Hybridization Cascade Plus Strand-Displacement Isothermal Amplification of RNA Target with Secondary Structure Motifs and Its Application for Detecting Dengue and Zika Viruses.

Biological RNA generally comprises secondary structure motifs which cause a problem for target RNA detection by isothermal amplification methods. The complexity of the secondary structures makes RNA targets inaccessible for probe hybridization, resulting in decreased sensitivity and selectivity. This is particularly important because the hybridization step of the isothermal amplification method requires a limited temperature range. A strand-displacement strategy can enhance the hybridization efficiency between the probe and target RNA with secondary structure motifs. A short, single-stranded segment within the secondary structure can be used as a toehold for initiating strand displacement. The strategy has been used to establish a highly sensitive isothermal amplification by a combination of a hairpin probe hybridization and strand-displacement amplification. The hairpin probe is placed on the single-stranded segment of the target RNA's secondary structure to initiate strand displacement. The probe's hybridization cascade provides a template for exponential amplification in two directions by strand-displacement amplification, designated hybridization cascade plus strand-displacement isothermal amplification (HyCaSD). The method requires no reverse transcription step. HyCaSD showed an excellent sensitivity with the limit of detection in the femtomolar (fM) range for synthetic targets as well as viral RNAs. Discrimination between DENV/ZIKV and JEV/CHIKV was successfully demonstrated using real viruses. Therefore, HyCaSD is a promising platform that can be further developed for diagnostic applications.