Ultrasensitive isothermal method to detect microRNA based on target-induced chain amplification reaction

We herein describe an ultrasensitive isothermal method to detect microRNA (miRNA) by utilizing target-induced chain amplification reaction (CAR). The hairpin probe (HP) employed in this strategy is designed to be opened upon binding to target miRNA. The exponential amplification reaction (EXPAR) template (ET) then binds to the exposed stem of HP and DNA polymerase (DP) promotes the extension reactions for both HP and ET, consequently producing intermediate double-stranded DNA product (IP) and concomitantly recycling target miRNA to open another intact HP. The IPs would produce a large number of target-mimicking probes (TMPs) and trigger probes (TPs) through the continuously repeated nicking and extension reactions at the two separated nicking sites within the IP. TMP triggers another CAR cycle by binding to intact HP as target miRNA did while TP promotes conventional EXPAR by independently binding to free ET. As a consequence of these interconnected reaction systems, a large number of final double-stranded DNA products (FPs) are produced, which can be monitored by measuring the fluorescent signal produced from duplex-specific fluorescent dye. Based on this unique design principle, the target miRNA was successfully determined down to even a single copy with high selectivity against non-specific miRNAs. The practical applicability of this method was also verified by reliably detecting target miRNA included in the total RNA extracted from the human cancer cell.