LAMP rapid detection method for magnaporthe oryzae in rice seeds and application
A rice seed and detection method technology, which is applied in the field of crop molecular genetics and breeding, can solve problems such as deadness and rice seedling disease spots, and achieve the effects of improving detection sensitivity, high detection sensitivity, and saving detection costs
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0034] (1) Materials and methods:
[0035] 1. Rice blast fungus strains; 9 groups of rice seeds produced by diseased valleys, 9 groups of rice seeds to be tested.
[0036] 2. DNA extraction method: NaOH solution was used to extract the genomic DNA of 9 groups of diseased grain rice seeds infected with rice blast race 2016-5A47 and the genomic DNA of 9 groups of rice seeds to be tested.
[0037] 3. In system A, a total of 10 tubes are set. Tube 1 is the negative control for adding water, tubes 2-10 are the genomic DNA templates for adding 9 groups of diseased rice seeds; in system B, a total of 10 tubes are set, 1 Tube No. 2-10 is the negative control for adding water, tube No. 2-10 is for adding 9 groups of rice seed genomic DNA templates to be tested.
[0038] 4. The LAMP reaction system in each tube is as follows: the volume is 25 μL, of which 20 μM FIP and BIP primers are 2 μL, 10 μM F3 and B3 primers are 0.5 μL, 10 μM LF primers are 1 μL, 10 mM dNTPs 3.5 μL, 100 mM MgSO ...
Embodiment 2
[0043] (1) Materials and methods:
[0044] 1. Rice blast fungus strain.
[0045] 2. DNA extraction method: The genomic DNA of the oryzae oryzae strain was extracted by CTAB method.
[0046] 3. Dilute the DNA of M. oryzae race 2016-5A47 by concentration gradient, and dilute to 100ng / μL, 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL respectively.
[0047] LAMP reaction system: volume of 25 μL, including 20 μM FIP and BIP primers 2 μL, 10 μM F3 and B3 primers 0.5 μL, 10 μM LF primers 1 μL, 10 mM dNTPs 3.5 μL, 100 mM MgSO 4 1μL, 2.5μL of 10×Isothermal Amplification Buffer, 4μL of 5M Betain solution, 2μL of 2mM HNB, 1μL of DNA template, 1μL of 8U / μL Bst DNA polymerase, added to the test tube in sequence, and added ddH 2 O to 25 μL and mix.
[0048] The reaction conditions were: under airtight conditions, constant temperature amplification at 64°C for 50min. Judging the reaction result according to the color change, if the reaction solution changes from violet to blue, it proves tha...
Embodiment 3
[0054] (1) Materials and methods:
[0055] 1. Rice blast fungus strains; Epichlo endophytes, Fusarium graminearum, Fusarium fujikuroi, Trichoderma xanthophyllum, Rhizoctonia graminis ), Ustilaginoideavirens uv-p1 (Ustilaginoideavirens uv-p1), Rhizoctonia zeae, Penicillium fungi, Alternaria fungi.
[0056] 2. DNA extraction method: The genomic DNA of each strain was extracted by CTAB method.
[0057] 3. LAMP reaction system: the volume is 25 μL, of which 20 μM FIP and BIP primers are 2 μL, 10 μM F3 and B3 primers are 0.5 μL, 10 μM LF primers are 1 μL, 10 mM dNTPs 3.5 μL, 100 mM MgSO 4 1μL, 2.5μL of 10×Isothermal Amplification Buffer, 4μL of 5M Betain solution, 2μL of 2mM HNB, 1μL of DNA template, 1μL of 8U / μL Bst DNA polymerase, added to the test tube in sequence, and added ddH 2 O to 25 μL and mix.
[0058] The reaction conditions were: under airtight conditions, constant temperature amplification at 64°C for 50min. The reaction results were judged according to the color ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More