DNA Detection Using Recombination Proteins

By O. Piepenburg, C. H. Williams, D. Stemple, N. Armes. PLoS Biology, 2006

2,150 citations recorded by the source corpus.

DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA), couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA. Reactions are sensitive, specific, and rapid and operate at constant low temperature. We have also developed a probe-based detection system. Key aspects of the combined RPA amplification/detection process are illustrated by a test for the pathogen methicillin-resistant Staphylococcus aureus. The technology proves to be sensitive to fewer than ten copies of genomic DNA. Furthermore, products can be detected in a simple sandwich assay, thereby establishing an instrument-free DNA testing system. This unique combination of properties is a significant advance in the development of portable and widely accessible nucleic acid–based tests.

Subfields: Biomedical Engineering, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/1c473f9370904ff77940d995437bc53ea0d47301

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Citations in the corpus

Recombinase Polymerase Amplification for Diagnostic Applications (2016, methodology): “Methicillin-resistant Staphylococcus aureus — <10 <30 rt/LF/AG Yes (4;3) Piepenburg et al.”
Nucleic acid detection with CRISPR-Cas13a/C2c2 (2017, neutral): “We targeted theV3 regionof the 16S ribosomalRNA (rRNA) gene, where conserved flanking regions allowuniversalRPAprimers tobeusedacrossbacterial species and the variable internal region allows for differentiation of species.”
dPCR: A Technology Review (2018, background): “A wide variety of isothermal nucleic acid amplification technologies have been developed such as rolling circle amplification (RCA) [111], nucleic-acid sequence-based amplification (NASBA) [112], loop-mediated amplification (LAMP) [113], multiple displacement amplification (MDA) [114], helicase-depe”
REVIEW: The detection of aquatic animal species using environmental DNA – a review of eDNA as a survey tool in ecology (2014, methodology): “2000) or recombinase polymerase amplification (Piepenburg et al. 2006) could be tested for their application to eDNA analysis to speed up the process and limit the amount of specialized equipment required.”
Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip (2017, methodology): “These sequences were designed, according to the original RPA publication (41), and ordered through Biosearch Technologies and Integrated DNA Technologies.”
Field-deployable viral diagnostics using CRISPR-Cas13 (2018, methodology): “DCB GFE Patient serum Patient saliva No Input Healthy Serum Healthy Saliva A Clinical sample RT-RPA Cas13 detection T7 transcription HUDSON SHERLOCK Nuclease °37-50 C, 5-20 min Viral inactivationinactivation °64-95 C, 5 min a total turnaround time of 1 hour for saliva despite lower viral titers tha”
Recombinase polymerase amplification: Basics, applications and recent advances (2017, methodology): “Basic Kit, SYBR Green I <30 37 <100 copies [4]”
In‐field molecular diagnosis of plant pathogens: recent trends and future perspectives (2018, methodology): “In 2006 a new isothermal amplification method for the detection of specific DNA sequences was reported (Piepenburg et al., 2006).”
Optimizing illumina next-generation sequencing library preparation for extremely at-biased genomes (2012, methodology): “We used recombinase polymerase amplification (RPA) technology as provided in the TwistAmp ™ DNA-amplifi-cation kit [11] to amplify 10 ng adapter-ligated library fragments.”