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Review
. 2016 Jul;62(7):947-58.
doi: 10.1373/clinchem.2015.245829. Epub 2016 May 9.

Recombinase Polymerase Amplification for Diagnostic Applications

Affiliations
Review

Recombinase Polymerase Amplification for Diagnostic Applications

Rana K Daher et al. Clin Chem. 2016 Jul.

Abstract

Background: First introduced in 2006, recombinase polymerase amplification (RPA) has stirred great interest, as evidenced by 75 publications as of October 2015, with 56 of them just in the last 2 years. The widespread adoption of this isothermal molecular tool in many diagnostic fields represents an affordable (approximately 4.3 USD per test), simple (few and easy hands-on steps), fast (results within 5-20 min), and sensitive (single target copy number detected) method for the identification of pathogens and the detection of single nucleotide polymorphisms in human cancers and genetically modified organisms.

Content: This review summarizes the current knowledge on RPA. The molecular diagnostics of various RNA/DNA pathogens is discussed while highlighting recent applications in clinical settings with focus on point-of-care (POC) bioassays and on automated fluidic platforms. The strengths and limitations of this isothermal method are also addressed.

Summary: RPA is becoming a molecular tool of choice for the rapid, specific, and cost-effective identification of pathogens. Owing to minimal sample-preparation requirements, low operation temperature (25-42 °C), and commercial availability of freeze-dried reagents, this method has been applied outside laboratory settings, in remote areas, and interestingly, onboard automated sample-to-answer microfluidic devices. RPA is undoubtedly a promising isothermal molecular technique for clinical microbiology laboratories and emergence response in clinical settings.

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Figures

Fig. 1.
Fig. 1.. RPA cycle.
The 3 core proteins, recombinase, SSB, and strand-displacing polymerase enable exponential DNA amplification without the need for thermal cycling or an initial chemical or thermal melting step. The complete reaction is performed at a single temperature from 25–42 °C depending on RPA kit formulation.
Fig. 2.
Fig. 2.. RPA probes.
Real-time detection (A) RPA-exo probe; (B) RPA-fpg probe. Exonuclease III and Fpg nucleases recognize and cut the internal abasic residue, thus generating fluorescence. (C), Post-RPA detection with LF probe. Amplicon detection is accomplished by capture of tags with anti-FAM and anti-Biotin antibodies generating a visual colored line on LF strips.

References

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