Recombinase Polymerase Amplification (RPA) is an isothermal nucleic acid amplification technology developed by Piepenburg et al. in 2006. By combining recombinase, single-stranded DNA–binding proteins, and strand-displacing DNA polymerase, RPA enables rapid nucleic acid detection at a constant temperature (37–42 °C), typically within 10–30 minutes.
Because it does not require thermal cycling, RPA is increasingly regarded as a powerful alternative to PCR.
This FAQ summarizes common technical questions and best practices for successful RPA experiments.
1. What are the key features of RPA?
|
Feature |
Description |
|
Multi-enzyme cooperative system |
RPA relies on coordinated action of multiple enzymes, including: • Recombinase (e.g., UvsX) • Accessory proteins (e.g., UvsY) • Single-stranded DNA-binding protein (SSB, e.g., GP32) • Strand-displacing DNA polymerase (e.g., Bsu DNA polymerase) |
|
Isothermal amplification |
• Reaction temperature: 37–42 °C • Reaction time: 3–20 minutes • No thermal cycler required |
|
High sample tolerance |
• Sensitivity down to single-copy or tens of copies • Broad tolerance to complex matrices • In some cases, crude samples (blood, nasal swabs, culture media) can be used directly without nucleic acid purification |
|
Broad template compatibility |
Suitable for both DNA and RNA targets |
2. What are the critical factors for a successful RPA reaction?
|
Factor |
Recommendation / Rationale |
|
Primer design |
• Recommended length: 30–35 nt • Short primers reduce speed and sensitivity • Overly long primers (>45 nt) increase secondary structure risk |
|
Amplicon length |
Optimal size: 100–250 bp |
|
Order of reagent addition |
Add template and magnesium acetate last to prevent premature initiation and non-specific amplification |
|
Mixing |
Thorough mixing is essential to ensure reaction homogeneity |
|
Post-amplification processing |
Treat products with Proteinase K or purify before gel electrophoresis to avoid protein-related interference |
|
Instrument setup |
• Turn off heated lid on PCR/qPCR instruments • Ensure lid temperature is <50 °C before inserting tubes • A hot lid (~105 °C) may partially or completely inactivate RPA enzymes |
3. Key principles for RPA primer design
|
Parameter |
Recommended Range / Rule |
|
Target region GC content |
40–60% |
|
Primer length |
30–35 nt |
|
Primer GC content |
30–70% |
|
Primer Tm |
50–70 °C |
|
Sequence constraints |
• Avoid repetitive sequences (≤5 identical bases in a row) • Avoid consecutive Gs at the 5′ end; C is preferred to promote recombination • GC-rich 3′ ends improve amplification performance |
|
Amplicon size |
80–500 bp (ideal: 100–200 bp) |
|
Secondary structure |
Minimize hairpins and primer–dimer formation |
4. How should primers be screened?
|
Stage |
Method |
Purpose |
|
Initial screening |
Agarose gel electrophoresis |
Rapid assessment of amplification efficiency and specificity |
|
Further optimization |
Fluorescent RPA kits (real-time fluorescence) |
Quantitative comparison and parallel testing of multiple primer/probe conditions |
|
Lateral-flow assay adaptation |
Strip-based (Nfo probe) RPA |
Probe design principles are similar to fluorescent RPA; primers optimized for fluorescence often translate well to strip assays |
5. Are TaqMan probes compatible with RPA?
No.
TaqMan probes rely on the 5′→3′ exonuclease activity of Taq DNA polymerase to release fluorescence.
Bsu DNA polymerase used in RPA does not possess this activity, making TaqMan probes incompatible.
6. Why are RPA products sometimes undetectable by agarose gel electrophoresis?
RPA reactions contain high concentrations of proteins and macromolecules, which can:
- Block gel wells
- Interfere with DNA migration
Solution: Purify amplification products before loading.
7. Why do false-positive results occur frequently?
|
Cause |
Description / Solution |
|
Aerosol contamination |
High-copy templates (≥10⁶ copies/µL) can generate aerosols that contaminate other samples. Handle carefully and minimize splashing. |
|
Laboratory zoning |
Separate areas for: • Reagent preparation • Sample handling • Reaction setup • AmplificationThis prevents cross-contamination between steps. |
|
Environmental decontamination |
Regularly clean workspaces and pipettes using nucleic acid decontamination solutions or 10% bleach to remove residual templates. |
|
Minimize tube opening |
Reduce post-amplification handling of tubes to prevent spreading amplified products. Consider using closed-tube detection methods whenever possible. |
8. Why is reagent addition order so important?
RPA reactions can initiate at room temperature.
If the activator (e.g., magnesium acetate) is added too early, it may trigger:
- Non-specific amplification
- Premature consumption of reaction components
Best practice: Add template and activator last to control reaction initiation and improve reproducibility.
