Ligation-dependent Cas14a1-Activated biosensor for one-pot pathogen diagnostic.

ABSTRACT

Pathogen identification requires nucleic acid diagnosis with simple equipment and fast manipulation. Our work established an all-in-one strategy assay with excellent sensitivity and high specificity, Transcription-Amplified Cas14a1-Activated Signal Biosensor (TACAS), for the fluorescence-based bacterial RNA detection. The DNA as a promoter probe and a reporter probe directly ligated via SplintR ligase once specifically hybridized to the single-stranded target RNA sequence, with the ligation product transcribed into Cas14a1 RNA activators by T7 RNA polymerase. This forming sustained isothermal one-pot ligation-transcription cascade produced RNA activators constantly and enabled Cas14a1/sgRNA complex to generate fluorescence signal, thus leading to a sensitive detection limit of 1.52 CFU mL-1E. coli within 2 h of incubation time. TACAS was applied in contrived E. coli infected fish and milk samples, and a significant signal differentiation between positive (infected) and negative (uninfected) samples was reached. Meanwhile, E. coli colonization and transmit time in vivo were explored and the TACAS assay promoted the understanding of the infection mechanisms of the E. coli infection, demonstrating an excellent detection capability.