Recombinase Polymerase Amplification - The Breakthrough Isothermal Alternative to PCR

TwistDx’s proprietary technology, recombinase polymerase amplification (RPA), is transforming our ability to amplify and detect nucleic acids in laboratory and resource-limited field settings.

TwistDx’s isothermal nucleic acid amplification technology, Recombinase Polymerase Amplification (RPA), represents a hugely versatile alternative to polymerase chain reaction (PCR) for the development of fast, portable, nucleic acid detection assays. Inherently adaptable to applications as diverse as infectious disease diagnostics and food contamination tests, RPA is ideally suited to field, point-of-care and other settings with minimal resources, and particularly to situations where speed is essential. Easily transportable, user friendly and highly sensitive, RPA is as specific as PCR amplification but is much, much faster. Results are typically generated within 3-10 minutes. And unlike PCR, the RPA reaction doesn’t require thermal or chemical melting, so there’s no need for an expensive thermoycler or any additional equipment or reagents.

RPA is very forgiving of operating temperature. The reaction works optimally at a temperature of around 37-42˚C, which is lower than for other isothermal approaches, but will also work over a wide range of ambient temperatures. The dry-formulated RPA reagents exhibit excellent stability at ambient temperatures typically at least 12 months*. Refrigeration for short-term transport is unnecessary. The lyophilized reagent pellet also involves a simple workflow that can be carried out without specialist training.

RPA can be used to replace PCR in a wide variety of applications. End-users can easily design their own ultra-sensitive assays using their own primers. RPA technology can be adapted to a range of microfluidic, lateral flow and other devices, and by adding reverse transcriptase to the reaction mix RPA technology can also be used to amplify and detect RNA.

Our customers tell us that the technology works brilliantly out of the box.


Figure 1: The RPA cycle


How does RPA work?

The RPA reaction exploits enzymes known as recombinases, which form complexes with oligonucleotide primers and pair the primers with their homologous sequences in duplex DNA. A single-stranded DNA binding (SSB) protein binds to the displaced DNA strand and stabilizes the resulting D loop. DNA amplification by polymerase is then initiated from the primer, but only if the target sequence is present. Once initiated, the amplification reaction progresses rapidly, so that starting with just a few target copies of DNA, the highly specific DNA amplification reaches detectable levels within minutes.

Why recombinase polymerase amplification?

Speed
RPA is fast, and at typical optimum reaction temperatures of 37-42°C just a few nucleic acid molecules can be amplified to detectable levels (typically) in 3-10 minutes, although this will depend on target size. In many cases, one person, with no specialist training, would be able to take a sample, prepare it, run the assay and get results within half an hour. Compare this with the 24-hour turnaround times that are currently typical for clinical samples that must be sent off for processing at central laboratories.

Sensitivity
RPA can detect single copies of DNA and tens of copies or fewer of RNA in complex samples, without the need for prior nucleic acid purification.

Specificity
RPA is so specific that the technology can detect and amplify a single molecule of DNA that is present in a sample containing potentially hundreds of nanograms of unrelated, complex genomic DNA from multiple, diverse species, including human DNA.

Low temperature operation
RPA operates at a constant, low temperature (optimally 37-42°C) and does not require initial melting of the sample DNA. For some applications the RPA reaction can even be supported by body heat, if necessary. The reaction is also robust at off temperatures and low temperature setups, and will even work, albeit more slowly, at the typically ambient temperature of 25°C. At this temperature results can still be obtained within an hour, as long as the biochemistry has been appropriately configured.

Sample tolerance
RPA is very forgiving of sample type, and in some instances can work directly on complex samples, such as blood, nasal swabs or culture media, which have not gone through nucleic acid purification. Often, it may be enough to subject the sample to basic pathogen lysis methods, such as treatment with heat or a weak alkali, to release the nucleic acids. The most appropriate sample preparation method required for each assay will be dependent upon factors such as pathogen titre, presence of inhibitors and lysis requirements, and will need to be designed into the assay procedure.This robustness of RPA to some complex sample types make the technology very well suited, in principle, for potentially wide-ranging field-based and point-of-care applications.

Broad applicability
RPA can be readily applied to any DNA or RNA target. End users have already reported developing ultra-high sensitivity, one-pot RNA detection assays by adding reverse transcriptase to the reaction mix.

Multiplexing
By combining multiple primers in the same reaction tube, a single RPA test can be used to amplify and detect several different targets.

Low cost burden
RPA requires only a basic detection device, which means that end users can use our products to develop user-friendly diagnostic assays and kits for a wide range of applications and settings.

Stabilised format
The reaction system is provided in a stabilized, dried format for easy transportation and storage without refrigeration for up to 12 months (application-specific variation in stability is possible and long-term storage under refrigeration is still recommended).

Multiple detection formats
RPA can be applied to a wide range of detection systems and instrumentation, including fluorescence, real time probes and sandwich assay formats.

*PF exo kit using internal tests