Molecular identification method for xiphinama brevicolle
A nematode and detection method technology, applied in the field of quarantine, can solve the problems such as the difficulty of molecular classification and identification, and achieve the effect of low detection limit and good specificity
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[0042] Below in conjunction with embodiment, further illustrate the present invention.
[0043] Prepare the extract: the extract contains 2.5 mmol / L of DTT, 1.125% Tween 20, 0.025% gelatin, 2.5 times of PCR buffer, and the balance is water. The PCR buffer contains 125 mmol / L KCl, 25 mmol / L Tris-HCl at pH=8.3, 3.75 mmol / L MgCl 2 , the balance is water;
[0044] Pre-cool the prepared extract to 4°C, and drop it onto the glass slide, and place the nematodes to be tested in the extract;
[0045] Cut the nematode into segments, pipette 8 μL of the segmented nematode suspension into an Eppendorf tube containing 10 μL of sterile water, then add 2 μL of pre-cooled 2mg / mL proteinase K to the tube to make the total volume 20 μL, and quickly Place in -70°C freezer for more than 10 minutes;
[0046] The frozen Eppendorf tube was kept at 65°C for 60 min, 95°C for 10 min, and centrifuged at 12,000 g for 0.5 min to obtain a nematode DNA suspension.
[0047] The total volume of the real-t...
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