(A) novel target RNA detection utilizing nicking/extension chain reaction system-based isothermal amplification

Abstract

We herein developed a novel target RNA detection method called Nicking/Extension chain reaction System-Based isothermal Amplification (NESBA). In principle, the presence of target RNA induces formation of double-stranded (ds) DNA containing nicking sites and T7 promoter sequence which are utilized for nicking/extension chain reaction- and transcription-mediated isothermal amplification, respectively. As a result, RNA amplicons bind to the molecular beacons (MBs) leading to highly enhanced fluorescent signal. Based on this novel strategy, we successfully detected target genomic RNA (gRNA) derived from influenza A (H3N2) virus down to 3.6 ag/$\mu$L with high specificity over other gRNAs from different subtypes of influenza A virus.