Primer and probe assembly and kit for detecting EGFR mutation sites as well as application of primer and probe assembly or kit

A technology of primer probes and mutation sites, which is applied in the fields of molecular biology and tumor detection, and can solve problems such as the inability to meet clinical needs.

Inactive Publication Date: 2018-10-12
PILOT GENE TECH HANGZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is heterogeneity in the molecular abnormalities of tumor tissues. Tumor patients with higher EGFR mutation "abundance"

Method used

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  • Primer and probe assembly and kit for detecting EGFR mutation sites as well as application of primer and probe assembly or kit
  • Primer and probe assembly and kit for detecting EGFR mutation sites as well as application of primer and probe assembly or kit
  • Primer and probe assembly and kit for detecting EGFR mutation sites as well as application of primer and probe assembly or kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0163] 1. The plasmid constructed by genetic engineering is used as the positive template, and the nucleic acid of the negative plasma sample is used as the negative control. The kit of the present invention contains mixed solution 1 (20×), mixed solution 2 (20×), mixed solution 3 (20×) and PCR premixed solution 4 (2×).

[0164] 2. System configuration:

[0165] Droplet digital PCR reaction system: 15ul, the system is as follows:

[0166] Reaction chamber 1, detection of L858R, L861Q, S768I mutation system:

[0167] 2 x master mix 4

7.5uL

20×mixture 1

0.75uL

Positive plasmids (10 each 4 copies / uL, 3 mixes)

1uL

Internal reference plasmid (10 -4 copy / uL)

0.5uL

wxya 2 o

2.25uL

total

15uL

[0168] Reaction chamber two, detection of T790M, G719X, 20ins mutation system:

[0169]

[0170]

[0171] Reaction chamber three, detection of 19Del mutation system:

[0172] 2 x master mix 4

7...

Embodiment 2

[0185] Embodiment 2. Sensitivity detection

[0186] For EGFR mutation detection, EGFR L858R, EGFR L861Q, EGFR T790M, EGFR S768I, EGFR G719X (G719S, G719A, G719C), EGFR 20ins (3 types), EGFR 19Del (19 types) and internal control plasmids were diluted, and the concentration gradient after dilution was sequential For: 10 5 、10 4 、10 3 、10 2 、10 1 、10 0 Copy, set 3 parallel repetitions for each gradient, and conduct sensitivity experiments.

[0187] The system configuration, droplet generation method, mutation site combination, amplification program and fluorescence reading method are the same as in Example 1. As a result, each mutation site can detect no more than 10 copies of the template at a minimum.

Embodiment 3

[0188] Example 3. Detection of the lowest mutation rate

[0189] The positive plasmids of each site were mixed with the negative plasma DNA samples to prepare nucleic acid samples with mutation rates of 0.05%, 0.1%, 0.5%, 1%, 5%, and 10% at 2ng / μL DNA concentration to detect the lowest mutation rate. The system configuration, droplet generation method, mutation site combination, amplification program and fluorescence reading method are the same as in Example 1.

[0190] As a result, the lowest detection limit of each site was less than or equal to 0.2% (when the total input DNA was 15ng, there were about 10 positive sites). The experiment was repeated 10 times, and all mutated genes were detected under the 0.2% mutation ratio of each site of EGFR. That is, this kit can detect 0.2% of EGFR gene mutations.

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PUM

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Abstract

The invention provides a primer and probe assembly for detecting specific mutation on 18, 19, 20 and 21 exons of EGFR genes, a kit which comprises the primer and probe assembly and is used for detecting EGFR mutation sites through multiple droplet digital PCR as well as an application and a use method of the primer and probe assembly or the kit. The primer and probe assembly or the kit can performhigh-throughput detection on 29 mutation sites of the EGFR genes, has high specificity, high sensitivity and good stability, has great significance in mutation detection of EGFR genes of NSCLC (non-small cell lung cancer) and can dynamically guide medication for patients.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to the field of tumor detection, and more specifically to a combination of primers and probes for detecting EGFR mutation sites, a kit and its application and usage method. Background technique [0002] Non-small cell lung cancer (NSCLC) accounts for about 80% of lung cancers, and most NSCLCs are in the advanced stage at the time of diagnosis. Targeted therapy is the standard treatment for patients with EGFR sensitive mutation genes . There is heterogeneity in the molecular abnormalities of tumor tissues, and tumor patients with higher EGFR mutation "abundance" may benefit more from TKIs. Only qualitative analysis of mutations to guide targeted therapy can no longer meet clinical needs. [0003] With the emergence of digital PCR (digital PCR, dPCR), next generation sequencing (next generation sequencing, NGS) and other methods, quantitative detection of EGFR mutations has b...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2537/143C12Q2563/159C12Q2545/101C12Q2545/113
Inventor 位玉玲宋小慧朱海涛
Owner PILOT GENE TECH HANGZHOU CO LTD
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