A noise-free, ultrasensitive and accurate miRNAs detection using streptavidin coated magnetic microsphere based stem-loop ligation PCR

Abstract

As important disease diagnostic markers, circulating miRNAs have been put on an urgent agenda in recent years with the focus on their highly sensitive and specific multiplex detection in one reaction. In this article, we proposed a unique miRNAs detection method based on tailored-designed stem-loop structure ligation strategy to realize ideal detection performance. The stem-loop ligation probe had a target miRNA mediated quick ligation, which the relative ligation efficiency of stem-loop structure was superior evidently to straight chain structure about 76% at 10 min on account of the preferentially formed partial dimer. Moreover, the streptavidin-coated magnetic microspheres combined with optimized ligation probes concentration at 10 nM were utilized to purify ligation products and completely eliminated nonspecific ligation substantially. Due to the stem-loop structure ligation products contained universal primer regions, let-7 family as model was used to evaluate the detection performance, demonstrating high sensitivity as the minimum detection limit of 2.5 fM with a five-orders of magnitude dynamic detection range. To some degree, the stem-loop structure-based ligation may inspire the flexible design strategy in the future application and provide a significant quick and efficient platform to miRNAs detection.

Keywords: Locked nucleic acid (LNA)-Modified blocker; Stem-loop ligation chain reaction; Streptavidin-coated magnetic microspheres; miRNAs.