Loop-mediated isothermal amplification (LAMP) primer combination, LAMP kit and LAMP method for detecting trichothecenes
A combination technology of trichothecenes and primers, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problem of unrelated reporting of trichothecenes, To achieve the effect of ensuring food safety and food safety, expanding the scope of detection, and improving efficiency
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Embodiment 1
[0045] Implementation example 1: specificity experiment of trichothecene toxin LAMP reaction
[0046] In order to verify the specificity of the LAMP method, 27 species of Fusarium were used as test objects, of which 18 species produced trichothecenes, 7 species produced fumonisins and other toxins, and 2 species did not produce toxins (Table 1).
[0047] Template DNA extraction: pick a small amount of hyphae on the surface of the sample with a sterilized toothpick or tip, put it into a PCR tube, add 50 μL Buffer A solution (100mM NaOH, 2% 20), incubate at 95°C for 10 minutes. Add 50 μL of BufferB solution (100 mM Tris-HCl, 2 mM EDTA), shake and mix, centrifuge at 12000 rpm for 15 s, and take 1 μL of the supernatant as a template for LAMP amplification.
[0048] LAMP reaction: Take 1 μL of DNA solution, add 5 μL of reaction solution, 5 μL of primer set mixture, and 14 μL of sterilized ultrapure water for LAMP reaction. The reaction program is 65°C, the time is 45 minutes, an...
Embodiment 2
[0050] Implementation example 2: Sensitivity experiment of trichothecene toxin LAMP reaction
[0051] In order to verify the sensitivity of the LAMP method, extract the trichothecene-positive Fusarium strain NRRL5883 (Table 1), and use a spectrophotometer to measure the DNA concentration (100 ng / μL), and perform 10-fold serial dilution with ultrapure water, Store at -20°C as a template. Take 1 μL of DNA solution of each concentration as a template, add 5 μL of reaction solution, 5 μL of primer group mixture, and 14 μL of sterilized ultrapure water for LAMP reaction. The reaction program is 65 ° C, the time is 45 min, and the color change of the amplified product is observed. . the result shows( figure 2 ), the reaction tubes added with 100ng, 10ng, 1ng, 100pg, and 10pg DNA were obviously sky blue, while the reaction tubes added with 1pg DNA were purple, indicating that the sensitivity of the LAMP reaction reached 10pg genomic DNA.
Embodiment 3
[0052] Example 3: Detection of trichothecenes in harvested wheat
[0053] Template DNA extraction: samples to be tested are harvested from 6 provinces of Jiangsu, Anhui, Zhejiang, Hebei, Henan, and Shandong
[0054] wheat. Add the wheat sample to be tested into a 2mL centrifuge tube, and add steel balls with a diameter of 4mm at the same time, freeze it in liquid nitrogen and grind it with a Biospec grinder at high speed for 1min, then add 200μL Buffer A solution (100mM NaOH, 2% 20), incubate at 95°C for 10 minutes. Add 200 μL of BufferB solution (100 mM Tris-HCl, 2 mM EDTA), shake and mix, centrifuge at 12000 rpm for 30 s, and take the supernatant as a template for LAMP amplification.
[0055] LAMP reaction: Take 1 μL of DNA solution, add 5 μL of reaction solution, 5 μL of primer set mixture, and 14 μL of sterilized ultrapure water for LAMP reaction. The reaction program is 65°C, the time is 45 minutes, and the color change of the amplified product is observed.
[0056] T...
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