Fig 1.
Schematic of amplification and ligation in the existing oligonucleotide ligation assay (OLA).
The pol gene of HIV DNA is amplified by nested PCR. Note that the HIV DNA gene map positions are not to scale. A portion of the amplicon is mixed with three oligonucleotide probes: a 5’ fluorescein (F) -conjugated mutant (MUT)-specific HIV probe; a 5’ digoxigenin (D) -conjugated wild-type (WT)-specific probe; and a 5’ phosphorylated, 3’ biotin (B) -conjugated common probe. When specific probes are complementary at the mutation site, they are ligated to the common probe to create a DNA strand with labels at both ends. Only ligated products are detected during the CDD procedure (surface capture, denaturation of oligonucleotide from target DNA, and enzyme-based detection).
Fig 2.
Plate and paper formats for Capture, Denaturation and Detection (CDD) in the oligonucleotide ligation assay.
(A) Plate CDD procedure. Products from the ligation step (both ligated and non-ligated products) are captured on a streptavidin-coated plate. Non-ligated probes are released during oligonucleotide denaturation, and ligated MUT and WT probes are then detected in sequential enzyme-based immunoassays (labeling by different detection antibodies, alkaline phosphatase yellow substrate development, optical density reading at 405nm, wash steps, tetramethylbenzidine (TMB) development, stop solution, and optical density reading at 450nm). (B) Paper CDD procedure. Similar to the plate CDD procedure, products from the ligation step (both ligated and non-ligated products) are captured. However, here the products are captured on paper strips by immobilized streptavidin. Non-ligated probes are released during oligonucleotide denaturation. Antibodies targeting the end-labels of the mutant (MUT) or wild-type (WT) probes have conjugated horseradish peroxidase (POD) that converts 3,3’ diazoaminobenzidine substrate (DAB) into brown precipitates. Signals were captured by the scanner (600 DPI). Reported signals represent capture spot intensity minus a background region from the strip.
Fig 3.
Comparison of paper Capture, Denaturation, and Detection (CDD) and plate CDD for analysis of a plasmid standard mixture series for mutation Y181C.
(A) Scanned images of paper CDD MUT (left) and WT (right) detection (B) Mutant (MUT) detection (C) wild-type (WT) detection. Left axes: Specimen optical density (OD) minus negative control OD analyzed in duplicate by plate CDD (mean ± SE). Right axes: Specimen capture intensity minus background intensity analyzed in triplicate by paper CDD (mean ± SE). The ratio of mean signal intensities for 2% MUT and 0% MUT was 2.00 for plate CDD and 2.74 for paper CDD.
Fig 4.
Analysis of clinical specimens and plasmid standards by paper capture, denaturation, and detection (CDD) and plate CDD for mutations K103N and Y181C.
Panels A and C show mutant (MUT) detection, and Panels B and D show wild-type (WT) detection. Sample optical density (OD) minus negative control OD (left y axis) for each specimen is shown in white/gray by rank along the x axis, from the lowest MUT OD, followed by the plasmid standards (0%, 5%, 50% MUT) performed in duplicate. Spot intensity minus background intensity (right y axis) for each specimen is shown in blue and green bars followed by the plasmid standards (0%, 5%, 50% MUT) performed in triplicate. Scanned images of the paper CDD detection strip are shown below each specimen’s signal data.
Fig 5.
Analysis of clinical specimens and plasmid standards by paper capture, denaturation, and detection (CDD) and plate CDD for mutations M184V and G190A.
Panels A and C show mutant (MUT) detection, and Panels B and D show wild-type (WT) detection. Sample optical density (OD) minus negative control OD (left y axis) for each specimen is shown in white/gray by rank along the x axis, from the lowest MUT OD, followed by the plasmid standards (0%, 5%, 50% MUT) performed in duplicate. Spot intensity minus background intensity (right y axis) for each specimen is shown in pink and orange bars followed by the plasmid standards (0%, 5%, 50% MUT) performed in triplicate. Scanned images of the paper CDD detection strip are shown below each specimen’s signal data.
Fig 6.
Correlation plot of MUT Ratios determined by paper capture, denaturation, and detection (CDD) versus plate CDD.
MUT and WT signals obtained by paper and plate CDD format OLA at codons K103N, Y181C, M184V and G190A from 26 clinical specimens were used to calculate MUT Ratios. The overall shape reflects the signal saturation of the paper CDD at high MUT concentration, as seen in Figs 3–5. Results correlate strongly across clinical specimens and plasmid standards’ data (see S4 Fig for fitted correlation).