Reverse transcription-based loop-mediated isothermal amplification strategy for real-time miRNA detection with phosphorothioated probes

Abstract

A novel reverse transcription-based loop-mediated isothermal amplification (LAMP) strategy for miRNA detection has been developed. This method consists of two stem-loop probes inspired by the dumbbell-shaped amplicons and inner primers used in conventional LAMP reactions. Termed "terminal hairpin formation and self-priming" (THSP), this reaction incorporates phosphorothioated (PS) modifications to achieve DNA folding and extension without primers. The final signal is monitored by a sequence-specific detection probe, which minimizes the background noise. We suggest that our rapid, facile, and reliable LAMP method will be a promising candidate for detecting miRNA in biomedical applications.

Keywords: Loop-mediated isothermal amplification; Phosphorothioated modifications; Self-priming; Terminal hairpin; miRNA detection.