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. 2022 May 13;13:811768. doi: 10.3389/fmicb.2022.811768

FIGURE 1.

FIGURE 1

Feasibility of the ERA/CRISPR–Cas12a system. (A) ERA/CRISPR Cas12a dual-system framework. The DNA of M. pneumoniae samples was extracted within 25 minutes, and the target sequence was amplified by ERA (15 minutes). The LbCas12a-gRNA complex recognized the amplification product and triggered the “collateral cleavage” activity, which cleaved ssDNA reporters (15–20 min). Qualitative analysis can be carried out by observing the fluorescence signal or chromatographic dipstick. T: test line; C: control line. (B) The selected sequence was the target fragment of the M. pneumoniae adhesion P1 sequence in this study. F was the forward primer of the ERA/CRISPR–Cas12a dual system, R was the reverse primer, PAM was the protospacer adjacent motif of the CRISPR array, and the immediately followed scribed fragment was the reverse complementary fragment of gRNA, which was the target sequence. (C) Real-time detection of Cas12a trans-cleavage activity using an F-Q reporter. NTC: no-template control. (D) M. pneumoniae fluorescence intensity compared with NTC within 10 min of reaction.