Multiplex Detection of Nucleic Acids Using Recombinase Polymerase Amplification and a Molecular Colorimetric 7-Segment Display

Abstract

Nucleic acid analysis has become highly relevant for point-of-care (POC) diagnostics since the advent of isothermal amplification methods that do not require thermal cycling. In particular, recombinase polymerase amplification (RPA) combined with lateral flow detection offers a rapid and simple solution for field-amenable low-resource nucleic acid testing. Expanding POC nucleic acid tests for the detection of multiple analytes is vital to improve diagnostic efficiency because increased multiplexing capacity enables higher information density combined with reduced assay time and costs. Here, we investigate expanding RPA POC detection by identifying a generic multiplex RPA format that can be combined with a generic multiplex lateral flow device (LFD) to enable binary and molecular encoding for the compaction of diagnostic data. This new technology relies on the incorporation of molecular labels to differentiate nucleic acid species spatially on a lateral flow membrane. In particular, we identified additional five molecular labels that can be incorporated during the RPA reaction for subsequent coupling with LFD detection. Combined with two previously demonstrated successful labels, we demonstrate potential to enable hepta-plex detection of RPA reactions coupled to multiplex LFD detection. When this hepta-plex detection is combined with binary and molecular encoding, an intuitive 7-segment output display can be produced. We note that in all experiments, we used an identical DNA template, except for the 5' label on the forward primer, to eliminate any effects of nucleic acid sequence amplification bias. Our proof-of-concept technology demonstration is highly relevant for developing information-compact POC diagnostics where space and time are premium commodities.

Figure 1

Lateral flow sandwich assay of single-plex RPA amplicons. (a) During the RPA reaction, (i) the 5′-labeled primer, reverse primer, and the 5′ FAM-labeled TwistAmp LF probe bind to DNA and (ii) the 3′ block (also known as blocker, e.g., C3-spacer) on the probe is removed by the enzyme Nfo, allowing (iii) extension by the Bst polymerase to create (iv) a dual-labeled double-stranded amplicon. Adapted from Li and Macdonald, Copyright 2014, with permission from Elsevier. (b) For single-plex LFD detection, a dual-labeled RPA amplicon is sandwiched between capture and detection antibodies via antigen–antibody binding. Capture antibodies (mouse anti-fluorescein antibody) are conjugated to AuNPs, which enables visualization of RPA amplicon binding through the appearance of a red colored test dot. Rabbit anti-mouse antibody, which can directly bind the capture antibody (mouse anti-fluorescein), is deposited in parallel as a control. (c) Results of single-plex RPA coupled with single-plex LFD detection using seven different labels incorporated on the 5′ forward RPA primer and with the corresponding anti-label antibody deposited as the detection antibody in the test zone of the LFD. (+) denotes that the RVFV S gBlock DNA template was added to the RPA reaction, whereas (−) indicates the negative control RPA reaction in the absence of a DNA template. (d) Representative analytical sensitivity of RVFV S RPA lateral flow sandwich assay showing the LOD using digoxigenin (NHS ester) as the 5′ molecular label. A representative photograph is shown, in which arrows indicate the position of control and test dots. The copy number of the serial diluted synthetic RFVF S template DNA is shown as gBlock DNA (copies/μL), and nuclease free water was used as no template control. The normalized black values (pixel densities) were used to calculate positives (indicated by *), negatives, and the cutoff (indicated by black dashed line). Positive results compared to the number of individual runs and analytical sensitivity as the percentage of correct results is displayed. Each assay was repeated at least twice, and a representative photograph is shown.

Figure 2

Specificity test results of all seven dual-labeled RPA amplicons on multiplexed LFDs. (a) Positioning of detection antibodies deposited upon multiplex LFDs. (b) Seven dual-labeled RPA amplicons (5 μL; with 5′ FAM and 3′ X, X = biotin, digoxigenin (NHS ester), TAMRA (NHS ester), Texas Red-X (NHS ester), Cascade Blue C6-NH, DNP-X C6-NH, or Dansyl-X C6-NH) were detected individually on multiplexed LFDs. The assay was repeated twice, and a representative photograph is shown.

Figure 3

Single-plex RPA in combination with multiplexed LFD detection. (a) Positioning of detection antibodies deposited upon multiplex LFDs (left); all seven dual-labeled RPA amplicons (3.0 μL of 5′ FAM/3′ biotin and 5′ FAM/3′ Dansyl-X C6-NH and 2.0 μL of 5′ FAM/3′ X, X = digoxigenin (NHS ester), TAMRA (NHS ester), Texas Red-X (NHS ester), Cascade Blue C6-NH, or DNP-X C6-NH) mixture was detected on the multiplexed LFD (right). (b) Positioning of the detection antibodies to form the 7 segments of the display. (c) Required number displays. (d) Addition of single-plex RPA dual-labeled amplicon signature mixtures (3.0 μL of 5′ FAM/5′ biotin and 5′ FAM/5′ Dansyl-X C6-NH and 2.0 μL of 5′ FAM/5′ X X = digoxigenin (NHS ester), TAMRA (NHS ester), Texas Red-X (NHS ester), Cascade Blue C6-NH, or DNP-X C6-NH) for defined number displays. (e) Resulting successful appearance of numbers (0–9). The assay was performed three times with similar results; a photograph of one test is shown.

Figure 4

Multiplex RPA in combination with multiplex lateral flow detection. (a) Positioning of the detection antibodies to form the 7 segments of the display. (b) Required number displays. (c) Addition of labeled RPA forward primer signature mixtures (5′ labeled with biotin, digoxigenin (NHS ester), TAMRA (NHS ester), Texas Red-X (NHS ester), Cascade Blue C6-NH, DNP-X C6-NH, or Dansyl-X C6-NH) with reverse primer, probe, and other RPA reagents in one reaction tube for defined number displays. (d) Resulting successful appearance of numbers (0−9) on the LFDs. The assay was performed three times with similar results; a photograph of one test is shown.