Introduction to Recombinase Polymerase Amplification (RPA) Technology

The revolution in nucleic acid detection: a sharp alternative to PCR - RPA

Recombinase Polymerase Amplification (RPA) is known as a nucleic acid detection technology that can replace PCR. The RPA technology relies primarily on three enzymes: a recombinase capable of binding to a single-stranded nucleic acid (oligonucleotide primer), a single-stranded DNA binding protein (SSB), and a strand displacement DNA polymerase. The mixture of these three enzymes is also active at room temperature, and the optimum reaction temperature is about 37 °C.

Introduction to RPA principle

A protein-DNA complex formed by the combination of a recombinase and a primer can find homologous sequences in double-stranded DNA. Once the primers have localized homologous sequences, strand exchange reactions occur and DNA synthesis is initiated, and the target region on the template is exponentially amplified. The replaced DNA strand binds to the SSB to prevent further substitution. In this system, a synthetic event is initiated by two relative primers. The entire process proceeds very quickly and generally yields detectable levels of amplification products in less than ten minutes.

The key to RPA analysis is the design of amplification primers and probes. PCR primers are mostly unsuitable because RPA primers are longer than typical PCR primers and typically require 30-38 bases. Short primers will reduce the rate of recombination, affecting the rate of amplification and detection sensitivity. When designing RPA primers, the denaturation temperature is no longer a key factor affecting the amplification primers. The primer and probe design of RPA is not as mature as traditional PCR, and users need to optimize their conditions.

What are the advantages of RPA technology?

Conventional PCR must undergo three steps of denaturation, annealing, and extension, and the PCR instrument is essentially a device that controls temperature rise and fall. The optimum temperature for the RPA reaction is between 37 ° C and 42 ° C without denaturation and can be carried out at room temperature. This will undoubtedly greatly speed up the PCR. In addition, because of the lack of temperature control equipment, RPA can truly achieve portable rapid nucleic acid detection.

According to reports, RPA detection is highly sensitive, capable of amplifying trace amounts of nucleic acid (especially DNA) templates to detectable levels, yielding approximately 1012 amplification products from a single template molecule. Moreover, RPA does not require complicated sample processing and is suitable for field detection of nucleic acids that cannot be extracted.

RPA can both amplify DNA and amplify RNA, and eliminates the need for additional cDNA synthesis steps. Not only can you perform endpoint detection on the amplified product, but you can also monitor the amplification process in real time and even read the results through the test strip (lateral flow chromatography strip LFD).

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