High-Throughput Ribozyme-Based Assays for Detection of Viral Nucleic Acids Original Research Article

Many reports have suggested that target-activated ribozymes hold potential value as detection reagents. We show that a “half”-ribozyme ligase is activated similarly by three unstructured oligoribonucleotides representing the major sequence variants of a hepatitis C virus 5′-untranslated region (5′-UTR) target and by a structured RNA corresponding to the entire 5′-UTR. Half-ribozyme ligation product was detected both in an ELISA-like assay and in an optical immunoassay through the use of hapten-carrying substrate RNAs. Both assay formats afford a limit of detection of approximately 1 × 106 HCV molecules (1.6 attomol, 330 fM), a sensitivity which compares favorably to that provided by standard immunoassays. These data suggest that target-activated ribozyme systems are a viable approach for the sensitive detection of viral nucleic acids using high-throughput platforms.