Ultrasensitive detection of nucleic acid based on a novel isothermal amplification

Abstract

The present study describes a novel technology referred to as reciprocal two single-stranded DNA (ssDNA) probe-mediated isothermal amplification (TPIA) that enables target nucleic acid detection. TPIA extends the ssDNA probe A into ultralong ssDNA products with tandem repeats of hairpin and ssDNA probe B to form double-stranded DNA hairpins of varying lengths via continuously repeated polymerization and displacement. The entire TPIA process was supervised in real time with the assistance of fluorescence techniques. By exploiting this new mechanism, the system can be used as an autocephalous module to provide a versatile tool for a biosensing program. The ssDNA probe B was relationally designed to form a stem-loop structure (HP), which can only unwind through specific recognition by the target nucleic acid. The HP stem region was then exposed and HP was rearranged to serve as the ssDNA probe B in the TPIA system after HP unfolding. The novel design strategy was exploited to specifically assay different target microRNAs (miR-21 and miR-221) with a low limit of detection on the 10 and 1 fM level, respectively. This method has great potential for nucleic acid detection in biomedical research and clinical diagnosis by rationally redesigning the template.