified exponentially as a template for the next round of amplification to obtain more two-tailed structure DNA products.

[0055] The present embodiment is designed for a conserved region of the EGFR gene L858R 3 pairs of primers, wherein in accordance with the isothermal amplification process of the present invention, after isothermal amplification of all primers, take 10 μL...

Embodiment 2

[0066] A method for non-diagnostic purposes to detect mutations in the EGFR gene L858R according to the kit, the specific steps are as follows:

[0067] (1) Extraction of ctDNA from the blood sample to be measured;

[0068] (2) RPA amplification is performed with ctDNA as a template according to the primers;

[0069] (3) Take out the required components of the kit 30min in advance, melt at room temperature, and mix well with shock;

[0070] (4) Add 10 μL of A liquid to each dry powder tube, that is, the reaction tube;

[0071] (5) Add 6 μL of C solution to each reaction tube separately;

[0072] (6) Add 2 μL of template to the reaction tube;

[0073] (7) Add 2 μL of B solution to the reaction tube and mix well;

[0074] (8) After mixing, the reaction solution is thrown (or quickly centrifuged) to the bottom of the tube, and then the reaction tube is immediately placed into the thermostatic reaction tank at 37 °C for 30 min;

[0075] (9) After the reaction is completed, the liquid i...

Embodiment 3

[0079] The sensitivity of the above method for detecting the L858R mutation of the EGFR gene was detected, and the mutant standard (purchased from Bioengineering (Shanghai) Co., Ltd.) was used for sensitivity detection.

[0080] composed Figure 7 It can be seen that after RPA isothermal amplification, the test strip is used for detection and the sensitivity can reach 600copies. In order to further verify the sensitivity of the detection method, different proportions of mutant standards were added to the wild-type standards (purchased from Bioengineering (Shanghai) Co., Ltd.) and amplified, and the detection sensitivity of the method was found to be up to 0.78%, of which there was a subtle background signal marked negative (see also). Figure 8 )。

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Abstract

The invention provides a primer probe for detecting the EGFR gene L858R mutation, which belongs to the technical field of gene detection. The upstream primer sequence of the L858R mutation is shown in SEQ ID NO: 1; the downstream primer sequence of the L858R mutation is shown in SEQ ID Shown in NO:2; the probe for the L858R mutation is a gold-labeled probe, and its sequence is shown in SEQ ID NO:3. The primer probe used in the present invention has high specificity, accuracy and sensitivity. The invention adopts the RPA amplification technology combined with the lateral flow chromatography test strip to detect the EGFR gene L858R mutation for the first time, and the detection time is short, the operation is simple and the cost is low.

Description

Technical field [0001] The present invention relates to the field of genetic detection technology, specifically to a primer probe for the detection of EGFR gene L858R mutations, a reagent strip and its application. Background [0002] The epidermal growth factor receptor (EGFR) is the expression product of the proto-oncogene, localized on the cell membrane, as a transmembrane receptor tyrosine kinase. EGFR gene mutations are mainly concentrated in the tyrosine kinase region (TKI) of the 18 ~ 21 exons, of which exon 21 point mutations, accounting for about 40 ~ 45% of all mutations, exon 21 point mutation is mainly located in the 858th codon T ~ G conversion, causing the amino acids in the protein to change from leucine to arginine, that is, L858R. [0003] At present, EGFR gene mutation detection is mainly divided into tissue biopsy technology and liquid biopsy technology. Tissue biopsy techniques are invasive, pose patient safety risks, and are inaccurate due to the inability to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/156C12Q2521/507C12Q2563/131C12Q2565/625
Inventor 刘国东董涛沈兵邱万伟钱立生张学记
Owner ENFIN BIOTECH (JIANGSU) CO LTD
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