Magnetic barcode platform for MTB detection

Figure 1a illustrates the magnetic barcode assay, designed to detect mycobacterial nucleic acids. In the current prototype, sputum samples are first processed off-chip to extract DNA from MTB. We adopted a simple mechanical method based on vigorous mixing with glass beads. Following the DNA extraction, the target DNA region is amplified through PCR. The amplicons are captured on polymeric beads (diameter: 1 μm) modified with complementary capture DNAs. Subsequently, the beads are rendered superparamagnetic by coupling MNPs (30 nm) to the opposite end of the amplicon. This scheme enhances detection specificity through the simultaneous tagging by the capture and the probe DNAs; it also offers fast binding kinetics (<1 min) as the labelling is performed in a small sample volume. The magnetic nanoparticles contain iron oxide cores encased by heat-resistant crosslinked dextran shells, to ensure high stability in varying salt conditions and the required nucleotide-annealing temperatures. After removing excess MNPs, samples are subject to NMR measurements. MNP-loaded beads produce local magnetic fields, which lead to faster relaxation of the 1H NMR signal. The decay rate is proportional to the MNP concentration (and thus initial DNA), enabling quantification of target DNAs.

Figure 1: Magnetic barcode assay for sensitive TB detection. (a) Assay procedure. From unprocessed sputum specimen, DNA is extracted though an off-chip mechanical stressing. Extracted DNA samples are then loaded into a fluidic device for on-chip processing. The target DNA sequences are amplified by asymmetric PCR and captured by polymer beads modified with capture DNA. MNPs are then used to specifically coat the beads via complementary sequence, and the samples are subjected to NMR measurements. The MNP-labelled beads accelerate the decay of NMR signal, providing analytical signal for nucleic acid detection. The entire assay time is ~2.5 h. (b) A fluidic cartridge was developed to streamline the assay. The device integrates PCR chambers, mixing channels and a microcoil for NMR measurements. The entire system was designed as a disposable unit to prevent cross-contamination of PCR-amplified products. Whole-genomic DNA, extracted from expectorated samples, capture beads and MNPs are loaded into inlet chambers gated by screw valves. After on-chip PCR, magnetic labelling of the beads takes place along the mixing channel. The magnetically barcoded beads are then purified and concentrated into the μNMR probe (microcoil) by the membrane filter. Scale bar, 1 cm. (c) Scanning electron microscopy confirmed the bead capture by the membrane filter. Scale bar, 1 μm. Transmission electron microscopy revealed that beads are efficiently labelled with MNPs (inset). Scale bar, 30 nm. Full size image

To streamline the assay procedure, we developed a microfluidic device for on-chip magnetic barcode assay (Fig. 1b). The device performs key functions of the assay: PCR amplification, magnetic labelling and NMR measurements (Supplementary Fig. S1). The assay uses sputum samples that are mechanically liquefied and loaded into the on-chip PCR chambers along with PCR reagents. MNPs and buffer solutions are loaded into separate chambers gated by valves. After target DNA sequences are PCR-amplified, the PCR products are combined with capture beads. The bead-DNA mixture and MNPs are then introduced into the extended mixing channel. The MNPs would only bind to the capture beads in the presence of the target amplicons. The MNP-labelled beads are purified by an in-line membrane filter (Fig. 1c) and concentrated into the miniaturized NMR (μNMR) chamber for detection (see Methods for detailed fluidic operations). Custom-designed portable NMR electronics are used to monitor and compensate for temperature drifts, which enables robust measurements across different temperature environments (4–50 °C)17.

Assay optimization

We first optimized the assay protocol to maximize the magnetic signal. Samples were prepared using synthetic 92-nucleotide (nt) single-stranded DNA (ssDNA) specifically found within the acyl-CoA dehydrogenase fadE15 gene of MTB19,20. Non-complementary 92-nt ssDNA was used for control samples. The transverse relaxation rate (R 2 ) of samples was measured, and the magnetic signal was defined as R 2 ratio between the target and the control samples (see Methods for details). When ssDNA and double-stranded DNA samples were compared (Supplementary Fig. S2a), ssDNA displayed higher signal; double-stranded DNA required additional denaturing and annealing steps, which lowered the binding stability of capture beads and MNP probes. For the ssDNA samples, the highest signal could be obtained when the target ssDNA was first captured by beads and then labelled with MNPs; this approach presumably minimized competitive binding to ssDNA between capture beads and MNPs21. Based on these results, we adopted the asymmetric PCR and sequential labelling strategy for target DNA production and its magnetic targeting, respectively.

