The RPA process employs three core enzymes – a recombinase, a single-stranded DNA-binding protein (SSB) and strand-displacing polymerase.
Recombinases are capable of pairing oligonucleotide primers with homologous sequence in duplex DNA.
SSB bind to displaced strands of DNA and prevent the primers from being displaced.
Finally, the strand displacing polymerase begins DNA synthesis where the primer has bound to the target DNA.
By using two opposing primers, much like PCR, if the target sequence is indeed present, an exponential DNA amplification reaction is initiated. No other sample manipulation such as thermal or chemical melting is required to initiate amplification. At optimal temperatures (37–42 °C), the reaction progresses rapidly and results in specific DNA amplification from just a few target copies to detectable levels, typically within 10 minutes, for rapid detection of viral genomic DNA or RNA, pathogenic bacterial genomic DNA, as well as short length aptamer DNA.
The three core RPA enzymes can be supplemented by further enzymes to provide extra functionality. Addition of exonuclease III allows the use of an exo probe for real-time, fluorescence detection akin to real-time PCR. Addition of endonuclease IV means that a nfo probe can be used for lateral flow strip detection of successful amplification. If a reverse transcriptase that works at 37–42 °C is added then RNA can be reverse transcribed and the cDNA produced amplified all in one step. Currently only the TwistAmp exo version of RPA is available with the reverse transcriptase included, although users can simply supplement other TwistAmp reactions with a reverse transcriptase to produce the same effect.
As with PCR, all forms of RPA reactions can be multiplexed by the addition of further primer/probe pairs, allowing the detection of multiple analytes or an internal control in the same tube.