1.3.3 CRISPR/CAST Package reaction
RPA reaction system (50 μl) : 2.4 μl of RPA
forward-and-reverse-direction primers F and R (10 μM), 29.5 μl of
primer-free rehydration buffer, 2.5 μl of MgOAc (280 mM), 1 μl of target
DNA, RNase-free water was replenished to 50 μl, place it in a 39°C
temperature box for the reaction for 15min. Cas12a digestion reaction
system (25 μl): 3 μl of 10 × LbCas12a buffer, 1 μl of LbCas12a (500
pmol), 1 μl of crRNA (10 μM), 0.5 μl of RNase inhibitor (5U/ μl), 0.5 μl
of szt-Probe (yg-Probe) (10 μM), The RPA amplified product, 3 μl,
refilling to 25 μl with RNase-free water; The prepared reaction tube was
centrifuged at 12,000 r/min for several seconds, at 42°C temperature for
10min. Test strip detection: transferred to a safe area after the Cas12a
cleavage reaction, makeup to 50 μl with RNase-free water, and insert the
test strip below the liquid level. After the test strip is infiltrated
completely, take out the test strip and observe the result within 10min
with naked eyes.