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Abstract
Although real-time PCR (RT-PCR) has become a diagnostic standard for rapid identification of bacterial species, typical methods remain time-intensive due to sample preparation and amplification cycle times. The assay described in this work incorporates on-chip dielectrophoretic capture and concentration of bacterial cells, thermal lysis, cell permeabilization, and nucleic acid denaturation and fluorescence resonance energy transfer assisted in situ hybridization (FRET-ISH) species identification. Combining these techniques leverages the benefits of all of them, allowing identification to be accomplished completely on chip less than thirty minutes after receipt of sample, compared to multiple hours required by traditional RT-PCR and its requisite sample preparation.
Highlights
Prompt public health investigation and response necessitates rapid identification of low bacterial concentrations
Presented here is a novel DNA-based diagnostic assay combining dielectrophoretic bacterial capture and concentration, on-chip thermal lysis, cell permeabilization and nucleic acid denaturation with fluorescence resonance energy transfer assisted in situ hybridization (FRET-ISH)
This approach dramatically reduces the time required for pan-enterobacterial detection to less than thirty minutes from receipt of sample (Table 1), compared to multiple hours needed for traditional real-time PCR (RT-PCR) and its requisite sample preparation
Summary
Prompt public health investigation and response necessitates rapid identification of low bacterial concentrations. Presented here is a novel DNA-based diagnostic assay combining dielectrophoretic bacterial capture and concentration, on-chip thermal lysis, cell permeabilization and nucleic acid denaturation with fluorescence resonance energy transfer assisted in situ hybridization (FRET-ISH) This approach dramatically reduces the time required for pan-enterobacterial detection to less than thirty minutes from receipt of sample (Table 1), compared to multiple hours needed for traditional RT-PCR and its requisite sample preparation. The result is a shift in emission correlated with the number of acceptor-labeled probe binding events, and a measure of the enterobacteria in the population Use of this method for FRET-ISH detection minimizes the influence of non-specific signals arising from unbound probes that limit traditional FISH assays. Off-chip measurements were carried out at the acceptor’s emission wavelength
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