A magneto-DNA nanoparticle system for rapid detection and phenotyping of bacteria - PubMed (original) (raw)

A magneto-DNA nanoparticle system for rapid detection and phenotyping of bacteria

Hyun Jung Chung et al. Nat Nanotechnol. 2013 May.

Abstract

So far, although various diagnostic approaches for pathogen detection have been proposed, most are too expensive, lengthy or limited in specificity for clinical use. Nanoparticle systems with unique material properties, however, circumvent these problems and offer improved accuracy over current methods. Here, we present novel magneto-DNA probes capable of rapid and specific profiling of pathogens directly in clinical samples. A nanoparticle hybridization assay, involving ubiquitous and specific probes that target bacterial 16S rRNAs, was designed to detect amplified target DNAs using a miniaturized NMR device. Ultimately, the magneto-DNA platform will allow both universal and specific detection of various clinically relevant bacterial species, with sensitivity down to single bacteria. Furthermore, the assay is robust and rapid, simultaneously diagnosing a panel of 13 bacterial species in clinical specimens within 2 h. The generic platform described could be used to rapidly identify and phenotype pathogens for a variety of applications.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. Magneto-DNA assay for the detection of bacterial 16S rRNA

a, Schematic of the assay procedure. Total RNA is extracted from the specimen, and the 16S rRNA is amplified by asymmetric real time-PCR. Single-strand DNA of the amplified product is then captured by beads conjugated to capture probes, before hybridising with MNPs to form a magnetic sandwich complex. Samples are subsequently analyzed using a _μ_NMR system. b, Hybridised probe complexes, as observed by transmission electron microscopy (left, size bar 100 nm), scanning electron microscopy (center, size bar 300 nm), and atomic force microscopy (right, size bar 100 nm).

Figure 2

Figure 2. Detection sensitivity of the magneto-DNA system

a, Serial dilutions of synthetic DNA or bacteria-derived DNA were used as detection targets. Bacteria-derived DNA molecules were obtained via asymmetric RT-PCR of S. aureus 16S rRNA (35 cycles); synthetic DNA had the same sequence as bacteria-derived DNA. The detection limit was ~ 0.5 pM. b, Bacterial detection by the magneto-DNA assay. Samples with varying numbers of S. aureus were used. Total RNA was extracted and target sequences were amplified by 30, 35, and 40 cycles of RT-PCR. Amplified target DNA were detected using the probe set in a. The observed detection limit was a single bacterium, and the dynamic range of detection could be controlled by changing the PCR cycle number. c, Bacterial detection in blood. Serial dilutions of E. coli were spiked into human blood and processed by first lysing the red blood cells and then extracting the RNA using the same procedure as described above. All experiments were done in triplicate. All Δ_R_2 values were obtained by subtracting the relaxation rate values of the hybridised probe complexes in the presence of target DNA (_R_2,target), with the relaxation rate values of the beads alone (_R_2,control). Data are expressed as mean ± SD.

Figure 3

Figure 3. Universal detection of bacteria using the magneto-DNA system

a, Sequences of universal probes targeting a conserved region of bacterial 16S rRNA. Two probe sequences with a single-base difference were blended and used for capture. (R, Y) = (A, T) for Staphylococcus, Escherichia, Pseudomonas, Klebsiella, Enterobacter, Haemophilus, Stenotrophomonas; (R, Y) = (G, C) for Streptococcus, Enterococcus, Acinetobacter, Proteus, Lactobacillus. b, Thirteen different bacterial species could each be detected using the universal probes. c, Mixtures containing different bacterial types were detected by the universal probes. The observed Δ_R_2 values were consistent with average Δ_R_2 value (dotted line) from single species. Data are expressed as mean ± SD. All samples for the assay were prepared in triplicate.

Figure 4

Figure 4. Differential detection using the magneto-DNA system

Probes targeting hypervariable regions of bacterial 16S rRNA sequences were used to specifically detect various bacterial types. RNA was extracted from bacterial cultures, amplified by asymmetric RT-PCR (35 cycles) using specific primers for each species, and detected using the corresponding probe conjugates. a, Probes specific for Staphylococcus were used for detecting S. aureus (DNA amount equivalent to 50,000 CFU). Target DNA from other bacterial species were added as controls to test off-target binding of the probes. b, Relaxation rates for differential detection of various bacterial types. Note the high specific signals and low background noise against other bacteria. Data are expressed as mean ± SD. All samples for the assay were prepared in triplicate. c,d, Heat maps comparing the specificity of the magneto-DNA assay with that of qPCR. Specificities in c were based on Δ_R_2 values from the magneto-DNA assay shown in b. Specificities in d are relative target amounts obtained from qPCR in Figure S5. Significant signals were marked as positive: positive signals for specific target bacteria were regarded as ‘true-positive’, while positive signals from non-targeted samples were classed as ‘false-positive’.

Figure 5

Figure 5. Diagnosis of clinical samples

Detection of pathogens by the magneto-DNA nanoparticle system using universal (a), and specific (c) probes for each bacteria type. b, Heatmap of obtained Δ_R_2 values for universal and specific detection. Clinical specimens (300 _μ_l for each sample) were processed to extract total RNA. This was followed by asymmetric RT-PCR (35 cycles) with universal and specific primers for all bacterial types. The PCR products (equivalent to 0.3 _μ_l volume of sample) were magnetically labeled and detected by _μ_NMR. a, Two of the 9 clinical samples tested negative, which correlated well with standard culture results. The other 7 samples were positive. ND represents samples with no pathogens detected. c, Identification of pathogen types within each sample. Note that some samples were infected with more than one pathogen, and the identified bacterial types correlated well with standard culture (see also Supplementary Table S3). Data are expressed as mean ± SD. All samples for the assay were prepared in triplicate.

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