Novel primer-less amplification method for detection of RNA molecules

As shown here, isothermal and primer-less amplification of specific padlock probes allows direct detection of SARS-CoV2 RNA without needing for a reverse transcription step. This simplified method of Hyperbranched Rolling Circle Amplification (HRCA) only requires three enzymes: SplintR (to ligate a specific padlock probe to its circular form, only when viral RNA is present), the unique DNA primase TthPrimPol (to generate de novo DNA primers), and an engineered and thermostabilized variant of bacteriophage phi29 DNA polymerase (phi29 DNApol), named Qx5. Qx5 is significantly more efficient than wild-type phi29 DNApol and shows an unexpectedly strong 3'-5' exoribonucleolytic activity capable of trimming the 3' polyA tail of the RNA target, thus enabling it as a primer for Qx5 to start Rolling Circle Amplification (RCA) of a nearby circularized padlock. The RCA step, that yields a long ssDNA concatemeric product (RCA product, RCP) is coupled to a second isothermal amplification step, assisted by TthPrimPol by synthesizing DNA primers on the RCP, that triggers exponential HRCA of the padlock sequence, catalyzed by Qx5. As a proof of concept, the application of this method for detection of SARS-CoV2 RNA rendered a significant amount of DNA in just 2 h at 37 °C, that can be easily evidenced by colorimetry, or even quantitated with a pocket fluorometer, and served for a quick diagnosis of SARS-CoV2 RNA infected versus non-infected samples.