Primers for detection of cucumber anthracnose pathogen lamp and its application
A cucumber anthrax detection primer technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of low accuracy, long time-consuming, long detection time, etc., to achieve strong specificity, Confident results with high specificity and sensitivity
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Embodiment 1
[0034] Example 1: Design of specific primer composition for the detection of cucumber anthracnose bacteria loop-mediated isothermal amplification (LAMP) and the specificity verification of the primers for cucumber anthracnose bacteria
[0035] 1. Extraction of genomic DNA of the tested strains
[0036] The genomic DNA of the tested strains (Table 1) was extracted by the CTAB method. The specific method is as follows: Take a small amount of mycelium powder in a 1.5mL centrifuge tube (it is better that the mycelium powder just covers the bottom of the semicircle), add 900 µL of 2% CTAB ( cetyltrimethylammonium bromide) extract (2% CTAB; 100 mmol / L Tris-HCl, pH 8.0; 20 mmol / L EDTA, pH 8.0; 1.4 mol / LNaCl) and 90 µL SDS (ten Sodium dialkylbenzene sulfonate) [Note: CTAB, SDS needs to be preheated at 60°C], use a shaker to shake and mix, 60°C water bath for 1h (DNA is released into the buffer), 12000 r min -1 Centrifuge for 15 min; take 700 µL of the supernatant, add an equal volume...
Embodiment 2
[0050] Example 2: Detection Sensitivity of Loop-Mediated Isothermal Amplification (LAMP) for Cucumber Anthracnose
[0051] 1. Preparation of genomic DNA at different concentrations
[0052] Genomic DNA of cucumber anthracnose bacteria was diluted with sterile ultrapure water, and prepared into a series concentration of 10 times order of magnitude for later use;
[0053] 2. Sensitivity determination and result observation of LAMP detection method
[0054] Genomic DNA of Cucumber anthracnose at different concentrations was used as a template, and primer combinations F3, B3, FIP and BIP were used for LAMP amplification. The LAMP detection reaction system was 25 μL, including 1.0 μL each of 5 μM F3 and B3, 40 μM FIP and BIP each 1.0 μL, LAMP reaction mixture 12.5 μL, 8 U Bst1.0 μL of polymerase, 1.0 μL of DNA templates with different concentrations, made up to 25 μL with sterilized ultrapure water; LAMP reaction conditions: incubate at 63.5°C for 60 min, inactivate at 85°C for ...
Embodiment 3
[0057] Example 3: LAMP detection of cucumber anthracnose bacteria in diseased leaves
[0058] Sample collection: Collect cucumber leaves with typical symptoms of anthracnose and healthy leaves from Fuzhou, Zhangzhou and Quanzhou in Fujian and bring them back to the laboratory for later use;
[0059] Fruit DNA extraction: DNA was extracted by NaOH rapid cracking method, the specific process is as follows: add 10µL 0.5 mol / L NaOH to each mg of plant tissue, fully grind the tissue into a paste in a mortar and transfer it to a 1.5mL centrifuge tube , centrifuge at 12,000 rpm for 6 min, take 5 µl of the supernatant and add 495 µL of 0.1 mol / L Tris-HCl (pH=8.0) to mix well, and take 1.0 µL as a PCR template for amplification.
[0060] Amplification detection and observation: Using the above-mentioned extracted DNA as a template, use primer combinations F3, B3, FIP and BIP for LAMP amplification. The LAMP detection reaction system is 25 μL, including 1.0 μL each of 5 μM F3 and B3, 40...
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