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Microfluidic-Based Amplification-Free Bacterial DNA Detection by Dielectrophoretic Concentration and Fluorescent Resonance Energy Transfer Assisted in Situ Hybridization (FRET-ISH).

Packard MM, Shusteff M, Alocilja EC - Biosensors (Basel) (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Nanobiosensors Laboratory, Michigan State University, East Lansing, MI 48824, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Quantitative Spectrofluorometry. Correlation of bacterial concentration and background subtracted (background = HEX probe signal with no bacterial DNA or donor dye present) FRET-ISH signal (acceptor dye emission at 560 nm) as recorded by the Nanodrop 3300.
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biosensors-02-00405-f002: Quantitative Spectrofluorometry. Correlation of bacterial concentration and background subtracted (background = HEX probe signal with no bacterial DNA or donor dye present) FRET-ISH signal (acceptor dye emission at 560 nm) as recorded by the Nanodrop 3300.

Mentions: Spectrofluorometry on the Nanodrop 3300 allowed quantification of HEX signal and associated labeled probe bound to bacterial DNA previously stained with SYTO®-9. Serial dilutions were tested to construct a calibration curve for the relative fluorescence signal intensity as a function of bacterial counts. An increase in HEX (acceptor) emission intensity at 560 nm was detectable for concentrations in the range of 101 to 108 cfu/mL (Figure 2). A small number of other bacterial species which also contain the ERIC sequence in their genome (including Salmonella enteritidis, Klebsiella pneumoniae, Streptococcus agalactiae, and Escherichia hermanii) were similarly tested off-chip, showing signal at 560 nm in the presence of the ERIC probe (data not shown).


Microfluidic-Based Amplification-Free Bacterial DNA Detection by Dielectrophoretic Concentration and Fluorescent Resonance Energy Transfer Assisted in Situ Hybridization (FRET-ISH).

Packard MM, Shusteff M, Alocilja EC - Biosensors (Basel) (2012)

Quantitative Spectrofluorometry. Correlation of bacterial concentration and background subtracted (background = HEX probe signal with no bacterial DNA or donor dye present) FRET-ISH signal (acceptor dye emission at 560 nm) as recorded by the Nanodrop 3300.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263560&req=5

biosensors-02-00405-f002: Quantitative Spectrofluorometry. Correlation of bacterial concentration and background subtracted (background = HEX probe signal with no bacterial DNA or donor dye present) FRET-ISH signal (acceptor dye emission at 560 nm) as recorded by the Nanodrop 3300.
Mentions: Spectrofluorometry on the Nanodrop 3300 allowed quantification of HEX signal and associated labeled probe bound to bacterial DNA previously stained with SYTO®-9. Serial dilutions were tested to construct a calibration curve for the relative fluorescence signal intensity as a function of bacterial counts. An increase in HEX (acceptor) emission intensity at 560 nm was detectable for concentrations in the range of 101 to 108 cfu/mL (Figure 2). A small number of other bacterial species which also contain the ERIC sequence in their genome (including Salmonella enteritidis, Klebsiella pneumoniae, Streptococcus agalactiae, and Escherichia hermanii) were similarly tested off-chip, showing signal at 560 nm in the presence of the ERIC probe (data not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Nanobiosensors Laboratory, Michigan State University, East Lansing, MI 48824, USA.

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.

No MeSH data available.


Related in: MedlinePlus