(An) ultrasensitive PDGF-BB assay based on the aptamer-mediated in vitro transcription linked with CRISPR/Cas13 system-based signaling

Abstract

We herein developed an ultrasensitive PDGF-BB assay based on the aptamer-mediated In vitro transcription linked with CRISPR/Cas13 system-based signaling. In principle, this method employs hairpin aptamers to detect PDGF-BB. In the presence of PDGF-BB, the 3' end of the hairpin aptamer is opened and amplified by the complementary Guide Strand (GS) and reverse transcriptase (RTase). In GS, there is a sequence complementary to the nicking site, and after amplification, a large number of single strands are generated by the nicking enzyme (NE). RNAs complementary to crRNA are generated via In vitro transcription reaction, using T7 RNA polymerase and primers having the T7 promoter and complementary to single strands. Then introducing CRISPR/Cas13 system, trans-cleavage activity cleaves the reporter probe and emits a fluorescent signal. With this method, we successfully detected PDGF-BB down to 190 fM, with high selectivity against other proteins. Furthermore, we successfully demonstrated the practical applicability of this sensing strategy by reliably detecting PDGF-BB in the human serum.