Strand displacement-based molecular probe for high-specificity detection of microRNA

Objective A new type of molecular probe design method was established to improve the sensitivity and specificity of microRNA detection. Methods This is an experimental study. The target hybridization sequence was designed on the stem side of the molecular beacon using the strand displacement principle and based on this, a new probe was designed by using the nucleic acid structure analysis software DNAman to optimize the secondary structure of the molecular probe, which was called as strand displacement molecular probe (MB-D) and MB-D plus. Taking microRNA-21 as an example, microRNA-21 and its related single nucleotide mutations were detected using conventional molecular probe (MB-C) and redesigned MBs (MB-D and MB-D plus) to analyze the differences on minimum detection limit, repeatability and specificity for microRNA detection among these three probes. Results The minimum detection limit of MB-C for microRNA-21 was 1 nmol/L, and the minimum detection limits for MB-D and MB-D plus were 0.1 nmol/L and 0.01 nmol/L, respectively. The established MB-D plus can significantly distinguish between miR-21 and single nucleotide mutations. Conclusion The molecular probe based on the principle of strand displacement and optimized by secondary structure can significantly increase the sensitivity and specificity of the probe for microRNA detection.(Chin J Lab Med, 2018, 41: 541-546) Key words: MicroRNAs; Molecular probes; Sensitivity and specificity