Primer, probes, PCR reaction liquid and kit for human EGFR (epidermal growth factor receptor) gene T790M site detection

A reaction solution and kit technology, applied in the field of molecular biology, can solve problems such as poor sensitivity, and achieve the effects of reducing strength, increasing specificity and sensitivity, and improving stability and affinity

Inactive Publication Date: 2018-11-02
GUANGZHOU FOREVERGEN BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Sanger sequencing, high-throughput sequencing, and ARMS-PCR detection are all qualitative detection, and the sensitivity is relatively poor

Method used

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  • Primer, probes, PCR reaction liquid and kit for human EGFR (epidermal growth factor receptor) gene T790M site detection
  • Primer, probes, PCR reaction liquid and kit for human EGFR (epidermal growth factor receptor) gene T790M site detection
  • Primer, probes, PCR reaction liquid and kit for human EGFR (epidermal growth factor receptor) gene T790M site detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0047] Example 2 Probe

[0048] A kind of embodiment of the probe of the human EGFR gene T790M site detection of the present invention, the probe described in this embodiment consists of the mutant probe (combined with the mutant DNA template) and the wild type probe for the human EGFR gene T790M site. Needle (binding to wild-type DNA template) composition. The mutant probe is Mut probe1: 5'-FAM-AGCTGC+ATGA+TGAGC-MGB-NFQ-3' ("+N" indicates that the base is modified by LNA) (as shown in SEQ ID NO:3) ; The wild-type probe is Wt probe1: 5'-VIC-AGCTGC+GTGATGAGC-MGB-NFQ-3' ("+N" indicates that the base is modified by LNA) (as shown in SEQ ID NO: 4). That is: the 5' end of the mutant probe is marked with FAM, the 3' end is marked with a non-fluorescence quencher group NFQ, and at the same time it is connected with a MGB modification group, and the 7th and 11th bases of the mutant probe are carried out. Locked nucleic acid modification; the 5' end of the wild-type probe is marked w...

Embodiment 3

[0050] Embodiment 3PCR reaction solution and kit

[0051] An embodiment of the PCR reaction solution for detecting the T790M site of the human EGFR gene of the present invention, the PCR reaction solution described in this embodiment is shown in Table 1. Among them, the PCR master mix is ​​MicroDrop from Guangdong Shunde Yongnuo Biotechnology Co., Ltd. TM The PCR premix Master Mix provided with the digital PCR instrument; the forward primer and reverse primer are as shown in Example 1; the mutant probe and wild-type probe are as shown in Example 2.

[0052] Table 1

[0053] Master Mix

10ul

Forward primer (20μm / L)

1.8μl

Reverse primer (20μm / L)

1.8μl

Mutant probe (10μm / L)

0.5μl

Wild type probe (10μm / L)

0.5μl

wxya 2 o

3.4μl

total capacity

18μl

[0054] During the experiment, the PCR reaction solution in Table 1 was mixed with 2 μl of DNA template to obtain a 20 μl PCR-specific amplification rea...

Embodiment 4

[0057] In this embodiment, the PCR reaction solution / kit described in Example 3 is used to detect the T790M site of the human EGFR gene. The specific detection method is:

[0058] (1) Genomic DNA extraction: Use Qiagen DNeasy Blood & Tissue Kit to extract normal peripheral blood genomic DNA and H1975 cell line DNA according to the kit instructions. The obtained genomic DNA was detected by NanoDrop 100 for DNA purity, gel electrophoresis for DNA integrity, qubit3.0 fluorometer for quantification, and DNA concentration ranged from 20 ng / μl to 50 ng / μl.

[0059] (2) The preparation of the PCR reaction solution for EGFR gene T790M mutation droplet digital PCR detection is as follows: PCR master mix 10 μl, 20 μm / L upstream primer 1.8 μl, 20 μm / L downstream primer 1.8 μl, 10 μm / L mutant probe 0.5μl, 10μm / L wild-type probe 0.5μl, ddH 2 O 3.4 μl, a total of 18 μl for one reaction.

[0060] (3) Preparation of PCR amplification system for EGFR gene T790M mutation droplet digital PCR d...

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PUM

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Abstract

The invention relates to a primer, probes, a PCR reaction liquid and a kit for human EGFR (epidermal growth factor receptor) gene T790M site detection and belongs to the field of molecular biology. The primer has the nucleotide sequences shown as SEQ ID NO:1 and SEQ ID NO:2. The probes comprise a mutant probe and a wild probe for a human EGFR gene T790M site, the mutant probe has the nucleotide sequence shown as SEQ ID NO:3, and the wild probe has the nucleotide sequence shown as SEQ ID NO:4; different fluorescent report genes are marked at 5' terminals of the mutant probe and the wild probe,a non-fluorescent quencher is marked at 3' terminals, and the 3' terminals are connected with MGB modification genes; at least one basic group of the mutant probe and the wild probe is modified with locked nucleic acid. The amplification specificity and the sensitivity of the primer are higher. The probe is an LNA modified TaqMan MGB (minor groove binder) probe, and an MGB can increase the Tm value of the probe by about 10 DEG C; by means of an LNA modified probe, the stability and the affinity of DNA molecules in a PCR (polymerase chain reaction) are improved, and the specificity and the sensitivity are increased.

Description

technical field [0001] The invention relates to a primer, a probe, a PCR reaction solution and a kit for detecting the T790M site of human EGFR gene, belonging to the field of molecular biology. Background technique [0002] Lung cancer is a general term for various lung malignancies. It is divided into two types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Non-small cell lung cancer accounts for about all lung cancer cases. More than 80% of the total, including two categories: 1) non-squamous carcinoma (including adenocarcinoma, large cell carcinoma and other subtypes); 2) squamous cell (epidermoid) carcinoma. Lung cancer is one of the malignant tumors with the highest morbidity and mortality in the world, ranking first among the causes of malignant tumor death in urban population in my country. [0003] The treatment of malignant tumors includes chemotherapy and targeted therapy. Traditional tumor chemotherapy has the characteristics of unclear t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/156
Inventor 赖炳权许少飞李伟琴罗景燕苏普霞李祎予
Owner GUANGZHOU FOREVERGEN BIOTECH CO LTD
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