Ascas12a

TwistAmp^@ Basic kit was purchased from TwistDx^™. The RPA primers, crRNA, AsCas12a, and fluorophore-quencher probes were all obtained from Integrated DNA Technologies, and detailed information about the synthetic oligonucleotides are listed in Table 2. The RPA primer sets were designed using PrimerQuest^™ Tool. Additionally, NEBuffer^™ r2.1 was purchased from New England Biolabs. The RPA reaction was conducted based on the instructions: A mixture of 29.5 μL of rehydration buffer, 11.2 μL of nuclease-free water, and 2.4 μL each of forward and reverse primers (10 μM) was added to the enzyme pellet. Then, 2 μL of purified DNA and 2.5 μL of MgOAc (280 mM) were added and mixed to achieve a total volume of 50 μL. The mixture was incubated at 37 °C for 20 min. Following the incubation, 2 μL of RPA amplicons were added to a pre-assembled CRISPR-Cas12a mixture comprising 50 nM of AsCas12a, 62.5 nM of crRNA , 10× buffer, and 2.5 μM of ssDNA-FQ probe, resulting in a final reaction volume of 20 μL. The reaction solution was incubated at 37 °C for 30 min. After the incubation, the mixture was excited by a blue light transilluminator (brand: SmartBlue, Part number: NEB-E4100, excitation wavelength of 465 nm) for naked-eye observation. Finally, 20 μL of nuclease-free water was added to 5 μL of the mixture, which was then characterized by an Agilent BioTek Cytation 5 imaging reader.