Stimulation of Cas12a produced trans-cleavage activity toward a non-specific methylene blue labeled DNA hairpin probe on the electrode surface. coli O157:H7 and complex formation by CRISPR/Cas12a and crRNA, respectively. coli O157:H7-specific aptamers and target repeated sequences, which allowed recognition and binding of E. Based on a sandwich-type immunoassay on magnetic beads, immuno-RCA generated long single-stranded DNA with abundant E. Herein, an electrochemical biosensor based on CRISPR/Cas12a combined with immuno-rolling circle amplification (immuno-RCA) was developed for detecting the pathogenic bacterium, Escherichia coli O157:H7. Rapid, simple, and sensitive detection of food-borne pathogenic bacteria is extremely important for preventing and controlling food-borne diseases. HPV Types with weak amplification by the primer pool are shown by box. Dots indicate the presence of nucleotides identical to the top sequence (GP6+). HPV genotypes are specified by numbers on the left. (E) Nucleotide sequence alignments of GP6+ (positions 5879 to 5903 according to HPV16 sequence, Genbank accession number NC_001526.4) to 13 HR-HPV genotypes. The RPA reaction was performed at 37 ☌ for 20 min and followed by Cas12a detection at 37 ☌ for up to 60 min. (D) The PGMY/GP6+ pool was tested using 10,000 copies of the indicated HPV plasmids for each RPA reaction.

For a single reaction, 5 μg of each individual HPV plasmid was used. (C) Real-time fluorescence detection of HPVs with the crRNA pool containing 11 crRNAs. (B) Plasmids with different copy numbers of HPV16 and HPV18 were amplified by RPA reaction using the PGMY/GP6+ primer pool.

(A) Schematic showing the relative position of the primer sets locating in the conservative L1 region of HPV.

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