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

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. 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. The final signal is monitored by a toehold-mediated strand exchange reaction (one-step strand displacement probe), which is able to minimize background noise. We hope this strategy can provide a highly selective and sensitive platform for miRNA detection. • A novel method of reverse transcription-based miRNA detection has been introduced. • It is based on the dumbbell-shaped amplicons and inner primers used in LAMP. • It has been termed as “terminal hairpin formation and self-priming” (THSP) reaction. • Self-folding efficiency was improved by phosphorothioated modification.