The invention relates to the technical field of
nucleic acid detection, and discloses a construction method of a PFLAMP-Cas12b one-tube method
reaction system based on single-stranded
stem loop mediation. The construction method comprises the following steps: designing an LAMP amplification primer; designing an sgRNA sequence of a single-stranded loop region in a targeted LAMP amplification product; and constructing a
reaction system containing the LAMP amplification primer, the Cas12b
protein, the sgRNA sequence and the
fluorescence report probe. A single-chain stem-loop structure formed in an LAMP amplification product is used as a specific activation target of the Cas12b
protein, PAM dependence of the Cas12b
protein on double-chain
DNA targeting in traditional detection is avoided, and the
bottleneck that sgRNA design is limited due to lack of natural PAM sites in a highly conserved
gene area is effectively overcome; and non-specific background amplification interference induced by artificially introducing a PAM sequence into the primer is eliminated, and the detection accuracy is improved.