The assay conditions for sequential labelling were further refined (Supplementary Fig. S2b). Four different sizes of capture beads ranging from 0.5 to 5 μm were tested to determine the most efficient substrate for DNA capture. After the capture beads were incubated in the ssDNA solution and labelled with MNPs, NMR measurements were performed under the same weight concentration for each bead size. The 1-μm capture beads provided not only large surface area for capturing and magnetic labelling but also had the lowest nonspecific binding, which resulted in the highest magnetic signal. The optimal incubation temperature was ~37 °C, agreeing with the melting temperature (t m =45 °C) of 92-nt fadE15 amplicons estimated by hairpin analysis22. With each MNP conjugated with >50 probe strands, the magnetic labelling of beads could be completed in <1 min, benefiting from the multiple binding valency of MNPs.

The developed assay protocol was applied to detect 92-nt segment of fadE15 ssDNA (Fig. 2a). Fluorescence imaging showed strong co-localization of the near-infrared fluorescent MNPs with green-fluorescent capture beads (Fig. 2b). Flow-cytometry analysis further showed highly specific MNP labelling on beads only in the presence of target ssDNA (Fig. 2c). On-chip μNMR measurements confirmed the specific detection of the target ssDNA (Fig. 2d) with similar signal-to-noise ratio as in flow cytometry. Note that the μNMR required much lower number of beads: ~106 beads in 1 μl volume compared with ~109 beads in 250 μl volume for flow cytometry. The sequence-specific hybridization between oligonucleotides further enabled highly selective amplification of MNP loading onto the bead surface, offering versatility in magnetic labelling. For example, by applying a pair of MNPs conjugated with complementary oligonucleotide sequences, we could form multiple MNP layers and thus amplified particle loading onto beads (Fig. 2e, left). The resulting NMR signal for target samples increased by nearly threefold, whereas control samples showed negligible increase in identically treated beads using non-complementary ssDNA (Fig. 2e, right).

Figure 2: Assay optimization and amplification process. (a) A segment (92-nt) of fadE15 ssDNA was used as a detection target. Capture beads and MNPs were conjugated with complementary oligonucleotides. (b) Following on-chip labelling, confocal microscopy of the magnetically barcoded beads were performed. The fluorescent polystyrene beads (green) co-localized with the near-IR fluorescence of the MNPs (red) in the presence of target fadE15 ssDNA. Scale bar, 5 μm. (c) Measurements by flow cytometry confirmed target-specific labelling of the beads. Non-complementary ssDNA samples showed low signal close to that of control. (d) Corresponding μNMR detection also displayed strong signal with the presence of fadE15 amplicons. (e) Sequential layering of the capture beads was performed using MNPs conjugated to alternating oligonucleotide sequences. Such layering amplified the number of MNP probes on the bead surface and therefore increased the overall magnetic signal. The error bars in d and e represent the s.d. of three replicates (n=3). Full size image

Detection sensitivity and specificity

The sensitivity of the magnetic barcode platform was comprehensively characterized using samples in different formats. We first used serially diluted fadE15 ssDNA samples, and determined the absolute detection limit of the magnetic barcode assay; without the PCR amplification step, the detection limit was ~1 nM ssDNA in 1 μl sample volume (Fig. 3). We next loaded genomic MTB DNA into the device, amplified the 92-nt segment of fadE15 via asymmetric PCR and performed the magnetic barcode assay. With PCR amplification of the target gene, the system could detect down to 1–5 genomic DNA in buffer solution (Fig. 4a), demonstrating the potential to detect a single bacterium.

Figure 3: Titration assay and PCR characterization of MTB genomic DNA. (a) The detection sensitivity of the magnetic barcode assay without PCR amplification was determined. Samples containing 92-nt fadE15 ssDNA were serially diluted and magnetically labelled. The detection limit was ~1 nM of ssDNA in 1 μl sample volume. The error bars in a represent the s.d. of three replicates (n=3). (b) Real-time PCR was used to correlate the amount of genomic DNA and to determine the detection sensitivity. Number of genomic copies was estimated using mass calculation, assuming molecular weight of 660 per base pair and MTB genome size of 4411529. Serial dilution of genomic DNA sample was performed to establish reverse transcription-PCR standard. Separate genomic DNA dilution was performed to contain 1, 10, 25, 100 and 1000 genome, and Ct values of the samples were compared with the standard. The sample dilution was used for μNMR measurements. Full size image