9. Which fluorescence channel is recommended for real-time RPA?
FAM channel: Excitation: 492 nm, Emission: 518 nm
10. What is the difference between RPA and RAA?
|
Feature |
RPA (Recombinase Polymerase Amplification) |
RAA (Recombinase-Aided Amplification) |
|
Fundamental principle |
Same isothermal amplification mechanism |
Same isothermal amplification mechanism |
|
Recombinase source |
T4 bacteriophage |
Bacterial or fungal origin |
|
Other notes |
Terminology differs only; reaction components and amplification strategy are essentially the same |
Terminology differs only; reaction components and amplification strategy are essentially the same |
11. Enzyme composition: PCR vs. LAMP vs. RPA
|
Amplification Method |
Core Enzyme(s) |
Enzyme Source |
Key Enzymatic Features |
Reaction Characteristics |
|
PCR |
Taq DNA Polymerase |
Thermus aquaticus |
• 5′→3′ DNA polymerase activity • High thermal stability • Often hot-start modified to suppress low-temperature activity |
• Requires thermal cycling • DNA denaturation by high temperature • High specificity with precise temperature control |
|
LAMP |
Bst DNA Polymerase |
Bacillus stearothermophilus |
• Strong strand-displacement activity • Lacks 3′→5′ and 5′→3′ exonuclease activity |
• Isothermal amplification • No thermal cycling required • Uses multiple primers to form loop structures |
|
RPA |
Recombinase + SSB + Bsu DNA Polymerase |
Recombinase (phage or microbial origin)Polymerase (Bacillus subtilis) |
• Recombinase–primer complexes search homologous sequences • Strand exchange enables primer invasion • SSB stabilizes displaced ssDNA |
• Rapid isothermal amplification (37–42 °C) • Multi-enzyme cooperative mechanism • No DNA denaturation step required |
12. How to choose an RPA kit?
|
RPA Kit Type |
Probe Type |
Detection Method |
Readout |
Typical Applications |
|
Basic (Gel-based) RPA |
None |
Agarose gel electrophoresis or CRISPR-based detection |
Endpoint |
• Target amplification only • Method development • CRISPR-Cas detection workflows |
|
Fluorescent RPA |
Exo probe |
Real-time fluorescence measurement |
Quantitative / Real-time |
• Quantitative analysis • High-throughput screening • Primer and probe optimization |
|
Lateral-flow RPA |
Nfo probe |
Lateral flow test strip |
Visual / Qualitative |
• Rapid on-site detection • Point-of-care testing (POCT) • Field diagnostics |
Yeasen RPA Product Selection Guide
|
Product Category |
Product Name |
Cat. No. |
Product Type / Application |
|
RPA Kits (Liquid Format) |
16702ES |
Fluorescent RPA kit (Exo probe–based) |
|
|
RNA Rapid Isothermal Amplification Kit (Fluorescent) |
16708ES |
Fluorescent RT-RPA kit (Exo probe–based) |
|
|
DNA Rapid Isothermal Amplification Kit (Chromatographic) |
16725ES |
Lateral-flow RPA kit (Nfo probe–based) |
|
|
RNA Rapid Isothermal Amplification Kit (Chromatographic) |
16726ES |
Lateral-flow RT-RPA kit (Nfo probe–based) |
|
|
DNA Rapid Isothermal Amplification Kit (Basic) |
16727ES |
Gel-based RPA kit |
|
|
RNA Rapid Isothermal Amplification Kit (Basic) |
16728ES |
Gel-based RT-RPA kit |
|
|
RPA Kits (Lyophilized) |
DNA Lyo Rapid Isothermal Amplification Kit (Fluorescent) |
16904ES |
Fluorescent RPA kit (Exo probe–based, in-tube lyophilized) |
|
RNA Lyo Rapid Isothermal Amplification Kit (Fluorescent) |
16905ES |
Fluorescent RT-RPA kit (Exo probe–based, in-tube lyophilized) |
|
|
DNA Lyo Rapid Isothermal Amplification Kit (Chromatographic) |
16906ES |
Lateral-flow RPA kit (Nfo probe–based, in-tube lyophilized) |
|
|
RNA Lyo Rapid Isothermal Amplification Kit (Chromatographic) |
16907ES |
Lateral-flow RT-RPA kit (Nfo probe–based, in-tube lyophilized) |
|
|
DNA Lyo Rapid Isothermal Amplification Kit (Basic) |
16908ES |
Gel-based RPA kit (in-tube lyophilized) |
|
|
RNA Lyo Rapid Isothermal Amplification Kit (Basic) |
16909ES |
Gel-based RT-RPA kit (in-tube lyophilized) |