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Primer probe and kit for detecting EGFR (epidermal growth factor receptor) gene L858R mutation and application of primer probe and kit

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 of next-generation sequencing technology, difficulty in meeting actual clinical needs, and high operation requirements, so as to shorten detection time, The effect of short detection time and complex operation procedures

Active Publication Date: 2021-09-07
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 and kit for detecting EGFR (epidermal growth factor receptor) gene L858R mutation and application of primer probe and kit
  • Primer probe and kit for detecting EGFR (epidermal growth factor receptor) gene L858R mutation and application of primer probe and kit
  • Primer probe and kit for detecting EGFR (epidermal growth factor receptor) gene L858R mutation and application of primer probe and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] 1. Detection of EGFR gene L858R primers and probes

[0054] Different from conventional RPA amplification primers, this example is RPA amplification based on 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 opposite primer Extend to form a double strand, and only two rounds of amplification are required to form a double-tailed DNA amplification product during RPA amplification (see figure 1 ), and then the double-tailed amplification product will be used as the template for the next round of amplification to exponentially amplify to obtain more double-tailed DNA products.

[0055] In this example, 3 pairs of primers were designed for the conserved region of the EGFR gene L858R. According to the isothermal amplification process of the present invention, after isothermal amplification of all the primers, 10 μL of the prod...

Embodiment 2

[0066] A non-diagnostic method for detecting the EGFR gene L858R mutation according to the kit, the specific steps are as follows:

[0067] (1) Extract ctDNA from the blood sample to be tested;

[0068](2) according to the primers, use ctDNA as a template to perform RPA amplification;

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

[0070] (4) Add 10μLA liquid into each dry powder tube, which is the reaction tube;

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

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

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

[0074] (8) After mixing, shake (or quickly centrifuge) the reaction solution to the bottom of the tube, then immediately put the reaction tube into a constant temperature reaction tank and incubate at 37°C for 30 minutes;

[0075] (9) After the reaction, oscillate and mix the liquid in the reaction tube, tak...

Embodiment 3

[0079] The sensitivity of the above-mentioned method for detecting the L858R mutation of the EGFR gene was tested, and a mutant standard product (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.) was used for sensitivity testing.

[0080] Depend on 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 the mutant standard were added to the wild-type standard (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.) and amplified for detection. It was found that the detection sensitivity of this method can reach 0.78%. , where there is a slight background signal caused by negative spikes (see Figure 8 ).

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Abstract

The invention provides a primer probe for detecting L858R mutation of an EGFR (epidermal growth factor receptor) gene, and belongs to the technical field of gene detection. An upstream primer sequence of the L858R mutation is as shown in SEQ ID NO: 1; a downstream primer sequence of the L858R mutation is as shown in SEQ ID NO: 2; and the L858R mutation probe is a gold-labeled probe, and the sequence of the L858R mutation probe is as shown in SEQ ID NO: 3. The primer probe used in the invention has high specificity, accuracy and sensitivity. According to the primer probe, an RPA amplification technology is combined with a lateral flow chromatography test strip to detect the EGFR gene L858R mutation for the first time, the detection time is short, the operation is simple and convenient, and the cost is low.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a primer probe for detection of EGFR gene L858R mutation, a reagent strip and an application thereof. Background technique [0002] Epidermal growth factor receptor (EGFR) is the expression product of proto-oncogene, located on the cell membrane, and is a transmembrane receptor tyrosine kinase. EGFR gene mutations are mainly concentrated in exons 18-21 of the tyrosine kinase region (TKI), of which exon 21 point mutations account for about 40-45% of all mutations, and exon 21 point mutations are mainly A T-G transition occurs at codon 858, causing the amino acid at this position in the protein to change from leucine to arginine, namely 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 their inabili...

Claims

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Application Information

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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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