Reverse transcription lesion-induced DNA amplification: An instrument-free isothermal method to detect RNA.

ABSTRACT

One challenge in point-of-care (POC) diagnostics is the lack of room-temperature methods for RNA detection based on enzymatic amplification and visualization steps. Here we perform reverse transcription lesion-induced DNA amplification (RT-LIDA), an isothermal amplification method that only requires T4 DNA ligase. RT-LIDA involves the RNA-templated ligation of DNA primers to form complementary DNA (cDNA) followed by toehold-mediated strand displacement of the cDNA and its exponential amplification via our isothermal ligase chain reaction LIDA. Each step is tuned to proceed at 28 °C, which falls within the range of global room temperatures. Using RT-LIDA, we can detect as little as ∼100 amol target RNA and can distinguish RNA target from total cellular RNA. Finally, we demonstrate that the resulting DNA amplicons can be detected colorimetrically, also at room temperature, by rapid, target-triggered disassembly of DNA-modified gold nanoparticles. This integrated amplification/detection platform requires no heating or visualization instrumentation, which is an important step towards realizing instrument-free POC testing.