Recombinase Polymerase Amplification (RPA) and Reverse Transcription RPA (RT-RPA)

1. Primer Recombination: Recombinase enzymes bind to single-stranded DNA primers, forming nucleoprotein complexes. These complexes actively scan double-stranded DNA to find the matching homologous sequence.
2. Strand Invasion: Upon locating the homologous site, the primer-recombinase complex invades the DNA duplex. This action separates the DNA strands and creates a D-loop structure, providing a binding site for DNA polymerase.
3. DNA Synthesis: A strand-displacing DNA polymerase binds to the primer and begins synthesis. As it extends the primer, it displaces the original template strand. This cycle repeats, resulting in the exponential amplification of the target DNA sequence.

This isothermal process is the key distinction when comparing RPA to PCR. The absence of high-temperature denaturation steps and thermal cycling simplifies the required instrumentation and significantly reduces amplification time.

Explore our RPA Endpoint Kit and RPA Real-Time Kit to learn more.

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.

Explore our RT-RPA Endpoint Kit and RT-RPA Real-Time Kit to learn more.

1. Prepare the Master Mix: Rehydrate the lyophilized pellet with the provided rehydration buffer.
• For RPA: The pellet contains all necessary enzymes (Key components including recombinase and DNA polymerase).
• For RT-RPA: The pellet contains all necessary enzymes (Key components including reverse transcriptase, recombinase, DNA polymerase).
2. Add Primers & Probes: Incorporate your optimized primer/probe set directly into the mix.
3. Introduce the Sample: Add your purified nucleic acid (DNA or RNA) to the reaction tube.
4. Isothermal Incubation: Incubate at a constant temperature (typically 37°C–42°C) using a simple heat block or water bath.
• RPA Time: 5–20 minutes.
• RT-RPA Time: 15–20 minutes (to allow for the integrated reverse transcription step).
5. Detection: Analyze results in real-time via fluorescence or at the endpoint using lateral flow strips.

Essential Best Practices

To ensure reliable and reproducible results across either assay, keep these three pillars in mind:

• Primer Optimization: Isothermal amplification is highly sensitive to primer design; always screen multiple sets to find the highest specificity.
• Contamination Control: Maintain a strict "clean-room" workflow to prevent aerosolized amplicon carryover.
• RNA Integrity: For RT-RPA, use RNase-free reagents and tips to prevent the degradation of your target template before the reaction begins.