Fluorogenic Templated Reaction Cascades for RNA Detection.

Abstract

Nucleic acids detection is essential to the study of biological processes and to diagnosis of pathological states. Although PCR is highly effective in vitro, methods that can function without prior sample preparation, thermal cycling, or enzymes are of interest due to their simplicity. Most current non-PCR detection methods rely on linear signal amplification, which hinders the detection of small amounts of genetic material. To address this limitation, we tested a new strategy for attaining higher-order signal amplification, in which a target sequence templates a chemical ligation, and the product of this reaction is in turn detected with a second templated reaction. The method is nonenzymatic, isothermal, and fluorogenic, allowing the direct detection of nucleic acids in complex matrices. Using this approach, as little as 500 attomoles (10 pM) could be detected with single nucleotide resolution. In a test of selectivity, single nucleotide substitutions and deletions could successfully be detected, including a deletion that is associated with tetracycline resistance in Helicobacter pylori. Compatibility with biological matrices was demonstrated by the direct detection of rRNA in bacterial lysate. Imaging and detection of target sequences on a solid support further illustrates the potential of the new approach for high-throughput analysis.