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Primers, probes and kit for detecting 19-Del mutation of epidermal growth factor receptor (EGFR) gene

A kit and gene technology, applied in the direction of biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of insufficient sensitivity, low sensitivity, and inability to meet clinical needs, so as to ensure sensitivity and specificity, Consistent detection sensitivity and improved amplification efficiency

Active Publication Date: 2019-11-12
HANGZHOU D A GENETIC ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some researchers in the early stage have developed a variety of 19-Del multiplex detection systems and methods based on ARMS, NGS and digital PCR technologies, but the systems and methods they developed have their limitations, and the methods based on NGS technology are relatively comprehensive in detecting mutations. , but the sensitivity is insufficient, the operation is complicated, difficult, and costly, which cannot meet clinical requirements; the sensitivity of ARMS technology is also low, and there are only a few to more than 20 detection sites, and there are many mutations that cannot be detected, resulting in missed detection It will bring serious consequences to the patient’s medication. The amplification block method is relatively harsh on the sample volume of clinical samples, and must be diluted to a certain concentration range. Too much sample volume will easily cause false positives, and too low will reduce its sensitivity and cause leakage. detection; ddPCR technology has high sensitivity and accurate quantification, but only 10-30 kinds of mutations can be detected in the current relevant literature and patents, covering 60-95% of the mutation incidence rate, and more than 1 / 3 of the types of mutations, about 5-15% of 19-Del mutation patients cannot be detected, and some missed detection cannot be avoided
Therefore, none of the existing products can effectively meet the current needs of clinical NSCLC patients for precise personalized medicine and companion diagnostics

Method used

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  • Primers, probes and kit for detecting 19-Del mutation of epidermal growth factor receptor (EGFR) gene
  • Primers, probes and kit for detecting 19-Del mutation of epidermal growth factor receptor (EGFR) gene
  • Primers, probes and kit for detecting 19-Del mutation of epidermal growth factor receptor (EGFR) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: The primers and probes for detecting 19-Del were synthesized by Bailige Biotechnology Co., Ltd., as shown in the following table:

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[0048]

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Embodiment 2

[0050] Example 2: Synthesis of positive mutant plasmids used to test each mutant primer. The mutant type that can be amplified with the same specific upstream primer only needs to synthesize one of the plasmids, and the insert sequence is designed and provided by Shanghai Sangon Bioengineering Co., Ltd. Synthesis, a total of 35 positive plasmids (pUC57 cloning plasmid + insert sequence), the specific primer positions match 53 common mutation types, covering more than 99% of the mutation incidence rate, as shown in the following table:

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[0052]

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[0055]

[0056] Note: *The mutation corresponding ID number and mutation incidence data come from the world's most comprehensive public tumor mutation database (https: / / cancer.sanger.ac.uk / cosmic)

Embodiment 3

[0057] Example 3: Preparation of EGFR gene 19-Del mutation detection kit.

[0058] (1) 2*supermix: The PCR reaction solution contains hot start DNA polymerase, Mg 2+ , PCR reaction buffer, dNTPmixture, betaine and tris(2-carboxyethyl)phosphine (TCEP), BSA, etc. Preferably, the concentration of hot-start DNA polymerase is 0.02-0.04U / μL, the concentration of Mg2+ is 7-8mM, the concentration of dNTP mixture is 500-1000μM, the concentration of betaine is 0.5-1M, and the concentration of TCEP is 0.5-1mM. Store at -20°C.

[0059] (2) Mutant primer-probe master mix (20*MUT): Dissolve the primers and probes shown in SEQ ID NO: 1-34 in double distilled water, the concentration of each primer is 100 μM, the concentration of the probe 10 μM, prepare 20*MUT premix; preferably, the concentration of primers 22 and 25 is 3-4 μM, the concentration of primers 1 and 32 is 7-8 μM, and the concentration of the remaining 29 primers is 1-2 μM, Probe No. 34 has a concentration of 4-6 μM and is st...

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Abstract

The invention discloses nucleic acid sequences and a kit for detecting 19-Del mutation of an epidermal growth factor receptor (EGFR) gene and belongs to the technical field of molecular diagnosis. Theprimers, the probes and the kit for detecting 19-Del mutation of the EGFR gene can detect 53 common mutation types (covering 99% of mutation occurrence rate or above) of EGFR-TKIs targeting drugs forclinical NSCLC patients with high sensitivity and high specificity. The invention also discloses the kit for detecting 19-Del mutation of the EGFR gene. The kit can realize quantitative detection ofmutation of 19-Del of the EGFR gene in one hole conveniently, quickly, sensitively, specifically and accurately, can be used as the reference basis of EGFR-TKIs targeting drugs and can be used for dynamically monitoring curative effects of drugs in real time.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a primer, a probe and a kit for detecting EGFR gene 19-Del mutation. Background technique [0002] In recent years, targeted drugs such as epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have become important in advanced non-small cell lung cancer (NSCLC). one of the treatment modalities. NCCN, ESMO, CSCO and other domestic and foreign tumor diagnosis and treatment and clinical practice guidelines recommend that all NSCLC patients with adenocarcinoma components must undergo EGFR gene mutation detection to guide the selection of EGFR-TKI targeted therapy. [0003] A large number of clinical studies have found that patients with non-small cell lung cancer cells carrying EGFR gene sensitive mutations, use EGFR tyrosine kinase inhibitors (EGFR-TKI) such as Iressa (gefitinib), Tarceva (erlotinib) Niger), compared with standard first-li...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q2563/159C12Q2563/107C12Q2525/117C12Q2545/113
Inventor 虞闰六任绪义
Owner HANGZHOU D A GENETIC ENG
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