Figure 4: MTB detection sensitivity using the magnetic barcode assay. (a) The detection limit of the platform with PCR steps was established. Genomic MTB DNA was loaded on the fluidic chip, and the 92-nt segment of fadE15 amplicons were prepared by asymmetric PCR. Titration experiments with initial DNA loading showed that the assay could detect down to a few genomic DNA in buffer solution. (b) Detection of MTB within sputum samples. Whole MTB cells were spiked into 0.5 ml aliquots of MTB-negative sputa to the final concentrations ranging from 0–107 CFU ml−1. Following the off-chip DNA extraction, samples were measured by the magnetic barcode assay. The sensitivity was 102−103 CFU ml−1. (c) Control samples containing non-MTB bacteria (106 CFU ml−1) spiked into the sputa were used to confirm the specificity of the primers and barcode assay. Samples measured from the non-MTB controls displayed baseline magnetic signal, similar to the blank sputa in which no MTB was present. (d) Clinical sputum specimens were analysed with the μNMR assay. Compared with the samples collected from MTB-positive patients, the samples collected from MTB/HIV-positive patients showed higher μNMR signals. MTB-negative sputa collected from healthy volunteers were used as negative controls. The error bars in a–d represent the s.d. from triplicate measurements. Full size image

To evaluate the overall detection sensitivity, we used sputum samples spiked with live MTB. To mimic clinical cases, we varied the final MTB concentration up to 107 colony forming unit (CFU) ml−1. Genomic DNA was first released off-chip through the mechanical disruption, and divided into two aliquots. The first half was used for conventional real-time PCR (Fig. 3b). The other half was processed by the magnetic barcode device; after the asymmetric PCR of 92-nt fadE15, we introduced capture beads into the PCR chamber to capture the amplicons, and then magnetically labelled the beads along the microfluidic channel. Titration measurements established that the detection limit was ~103 spiked MTB in 1 ml sputum (Fig. 4b). The observed sensitivity was lower than with pure genomic DNA samples. This could be attributed to suboptimal DNA extraction with spiked samples. Additional sample loss presumably happened during the off-chip DNA extraction and transfer processes. The current magnetic barcoding assay, however, was superior to smear test (detection threshold ~104 CFU in 1 ml of sputum) and was considerably faster (2.5 h) than culture-based method that requires weeks23,24. To improve the sensitivity, we intend to combine the mechanical extraction process with chemical treatment and DNA purification. Note that the barcode assay also showed a good correlation with separate real-time PCR of whole-genomic extracts (Fig. 3b).

Signals from sputum samples containing high concentration (106 CFU ml−1) of clinically relevant non-MTB species (Streptococcus pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Haemophilus influenzae) were nearly identical to those from control sputa (Fig. 4c), indicating that nonspecific amplification and/or binding of the primers was negligible. Such high specificity would be crucial to achieve accurate MTB detection from complex specimens, which may contain multiple bacterial strains.

TB detection in clinical samples

To evaluate the clinical utility of the magnetic barcode platform, we analysed clinical sputum samples from MTB smear-positive patients, and as a negative control, sputa from healthy patients without a diagnosis of MTB infection. The magnetic barcode assay detected the presence of MTB in all MTB-positive patient samples (Fig. 4d). With control samples, the signal was at the baseline level, confirming the specificity of the assay. Interestingly, the assay performed well in patients with MTB/HIV co-infection. Diagnostics for these patients remain unclear because the standard approaches often perform poorly, yet studies have shown that HIV co-infection induces greater bacterial burden by accelerating the growth of MTB11,25,26. This finding supports our measurement of a higher bacterial burden in co-infected patient sputum.

Analysis of single-nucleotide mutation

Drug-resistant TB strains can be detected by identifying specific genetic mutations. For instance, over 95% of RIF-resistant MTB strains have mutations within the 81-nt core region of the rpoB gene (Fig. 5a, top), whereas the mutation is absent in nearly all RIF-susceptible strains2. Approximately 90% of RIF-resistant MTB is also resistant to isoniazid, which makes the mutant rpoB gene as a potential surrogate marker for multi-drug-resistant TB27.

