Molecular Biology

Recombinase Polymerase Amplification & Lateral Flow

A Short Intro­duction

Recom­binase Polymerase Ampli­fi­cation (RPA) is a very sensitive, isothermal DNA ampli­fi­cation method that is extremely robust against common inhibitors and runs at compar­a­tively low temper­a­tures. The RPA can be directly combined with reverse transcription, which allows the sensitive detection of defined RNA sequences.

Due to the isothermal character, the robustness against inhibitors and the ampli­fi­cation speed, the RPA has developed to the most relevant DNA ampli­fi­cation method in the field of Point-​​of-​​Care appli­ca­tions under resource-​​limited settings.“

History of the Recom­binase Polymerase Ampli­fi­cation

The RPA was initially described in 2006 by Niall Armes and Olaf Piepenburg. The authors of the publi­cation with the title: „DNA Detection Using Recom­bi­nation Proteins” were already aware of the impor­tance of their innov­ative DNA ampli­fi­cation method for point-​​of-​​care appli­ca­tions. Even then, an RPA method was presented that could be combined with lateral flow analysis using the Milenia HybriDetect. 15 years after publi­cation of this landmark paper, RPA has developed to one of the most frequently used DNA-​​amplification techniques in combi­nation with universal lateral flow immunoassays.

Recombinase Polymerase Amplification - Workflow and Proof of Concept
Workflow of the Recom­binase Polymerase Ampli­fi­cation

General Mechanism of the Recom­binase Polymerase Ampli­fi­cation

The RPA works at temper­a­tures around 37 to 42 °C. In order to achieve exponential ampli­fi­cation at lower temper­a­tures, compo­nents of DNA repli­cation or recom­bi­nation are used to mimic natural processes. Viral proteins, that are involved in DNA-​​recombination, –repair, and –repli­cation in vivo, are added to the RPA– reaction to allow primer invasion. A suitable DNA-​​polymerase with strong strand displacement activity ensures elongation and thus efficient ampli­fi­cation of the RPA product.

General Mechanism - Recombinase Polymerase Amplification
Mechanism of the Recom­binase Polymerase Ampli­fi­cation

Modifi­cation of a RPA for Lateral Flow based Readout

The most important supplier of RPA reagents is TwistDx, which is part of Abbott since 2018. In order to avoid the detection of non specific RPA-​​products via Lateral Flow, it is highly recom­mended to add an additional specificity-​​generating step to the RPA. Therefore, TwistDx offers a ready-​​to-​​use mix.

Nfo – Endonu­clease IV

The use of an additional enzyme was already described in the first RPA publi­cation in 2006. Armes and Piepenburg described the addtion of the Endonu­clease IV (nfo) to the RPA reaction, which solved the problem of non specific ampli­fi­cation during RPA. The nfo-​​mediated, site-​​specific cleavage of an elongation blocked probe was neccessary to initiate the efficient ampli­fi­cation of dual labelel amplicons, which were detectable with the universal Lateral Flow Device, Milenia HybriDetect 2T. This addition was essential for a suffi­cient speci­ficity of the RPA-​​Lateral Flow rapid test. Never­theless, this method­ological detection strategy compli­cates the general RPA assay desgin.

Recombinase Polymerase Amplification - nfo dependent mechanism
Mechanism of the nfo-​​dependent Recom­binase Polymerase Ampli­fi­cation & Lateral Flow

Finding the perfect primer-​​/​probe-​​combination

In order to achieve a satis­fying RPA sensi­tivity, the search for the perfect primer-​​/​ probe-​​combination can be quite complex. The rules for primer design are compa­rable to those for PCR, except that the primers should be made signif­i­cantly longer (30 – 36 bases) and the amplicons should be kept small. The use of an additional nfo probe (approx. 45 bases) further compli­cates the assay design. However, this should not be a reason to opt out of the RPA. Ultimately, it is only a matter of investing more time and energy in a meaningful primer-​​probe-​​screening system. As a reward, very sensitive, specific ampli­fi­cation reactions can succeed, which run with an impressive robustness and speed even under limited-​​resource settings.

RPA & Multi­plexing

Generally RPA is an isothermal DNA-​​amplification technique that allows extremely rapid and robust analysis. But RPA is not known to be the perfect technique for multi­plexing. Never­theless, it is possible to develop multiplex RPA assays that are perfectly compatible with the multiplex Lateral Flow Device – Milenia HybriDetect 2T. Multiplex RPA assays have been success­fully developed, published and thus provide multiple infor­mation under low resource settings. As described recently by Mancuso et al., Anal. Chem. 93, 2021 even (semi-​​) quanti­tative analysis is possible due to a special duplex assay design. Moreover, stronger multi­plexing can be achieved in combi­nation with additional post ampli­fi­cation techniques, such as nested ampli­fi­cation, simple probe hybridization or CRISPR-​​Cas-​​dependent amplicon recog­nition assays.

Learn more about (RPA) Multi­plexing & Lateral Flow

Limita­tions of the RPA

In principle, the RPA is suitable for multiplex appli­ca­tions, but assay design and the general RPA perfor­mance noticeably limit the multi­plexing ability. In addition to the problem of potential carryover conta­m­i­na­tions, the non-​​specific ampli­fi­cation of unwanted products is one of the most important RPA-​​characteristic limita­tions. A Methodical adaptation such as nfo-​​dependent RPA or final hybridization with an amplicon-​​specific probe is often unavoidable.

Benefits of the RPA

Recom­binase polymerase ampli­fi­cation is one the most important isothermal ampli­fi­cation techniques for a new gener­ation of molecular point-​​of-​​care diagnostics. RPA can be incredibly fast. Results in less than 10 minutes are possible with sensi­tiv­ities that are compa­rable to estab­lished laboratory methods. The isothermal character at temper­a­tures around 37°C, the compat­i­bility with the lateral flow technology and the robustness against inhibitors make the RPA one of the most important tools for sensitive and accurate rapid testing in a true Point-​​of-​​Need setting.

Method Comparison Table
Method combined with LFASensi­tivitySpeci­ficitySpeedreverse Transcription (one pot)RobustnessMethod. VarietySimple Assay DesignMulti­plexing AbilityPrevention Carryover Cont.
PCR
LAMP
RPA
CRISPR (Label-​​Sep.)
CRISPR (Label-​​Inc.)