Recombinase-mediated Isothermal Amplification Technology
Recombinase-aided amplification (RAA) is a novel type of isothermal amplification technology based on recombinase polymerase amplification (RPA) technology. RAA technology uses recombinase, single-strand binding protein (SSB), and DNA polymerase to amplify nucleic acids under isothermal conditions. During the RAA reaction, the recombinase, SSB, and primers form a complex to scan the double-stranded DNA and cause the double-stranded DNA to unravel at the sequence homologous to the primer. The SSB binds to the single-stranded DNA to prevent it from re-forming a double-stranded structure. With the presence of energy and dNTP, the DNA polymerase completes chain extension. Under isothermal conditions, within 30 minutes, rapid nucleic acid amplification can be achieved through the RAA reaction within 30 minutes, and no specialized thermal cycling equipment is required.
Fluorescent RAA
After introducing specific fluorescent probes into the RAA system, as the amplification products increase, the fluorescence signal will be enhanced. The changes in the signal can be monitored in real time to determine whether the target nucleic acid exists or to perform quantitative analysis. Fluorescent RAA is used for the detection of DNA of interest. In the fluorescent reverse transcription RAA (RT-RAA) reaction, an additional step is added before the RAA amplification. The additional step of adding reverse transcriptase enables the conversion of RNA into cDNA for amplification, thereby allowing the detection of the target RNA.
Why Choose Fluorescent RAA
Compared with traditional amplification techniques, RAA technology exhibits a rapid reaction rate at low temperatures without the thermal cycling process, making it highly suitable for portable testing.
| Technologies |
Fluorescent RAA |
qPCR |
LAMP |
| Temperature |
Isothermal (37-42 °C) |
3‑step cycling (50-95 °C) |
60-65 °C |
| Time-to-result |
~10-30 min |
60-120 min |
15-45 min |
| Primer/probe |
2 primers + 1 exo probe |
2 primers + probe |
4-6 primers (+ optional probe) |
| Instrument |
Simple heater/portable fluorimeter |
Calibrated thermocycler |
Simple heater; basic fluorimeter or color reader |
| Sensitivity |
High |
Very high |
High |
| Quantitation |
Semi‑quantitative |
Gold‑standard quantitation |
Semi‑quantitative |
| On-site Testing |
Excellent (low heat, fast) |
Moderate (power, thermal cycling) |
Excellent (but higher temp) |
RAA Rapid Detection Kits
Amerigo Scientific offers a variety of easy-to-use fluorescent RAA nucleic acid detection kits. Under isothermal conditions, the amplification of pathogen nucleic acids can be completed within 15-30 minutes using our kits, thereby enabling the detection of the target pathogen.
Application
RPA and RAA are emerging as rapid, specific and highly sensitive methods for identifying various pathogens. African swine fever (ASF) is a fatal and highly contagious infectious disease that affects domestic and wild pigs, caused by the African swine fever virus (ASFV). 152 porcine samples suspected for ASFV were tested. The results of testing multiple samples showed that the consistency between RPA, RAA and the real-time fluorescent quantitative PCR method was high.
Figure 1. Comparison of detection performance between the threshold time of ASFV real-time RPA, RAA, and Ct value of real-time PCR (y axis) on positive field samples (n = 88) (doi: 10.3389/fmicb.2020.01696.)
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