Pine wood nematode pcr detection kit and its detection method
A detection kit and technology of pine wood nematode, which is applied in the field of microbial detection, can solve the problems that it is difficult to meet the rapid quarantine requirements of forest plant quarantine checkpoints, the detection accuracy needs to be improved, and the detection specificity is not strong, and the results are objective and reliable. DNA Good extraction effect and high specificity
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Embodiment 1
[0058] The raw material of kit of the present invention and preparation:
[0059] 1) Tris: analytically pure, content 99.7%, infrared qualified, pH (5% water) 10.3-10.9, moisture 0.3%, melting point 167-171°C, qualified absorption system, qualified maximum impurity content.
[0060] 2) MgCl 2 : Analytical pure, the content is not less than 99%, the aqueous solution reaction is qualified, and the highest impurity content is qualified.
[0061] 3) EDTA: analytically pure, white crystalline powder, soluble in water, the solution is acidic, insoluble in alcohol, the content is not less than 99.5%, the aqueous solution reaction is qualified, the complexing force test is qualified, and the highest content of impurities is qualified.
[0062] 4) HCl: analytically pure.
[0063] 5) Purified water: pure water, treated by a pure water machine, with a resistivity of 18.2MΩ.
[0064] 6) Synthetic primers and probes:
[0065] Conserved sequence of pine xylophilus exotoxin A gene, that ...
Embodiment 2
[0099] 1. Reagent preparation:
[0100] 1. Take the kit out from -20°C and equilibrate at room temperature for 20 minutes.
[0101] 2. Centrifuge the hot-start Taq enzyme and UNG enzyme briefly, and put them at -20°C for later use.
[0102] 3. After determining the number n of reaction tubes to be carried out, take out the PCR reaction solution of pine wood nematode, add n×44.3 μl pine wood nematode PCR reaction solution, n×0.5 μl hot-start Taq enzyme and n×0.2 μl UNG enzyme to one Put it in a centrifuge tube and vortex to mix well. After instantaneous centrifugation, dispense 45 μl into each PCR reaction tube, cover the tube cap and store in a 4°C refrigerator in the dark for later use.
[0103] 4. Put the positive control, negative control, and internal reference in a 4°C refrigerator for later use.
[0104] 2. Processing of samples to be tested:
[0105] Applicable specimen type: wound swab
[0106] 1. Add 1ml of normal saline to the 1.5ml centrifuge tube, fully wash th...
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