Micro-NMR Platform: Genetic Detection of Infectious Pathogens

  • Fig. 1: Schematic of the magnetic barcode assay for detection of infectious pathogens. (a) The nucleic acid is extracted from the bacteria directly from blood or sputum specimen, and amplified by asymmetric PCR. The amplicons are then captured by polymer beads, hybridized with MNPs via complementary sequence, and analyzed by a μNMR system. (b) A disposable, fluidic cartridge was developed by integrating PCR chambers, mixing channel and a microcoil for μNMR measurements. Following on-chip PCR, beads are magnetically barcoded along the mixing channel, and concentrated into the microcoil by the membrane filter (scale bar: 1 cm). (c) Electron microscopy analysis confirmed the bead capture on the membrane filter (scale bar: 1 mm) and labeling by the MNPs (inset, scale bar: 30 nm).Fig. 1: Schematic of the magnetic barcode assay for detection of infectious pathogens. (a) The nucleic acid is extracted from the bacteria directly from blood or sputum specimen, and amplified by asymmetric PCR. The amplicons are then captured by polymer beads, hybridized with MNPs via complementary sequence, and analyzed by a μNMR system. (b) A disposable, fluidic cartridge was developed by integrating PCR chambers, mixing channel and a microcoil for μNMR measurements. Following on-chip PCR, beads are magnetically barcoded along the mixing channel, and concentrated into the microcoil by the membrane filter (scale bar: 1 cm). (c) Electron microscopy analysis confirmed the bead capture on the membrane filter (scale bar: 1 mm) and labeling by the MNPs (inset, scale bar: 30 nm).
  • Fig. 1: Schematic of the magnetic barcode assay for detection of infectious pathogens. (a) The nucleic acid is extracted from the bacteria directly from blood or sputum specimen, and amplified by asymmetric PCR. The amplicons are then captured by polymer beads, hybridized with MNPs via complementary sequence, and analyzed by a μNMR system. (b) A disposable, fluidic cartridge was developed by integrating PCR chambers, mixing channel and a microcoil for μNMR measurements. Following on-chip PCR, beads are magnetically barcoded along the mixing channel, and concentrated into the microcoil by the membrane filter (scale bar: 1 cm). (c) Electron microscopy analysis confirmed the bead capture on the membrane filter (scale bar: 1 mm) and labeling by the MNPs (inset, scale bar: 30 nm).
  • Fig. 2: Detection using the magnetic barcode assay. (a) Probes targeting a short segment of a gene or the hypervariable regions of bacterial 16S rRNA sequences were used to detect specific pathogens. (b) The designed probes for each bacterial type showed high selectivity with minimal off-target binding. Samples containing DNA from control bacterial species displayed baseline μNMR signal.
  • Fig. 3: Analysis of patient samples. (a) The samples collected from MTB/HIV-positive patients showed higher μNMR signals compared to the samples collected from MTB-positive patients. (b) Heat map of μNMR signals obtained from the clinical specimens correlated well with standard culture results.
  • Fig. 4: Magnetic detection of antibiotic-resistant strains. (a) Magnetic barcode assay was optimized to detect mutation in codon 531 in rpoB gene that confer resistance to rifampin. In the presence of the S531L mutant DNA, both magnetic and fluorescence measurements showed higher signals. (b) Antibiotic resistance genes mecA and VPL were analyzed in several bacterial strains. Methicilin-resistant S. aureus strains BAA-1720 is positive for mecA and strain BAA-1707 is positive for VPL and mecA. The other bacterial strains are negative for the resistance genes.

Infectious pathogens continue to be a significant challenge in public health due to the lack of fast and sensitive detection technology. Here, we introduce a new diagnostic platform that is capable of rapid and specific profiling of pathogens directly in clinical samples. The platform detects nucleic acids based on a nuclear magnetic resonance (NMR) assay. We utilized this platform to detect various clinically relevant bacterial strains in whole blood and sputum, with sensitivity down to single bacterium within 2.5 hours [1,2].

Assay design
For the specific detection of bacteria, we targeted a short component on either the genomic DNA or the 16s rRNA region, and amplified the sequence using polymerase chain reaction (PCR) to produce large numbers of single-stranded DNA. Following the nucleic acid extraction and amplification process, the resultant DNAs (amplicons) are captured on polymeric beads conjugated with complementary capture-DNAs (Fig. 1a). Subsequently, the beads are rendered superparamagnetic by coupling magnetic nanoprobes (MNPs) to the opposite end of the amplicon. These magnetically barcoded beads increase the transverse relaxation rate (R2) of the sample, which is detected by a miniaturized NMR (μNMR) probe.

