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.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
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.
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com