RPA (recombinase polymerase amplification) technology principle and comparison of RPA and LAMP

The British company TwistDx (formerly ASM Technology Co., Ltd.) was founded in 1999, based on the biotechnology company established by the Babraham Institute in Cambridge, England. The company's patented recombinase polymerase amplification technology (RPA), developed by breaking through isothermal DNA amplification, is hailed as a revolutionary innovation in the field of DNA diagnostics. (http://)

What is RPA?

Concept: 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.

RPA technology 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.

Who uses RPA?

Based on this, TwistAmp® nucleic acid amplification products can perform single-molecule nucleic acid detection at room temperature within 15 minutes. This technology has low requirements for hardware equipment, and is especially suitable for in vitro diagnostics, veterinary, food safety, biosafety, Agriculture and other fields.

What are the characteristics of RPA technology:

RPA features:

Rapid detection of 15 min for single molecule detection test

Simple packaging stable freeze-dry form reagent

Eliminate any instrumental constraints without the need for a thermal cycling process

Low requirements for portable devices and poor detection

Can RPA be multiplexed?

can. RPA technology supports simultaneous multiple amplification reactions in the same tube. However, multiple primer combinations need to be carefully designed so that each primer works equally well. It should be noted that the total amount of primers (nmol) of the RPA reaction is preferably not excessive. If more than two amplification primers are used in one reaction, the amount of different primers should be controlled. In addition, we should consider the compatibility of probes, devices, and fluorophores that detect multiple amplification events in advance.

Can you quantify the template?

can. The time at which the amplicon reaches a detectable level depends on the amount of template at the start of the reaction. The more copies of the initial template, the faster the amplicon reaches the detectable level. However, this quantification requires careful experimental arrangements to ensure that the control reactions begin simultaneously. For example, the addition time of magnesium ions can be utilized for control. As the magnesium ions enter the system, the RPA reaction begins.

In addition, a slower amplification process facilitates more accurate quantification. We can slow down the reaction rate of RPA by adjusting the reaction conditions or primer design.

Can fluorescence endpoint analysis be performed?

can. This compares the fluorescence at the start of the reaction with the fluorescence at the end of the reaction, and the increase in fluorescence means that amplification has occurred.

Can support biotin or fluorescently labeled oligonucleotides?

stand by. In the RPA reaction, primers with biotin or fluorophores behave similarly to unmodified primers. It is reported that no difference has been found.

Need to recycle RPA products before running rubber?

need. Substances in the RPA reaction interfere with agarose electrophoresis, and if the product is not recovered, the run-out band will be tailed.

Constant temperature amplification

Nucleic acid amplification instead of PCR

Field: Widely used in clinical diagnosis, food safety testing, animal disease testing, etc.

Features: fast, efficient, specific, no special equipment required

RPA vs. LAMP

LAMP: Loop-mediated isothermal amplification is characterized by designing four different primers for six segments on the target DNA strand and then performing the reaction at a certain temperature using a strand displacement reaction. The reaction only needs to put the gene template, the primer, the strand displacement DNA synthetase, the substrate and the like together at a certain temperature (60-65 ° C), and can be completed in one step.

RPA: mainly relies 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.

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.

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