Transgenic oilseed plant GT73 detecting method based on annular mediation isothermal amplification extinction technology
A technology of GT73-F3 and GT73-B3, which is applied in the detection field of transgenic rapeseed GT73, can solve problems such as cross-contamination, and achieve the effect of fast response, fast response and high specificity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0042] Example 1 Design and Synthesis of LAMP Primers Used to Detect Transgenic Rapeseed GT73
[0043] Primers were designed for the partial region (Seq ID No: 5) of the transgenic rapeseed GT73 exogenous gene Epsps through the loop-mediated primer online design software PrimerExplorerV5 (https: / / primerexplorer.jp / lampv5 / index.html) of Rongyan Co., Ltd., Japan, The primers were synthesized by Shanghai Yingwei Jieji Co., Ltd., and the primers were diluted to 10 μmol / L. The primer sequences are as follows:
[0044] Outer forward primer GT73-F3: 5'-CTGGGAGGGAGGGAGGGCGACGTCACCATCCTTAACG-3;'
[0045] Outer reverse primer GT73-B3: 5'-CTGGGAGGGAGGGAGGGCAGCTGCAACAGCGAGAA-3;' and
[0046] Inner forward primer GT73-FIP: 5'-AGCAAGACGTGGGTTGATCACT CTGGGAGGGAGGGAGGGCCAACCCGTACTGGTCTCA-3;'
[0047] Inner reverse primer GT73-BIP: 5'-GGAGAAGACGTGGCTGACTTGC CTGGGAGGGAGGGAGGGGAAGGAGCACGGTCTTCTGG-3'.
[0048] Compared with ordinary LAMP primers, the inner primers of the present invention are...
Embodiment 2
[0050] Example 2 Method for detecting transgenic rapeseed GT73 based on ring-mediated isothermal amplification extinction technology
[0051] 1. Experimental method
[0052] ①Collect transgenic rapeseed GT73 seeds, grind the sample into powder in liquid nitrogen, weigh 100mg±10mg sample, extract the genome by CTAB method, use it as a DNA template, and dilute the template to 50ng / μL;
[0053] ② Color reaction: mix the hemin working solution with the LAMP primer combination in Example 1, incubate at 40°C for 30 minutes, then add ABTS color solution and H 2 o 2 , Immediately observe the color with the naked eye or use a UV spectrophotometer to measure the OD 419nm value;
[0054] The reaction system for color reaction is: 90 μL of 50 μM hemin working solution, 0.5 μL of 10 μM primers GT73-F3 and GT73-B3, 4 μL of 10 μM primers GT73-FIP and GT73-BIP, 30 μL of 36 mM ABTS chromogenic solution, 30% H 2 o 2 1 μL with ddH 2 O was made up to 130 μL of the total system; among them,...
Embodiment 3
[0060] Example 3 Ring-mediated isothermal amplification extinction reaction system and optimization of reaction conditions
[0061] In order to ensure the best color development system, the present invention optimizes the following reaction factors.
[0062] 1. Optimization of color development time
[0063] Prepare reagents: 10 μM primers GT73-F3, GT73-B3, GT73-FIP and GT73-BIP, 40 μM hemin working solution, 30 mM ABTS chromogenic solution, 30% H 2 o 2 .
[0064] Experimental method: Add 0.5 μL of primers GT73-F3 and GT73-B3 and 4 μL of primers GT73-FIP and GT73-BIP to 90 μL of hemin working solution, and incubate at 35°C for 20 minutes. Then add 30 μL ABTS chromogenic solution and 1 μL H 2 o 2 , using a UV spectrophotometer to measure the OD of the resulting reaction mixture at 0s, 30s, 1min, 2min, 5min, 10min, 30min 419nm value. Negative control does not add primers, use ddH 2 O alternative. According to the results in Table 1, it can be seen that with the prolonga...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More