Figure 5: Magnetic detection of single-nucleotide polymorphism. (a) Magnetic barcode assay was optimized to detect point mutations in rpoB gene (Q513E, C-to-G; H526Y, C-to-T; S531L, C-to-T), which confer drug (RIF) resistance to MTB. When the capture beads are complementary to the rpoB WT ssDNA, both fluorescence and magnetic readouts were indiscernible among rpoB alleles (left). When the capture DNA sequence was modified to fully match the single-nucleotide polymorphism in S531L-mutant strand, the magnetic barcode assay could selectively detect the target gene (right). (b) The optimized set of magnetic barcode probes (Supplementary Fig. S5a) could effectively detect the presence of MTB (via fad15E probes) as well as analyse the mutational status (via rpoB probes). For each probe, the signal levels were normalized against that of a WT sample. The data in a and b are displayed as mean±s.d. from triplicate measurements. (c) Magnetic profiling on a panel of MTB strains in sputum samples. RIF-resistant W-Beijing strains, and WT H37Rv strains were spiked in sputa (104 CFU ml−1); non-MTB mixed bacteria was used as the control. Following the DNA extraction, samples were loaded onto the device for PCR and magnetically labelled. The heat map showed the universal MTB detection using the fadE15 probes, as well as the sequence-specific identification of RIF-resistance with the rpoB probes. Full size image

We set out to optimize the magnetic barcode assay for fast detection of single-nucleotide polymorphism on the segments of the rpoB gene. We initially focused on detecting the C-to-T mutation in codon 531 (S531L), which is the most common amino-acid substitution responsible for RIF resistance. This mutation is observed in 70% of drug-resistant strains found in the clinics14, and these strains are reported to be the most transmissible and have the highest fitness level (that is, survival under pressure)28. Two types of capture 20-nt oligonucleotide for the rpoB amplicons were designed, one fully complementary to wild-type (WT) strand and the other to the S531L-mutant strand. The 81-nt rpoB ssDNA assumed a high hairpin melting temperature at ~68 °C (ref. 22), requiring higher incubation temperate (60 °C) during the magnetic labelling. With the WT-capture beads, the barcode assay universally detected rpoB strands regardless of their mutational status (Fig. 5a, left). Capture beads that fully complement with S531L mutation, on the other hand, were able to distinguish the specific mutation (Fig. 5a, right); magnetic signals were >400% higher with S531L-mutant strands than with WT or other rpoB mutant strands (Q513E, C-to-G mutation in codon 513; H526Y, C-to-T mutation in codon 526). Interestingly, when a similar strategy was applied to the MNPs, that is, having the oligonucleotide on the MNPs match the mutant rpoB ssDNA, no difference in binding affinity was observed between the target mutant and other strands (Supplementary Fig. S3). It is likely that the multiple binding valency of MNPs resulted in higher binding affinity with the rpoB strands and prevented the discrimination of the single-nucleotide mismatch29.

Capture oligonucleotides probes were further developed to detect other rpoB mutations that lead to clinically relevant amino-acid substitutions, H526Y and Q513E. Unlike the case with S531L, initial 20-nt probes for H526Y and Q513E were ineffective in discriminating target mutant strands (Supplementary Fig. S4). Analysis of the rpoB ssDNA revealed that the binding region for the S531L capture probe contains higher G/C contents and forms stable hairpin structure (t m =80 °C), whereas Q513E and H526Y regions have less stable hairpin structure (t m =50–60 °C). We thus hypothesized that reducing the length of capture probes would improve the specificity to Q513E and H526Y by decreasing the thermodynamic stability for single-nucleotide mismatch binding. Indeed, when the capture sequence was reduced to 15 nt, high selectivity for corresponding mutant strands could be achieved (Supplementary Fig. S4). These probes displayed consistent, high signal-to-noise ratios (~300%) and complemented the fadE15 probe for comprehensive MTB analyses (Fig. 5b).

We finally tested the developed platform to detect RIF-resistant and WT strains of MTB. RIF-resistant MTB colonies were cultured and screened for specific rpoB mutations through sequencing. Samples were prepared by spiking MTB in sputa. Extracted DNA samples were amplified for 126-nt segment of rpoB and 92-nt segment of fadE15 regions, followed by the labelling with the optimized probe set (Supplementary Fig. S5a). Although the WT strain yielded only positive signal for the fadE15 probes, the Q513E, H526 and S531L-mutant isolates yielded positive signals for both the fadE15 and the corresponding mutant rpoB probes (Fig. 5c and Supplementary Fig. S5b). We further profiled a mixed population of WT and RIF-resistant strains (Fig. 6). Through multi-channelled measurements using both WT and mutant-specific probes, we could determine the ratio between RIF-resistance and susceptible MTB; such a capacity could potentially be used to study the bacterial mutation rate in culture during antibiotic treatments30. One of the methods that could be implemented to detect low ratios of resistant strain in mixed population would be to combine the magnetic barcode assay with droplet-based PCR.31