Fig 1.
Solution-based versus surface-based hybridization capture.
(A) Solution-based hybridization hybridizes probes to target in solution, followed by capture of probe-target complexes on magnetic beads. (B) Surface-based hybridization hybridizes target to probes that are pre-immobilized on magnetic beads.
Table 1.
Previous uses of sequence-specific hybridization capture for sample preparation without bacterial culture, pre-purification, or pre-amplification.
Table 2.
Probe, primer, and target sequences.
Fig 2.
Hybridization capture probe design and procedure.
(A) Schematic illustrating hybridization capture probe design. Two short, sequence-specific probes, one complementary to each strand of the double-stranded target region, are immobilized on streptavidin-coated magnetic beads via dual biotin linkers. (B) Overview of hybridization capture procedure. Beads with pre-immobilized capture probes are added to 10 mL urine (along with 1 M NaCl and 0.1% Tween-20), heat denatured (15 minutes at >90°C), hybridized to target DNA (30 minutes at room temperature), and washed to remove non-target DNA (2X high-salt wash, 1X low-salt wash). Purified target-specific DNA is eluted under basic conditions, neutralized, and amplified by short-target qPCR.
Fig 3.
Analytical performance of the hybridization capture method.
(A) The hybridization capture method recovers nearly all target-specific dsDNA (103 copies of 50 bp positive control) spiked into 10 mL urine (mean ± SD, n = 6 independent experiments processed on different days). (B) Representative calibration curves for DNA recovered by hybridization and qPCR standards overlap across concentrations from 0.5–104 copies/mL 50 bp dsDNA in urine (mean ± SD, n = 3). (C) The hybridization method reliably distinguishes 5 copies of 50 bp dsDNA spiked into 10 mL urine (0.5 copies/mL) from negative controls (mean ± SD, n = 6). (D) Hybridization has high recovery across fragment lengths from 25–150 nt (mean, n = 3 independent experiments processed on different days, 103 copies ssDNA input). Each dot represents the mean of 3 technical replicates performed for each independent experiment. One experiment was excluded for the 80 nt fragment length due to a calculated recovery of >200% caused by delay in amplification of the PCR standards. ns indicates not significant (one-way ANOVA with post-hoc Tukey test). (E) Hybridization is tolerant to variations expected in clinical urine samples, including pH (5–8), non-target DNA (0–10 μg), and salt (0–500 mM) (n = 1, 103 copies 50 nt ssDNA input). (F) Hybridization enables amplification of entire output from 10 mL urine in a single qPCR well without inhibition. Eluate extracted from pooled urine (no added target) was spiked into qPCR containing a constant target concentration (103 copies 50 nt ssDNA). Resistance to qPCR inhibition is indicated by the lack of increase in quantification cycle (Cq) despite increasing fraction of eluate (mean, n = 3).
Fig 4.
Two probe system enables recovery of both strands of double-stranded DNA.
(A) Rationale for two-probe system. When using a single capture probe (left column), only one strand of the dsDNA target is recovered by hybridization; the other strand is discarded. When a second capture probe is added to target the complementary strand (right column), both strands of dsDNA are recovered, doubling the concentration of detectable DNA. (B) Schematic of two-probe system design. To minimize target footprint, probes are truncated versions of the forward and reverse qPCR primers (15–20 nt). Probes hybridize to different sub-regions of the target sequence so that opposite probes do not hybridize to each other. (C) Using both capture probes results in an approximate 2-fold increase in detected dsDNA compared to either single probe (mean, n = 3 technical replicates from the same experiment; 103 copies 50 bp dsDNA input). Each target strand was quantified by a different standard curve, so the minor difference observed in calculated percent recovery for BP1 and BP2 is unlikely to be meaningful (see S1 Fig; same applies for panels D-E). (D) Addition of a second probe does not affect recovery of ssDNA by the opposite probe (mean, n = 3 technical replicates from the same experiment; 103 copies 50 nt ssDNA input). (E) Re-hybridization of complementary dsDNA target strands is negligible compared to probe-target hybridization and does not affect recovery (mean, n = 3 technical replicates from the same experiment; 103 copies 50 nt ssDNA or 50 bp dsDNA input). Because plots in this figure show technical replicates from the same experiment, the data are not independent and statistical analysis was not conducted.
Fig 5.
Dual biotinylated capture probes improve hybridization performance compared to single biotinylated capture probes.
(A) Dual biotinylated probes (in orange) facilitate optimal spacing and density of probes on the bead surface, increasing recovery and reducing reliance on the probe concentration used during bead functionalization compared to single biotinylated probes (in black) (n = 3 independent experiments on different days; 103 copies 50 nt ssDNA input). (B) Beads functionalized with dual biotinylated probes are thermostable up to at least 90°C, while beads functionalized with single biotinylated probes lose function after heating to 90°C. Grey columns labeled as “+” were heated to >90°C for 15 minutes prior to hybridization; white columns labeled as “-”were unheated controls (mean, n = 3 independent experiments on different days; 103 copies 50 nt ssDNA input). (C) Beads functionalized with dual biotinylated probes tolerate incubation with free biotin, while beads functionalized with single biotinylated probes have reduced recovery after incubation with free biotin. Grey columns labeled as “+” were incubated for 15 minutes with 1 μM free biotin; white columns labeled as “-”were controls not incubated with free biotin (mean, n = 3 independent experiments on different days; 103 copies 50 nt ssDNA input). ** indicates P value of 0.001 to 0.01, **** indicates P value of <0.0001, ns indicates not significant (one-way ANOVA with post-hoc Tukey test).