DNA detection using recombination proteins

Abstract

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.

Figure 1. Schematic of the RPA Process and the Recombinase/Primer Filament Formation

(A) Recombinase/primer filaments scan template DNA for homologous sequences (red/blue). Following strand exchange, the displaced strand is bound by gp32 (green), primers are extended by Bsu polymerase (blue). The net result of two opposing primer binding/extension events is the generation of one complete copy of the amplicon in addition to the original template. Repetition of the process results in exponential DNA amplification. (B) In the presence of ATP, uvsX (gray) binds cooperatively to oligonucleotides (red, top). Upon ATP hydrolysis, the nucleoprotein complex disassembles (left) and uvsX can be replaced by gp32 (green, right). The presence of uvsY and Carbowax20M shifts the equilibrium in favour of recombinase loading.

Figure 2. RPA Amplifies Specific Target Regions from Complex DNA Templates in less than 30 Minutes

(A) Acrylamide gel electrophoresis of RPA products using primers for three human markers (apoB, Sry, PBDG). Water (−) or 1,000 copies of genomic DNA (+) served as template. Ten percent of each reaction is loaded on the gel. The expected product sizes are 305, 371, and 353 base-pairs for apoB, Sry, and PBDG, respectively. (B) Real-time RPA using primers for the B. subtilis SpoB locus. Fluorescence upon intercalation of SybrGreenI into nascent product is detected. B. subtilis DNA served as template in triplicate reactions at 10 ⁵ (black), 10 ⁴ (red), 10 ³ (yellow), or 100 copies (green) or water (blue). The onset of amplification depends linearly on the logarithm of the starting template copy number [see inset; time in minutes (midpoint of growth curve) versus log {template concentration in copy number)].

Figure 3. Schematic of the Probe-Based RPA Detection Method

(A) Signal generation by separation of a fluorophore and a quencher depends on cutting of the probe by double-strand specific Nfo. (B) Arrangement of primers and probes relative to the targets used in Figures 4 and 5. See Protocol S1 for sequences of MRSA isoforms. A PCR fragment that fused an unrelated sequence to the target sites sccIII and orfX served as internal control.

Figure 4. RPA Enables Specific DNA Amplification from Few Template Molecules

(A) Probe signal of RPA reactions using the primer set orfX/sccIII (see Figure 3). MRSAIII DNA at 10 ⁴ (black, reactions 1–3), 10 ³ (red, 4–6), 100 (yellow, 7–9), ten (green, 10–12), or two (purple, 13–17) copies or water (blue, 18–20) served as template. (B) A plot of the onset time of amplification (defined as passing the 2.5 threshold) in reactions 1 to 12 in Figure 4A against the logarithm of the template copy number reveals a linear relationship.

Figure 5. A Multiplex RPA Approach Enables Detection of Different MRSA Alleles and an Internal Control in the Same Reaction

(A) MRSAI (green), MRSAII (dark blue), MRSAIII DNA (red) at ten copies, or MSSA DNA at 10 ⁴ copies (blue, negative control) or water (yellow, turquoise, negative controls) served as a template (in triplicate for each template condition). See Figure 3 for the arrangement of primers and probes used. (B) Detection of 50 copies of internal control DNA included in the reactions in (A). Note that two of the negative control sets (blue, yellow) included internal control template, whereas one set of reactions (turquoise) contained water only and served as “double negative” control.

Figure 6. RPA for MRSA Detection Can Be Combined with an Instrumentation-Free Read-out System

(A) Schematic of the principle of product generation and lateral-flow strip detection (see text for details). FAM label (green), biotin label (red), α-FAM-gold (purple), α-biotin antibody (Y, detection line, filled arrowhead), species-specific α-[α-FAM-gold] antibody (T, control line, open arrowhead). (B) Schematic of the lateral-flow probe design (top, here Lfs1) and its arrangement relative to amplification primers (bottom, here orfX and sccI/II). FAM label (green), biotin label (red). (C) Lateral-flow strips used for the detection of RPA products. Reactions contained (left to right) ten copies of MRSAIII, ten copies of MRSAII, ten copies of MRSAI, or 10,000 copies of MSSA (negative control) as template. Positive signals are generated in the first three reactions (filled arrowhead). The MSSA control reaction only produced a flow-control signal.