To prevent cross-contamination between samples, as well as to streamline the assay, we developed a microfluidic cartridge that performs key functions of the assay procedure (Fig. 1b). The processed sample and PCR reagents along with the capture beads and MNPs were contained within individual chambers on the cartridge. After PCR amplification, the bead-DNA mixture and MNPs were flowed along the mixing channel, purified by an inline membrane filter, and concentrated into the μNMR chamber for measurement. The MNPs would only hybridize with the beads and generate the signal in the presence of the target amplicons (Fig. 1c). Since the R2 is proportional to the concentration of MNPs bound to the beads, the concentration of nucleic acids could be quantified.

Performance and Sensitivity
We amplified a sequence within the acyl-CoA dehydrogenase fadE15 gene for the detection of Mycobacterium tuberculosis (MTB), and targeted a hypervariable region within the 16S rRNA for the identification of different bacterial species (Fig.

2a). By designing a specific combination of the capturing and labeling probes, the amplified DNA from target pathogens could be detected with negligible background signals from other species (Fig. 2b).

Combining the assay with PCR amplification, the device could detect as few as 1–2 bacteria per 10 ml of blood, and a range of 102–103 bacteria per 1 ml of sputum. The detection through 16s rRNA amplification could potentially give higher sensitivity than through genomic DNA due to the larger abundance of the target nucleic acids. The decrease in sensitivity for MTB could be caused by the thick mycobacterial cell wall that required more stringent nucleic acid extraction process.

To evaluate the clinical utility of the μNMR platform, we analyzed patient specimens. In the first part, we analyzed sputum samples from MTB smear-positive patients and compared the results with sputa from healthy patients (Fig. 3a). The DNA-μNMR assay confirmed the presence of MTB in all MTB-positive patient samples, whereas the negative control samples showed negligible signals. The results also showed higher signals in sputa from patients with MTB/HIV co-infection, and supported the studies which have shown that HIV coinfection induces greater bacterial burden by accelerating the growth of MTB [3]. In the second part, we analyzed blood aspirate samples from patients with suspected infections (Fig. 3b). The magnetic assay not only accurately detected all bacterial species identified by standard culture, but also identified other species that were undetectable by culture methods. These false negative results could have been caused by the growth inhibition that occurred during culture.

Drug-resistant bacteria strains can be identified by designing probes that detect specific genetic mutations or target mRNA. For the identification of rifampin-resistant MTB, nucleic acid probes were designed to detect single- nucleotide polymorphisms on the core region of the rpoB gene [4]. The combination of capture beads and labeling probes designed to be fully complementary with the target mutation showed higher signals in the presence of the target strain (Fig. 4a). By expanding the assay with reverse transcription, we could identify the mRNA of mecA and Panton–Valentine leukocidin genes and differentiate the methicillin-resistant Staphylococcus aureus (Fig. 4b) [5].

Summary
We have developed a magnetic barcode assay for fast and portable detection of infectious pathogens by integrating PCR, fluidics and NMR probe into a portable, easy-to-use device format. This detection method is highly sensitive and robust through the three steps of signal amplification: (i) PCR amplification of the target nucleic acids; (ii) bead capture and enrichment of amplicons; and (iii) magnetic amplification through surrounding water protons. The μNMR platform is a versatile technology that could be readily applied to other studies and diseases.

References
[1] Chung H. J. et al.: Nat. Nanotechnol. 8, 369-375 (2013)
[2] Liong M. et al.: Nat. Commun. 4, 1752-1760 (2013)
[3] Pawlowski A. et al.: PLoS Pathog. 8, e1002464 (2012)
[4] Musser J. M.: Clin. Microbiol. Rev. 8, 496-514 (1995)
[5] Wada M. et al.: J. Appl. Microbiol. 108, 779-788 (2009)

Authors
Monty Liong, Hyun Jung Chung, Jaehoon Chung, Changwook Min, Hyungsoon Im, Huilin Shao, Ralph Weissleder, Hakho Lee, Center for Systems Biology, Massachusetts General Hospital-Harvard Medical School

 

Contact

Massachusetts Gen. Hospital - Harvard Medical School
185 Cambridge St.
Boston, PA 02114
USA

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