A novel SARS-CoV-2 detection method based on trans-acting ribozymes and FRET of DNA-hairpins in a hybridization chain reaction

Abstract

The diagnosis of SARS-CoV-2 by real-time detection of the viral genome using RT-qPCR (Reverse Transcriptase quantitative Polymerase Chain Reaction) and serological methods were essential to control its rapid spread during the pandemic. However, RT-qPCR, which is the gold standard method to identify SARS-CoV-2 at the initial phase of infection, has high costs and other limitations such as refrigeration and specialized equipment and professionals. Therefore, a sensitive, not time consuming, not costly, not laborious, and free of refrigeration and specialized equipment is of utmost importance in view of the threat of future epidemics. Here, we propose a detection method where SARS-CoV-2 RNA is recognized and cleaved by ribozymes releasing an initiator fragment. This fragment triggers a hybridization chain reaction (HCR) with DNA hairpins containing fluorophores which leads to a Fluorescence Resonant Energy Transfer (FRET) interaction. First, a consensus SARS-CoV-2 RNA sequence was identified. This target viral RNA fragment and ribozymes were in vitro transcribed, followed by cleavage reactions. DNA hairpins with Cy3/Cy5 fluorophores were designed and synthesized for FRET-HCR assays that were performed to target the RNA fragment sequences obtained after ribozyme cleavage. Our results indicated that two out of the three designed ribozymes cleaved the target RNA. Furthermore, DNA hairpins with Cy3/Cy5 pairs were efficient in target RNA detection and in triggering FRET-HCR detectable reactions. Altogether, the results of this study laid the cornerstone, as a proof of concept, for the joint use of ribozymes and DNA hairpins with Cy3/Cy5 in FRET-HCR reactions to detect SARS-CoV-2 virus.