DETECTR assay was performed as previously described,15 (link) including 200 ng Cas12a protein, 25 nM FAM-ssDNA-BHQ (Sangon, China), 1 μM crRNA, 2 μL sample, and 10x NEBuffer 2.1 in a 20 μL reaction. QPCR machine was used for recording fluorescence signal at 37°C (5′-FAM-ssDNA-BHQ-3′ reporter, λex: 485 nm; λem: 535 nm). LtCas12a-based all-in-one system combined the RAA reaction with Cas12a cleavage in a one-pot reaction. The 50 μL system consisted of 200 ng Cas12a protein, 25 nM FAM-ssDNA-BHQ (Sangon, China), 1 μM crRNA, 4 μL NEBuffer 2.1, 0.36 μM forward and reverse primers, and 1.06 μL of MgOAc.
Chen Y., Meng L., Lang B., Li L., Liu J., Wang Y., Huang Z., Tian X., Tian R, & Hu Z. (2023). A Cas12a ortholog with distinct TTNA PAM enables sensitive detection of HPV16/18. Cell Reports Methods, 3(4), 100444.
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