Reverse Transcriptase Recombinase Polymerase Amplification (RT-RPA)

1. Reverse Transcription: A reverse transcriptase enzyme synthesizes a single strand of cDNA from the target RNA molecule using a specific primer. This initial step converts the RNA target into a stable DNA template for amplification.
2. Primer Recombination: Recombinase enzymes coat the RPA primers, forming nucleoprotein filaments that scan the newly synthesized cDNA for homologous sequences.
3. Strand Invasion and Amplification: The primer-recombinase complex invades the target DNA sequence and a strand-displacing DNA polymerase then initiates the synthesis and elongates the primer. Repeated cycles of this reaction lead to exponential amplification of the cDNA template. The entire reaction occurs at a single, low temperature, a key differentiator in the RT-RPA vs RT-PCR comparison.

1. Prepare the Reaction Mix: Rehydrate the lyophilized RPA pellet, which contains all necessary enzymes (reverse transcriptase, recombinase, DNA polymerase), and add your primers and probes.
2. Add the RNA Sample: Introduce the target RNA sample directly into the master mix.
3. Initiate the Reaction: Briefly mix and incubate the reaction at a constant temperature, typically between 37°C and 42°C, for as little as 15-20 minutes.
4. Detect the Results: Analyze the amplified product in real-time using a portable fluorometer or at the endpoint with lateral flow strips.

For optimal results, proper RT-RPA optimization is critical. Test multiple primer and probe designs to ensure specificity and efficiency. Additionally, it is critical to establish clean workflows to avoid contamination and maintain RNA integrity.