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Optical Imaging of Paramagnetic Bead-DNA Aggregation Inhibition Allows for Low Copy Number Detection of Infectious Pathogens.

DuVall JA, Borba JC, Shafagati N, Luzader D, Shukla N, Li J, Kehn-Hall K, Kendall MM, Feldman SH, Landers JP - PLoS ONE (2015)

Bottom Line: The fragments generated via LAMP fail to induce chaotrope-mediated bead aggregation; however, due to their ability to passivate the bead surface, they effectively inhibit bead aggregation by longer 'trigger' DNA.We also show the concentration dependence of aggregation inhibition, suggesting the potential for quantification of target nucleic acid in clinical and environmental samples.Lastly, we demonstrate the ability to rapidly detect infectious pathogens by utilizing a cell phone and custom-written application (App), making this novel detection modality fully portable for point-of-care use.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Virginia, Charlottesville, VA, United States of America.

ABSTRACT
DNA-paramagnetic silica bead aggregation in a rotating magnetic field facilitates the quantification of DNA with femtogram sensitivity, but yields no sequence-specific information. Here we provide an original description of aggregation inhibition for the detection of DNA and RNA in a sequence-specific manner following loop-mediated isothermal amplification (LAMP). The fragments generated via LAMP fail to induce chaotrope-mediated bead aggregation; however, due to their ability to passivate the bead surface, they effectively inhibit bead aggregation by longer 'trigger' DNA. We demonstrate the utility of aggregation inhibition as a method for the detection of bacterial and viral pathogens with sensitivity that approaches single copies of the target. We successfully use this methodology for the detection of notable food-borne pathogens Escherichia coli O157:H7 and Salmonella enterica, as well as Rift Valley fever virus, a weaponizable virus of national security concern. We also show the concentration dependence of aggregation inhibition, suggesting the potential for quantification of target nucleic acid in clinical and environmental samples. Lastly, we demonstrate the ability to rapidly detect infectious pathogens by utilizing a cell phone and custom-written application (App), making this novel detection modality fully portable for point-of-care use.

No MeSH data available.


Related in: MedlinePlus

Lower limit of Escherichia coli O157:H7 detection via PiBA.Limit of detection was determined to be conservatively 20 copies (2 out of 3 analyses were positive using 2 genomic copies of template DNA). Each starting template amount was tested three times, n = 3. The LOD was determined using Escherichia coli O157:H7 strain EDL933 isolated from ground beef.
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pone.0129830.g004: Lower limit of Escherichia coli O157:H7 detection via PiBA.Limit of detection was determined to be conservatively 20 copies (2 out of 3 analyses were positive using 2 genomic copies of template DNA). Each starting template amount was tested three times, n = 3. The LOD was determined using Escherichia coli O157:H7 strain EDL933 isolated from ground beef.

Mentions: Having shown (at least with the targets tested) that PiBA had the desired specificity for infectious pathogen detection, the sensitivity of the assay was evaluated using the primer set specific for E. coli O157:H7 stx2 gene sequences (S1 Table). Solutions prepared through serial dilution provided starting template concentrations that varied from less than 1 to a high of 2000 genomic copies (per amplification reaction). Fig 4 (corresponding to S6 Fig) shows that the aggregation inhibition with template concentrations from 2000 copies down to 20 copies was unequivocal. At a template concentration of 2 copies, significant aggregation inhibition was observed in 2 of 3 analyses. Further dilution to less than 1 copy per reaction (0.2 copies) produced no observable aggregation inhibition. Thus, we conclude that the limit of detection lies around 20 starting copies of genomic DNA. Research has shown that E. coli contains approximately 4 genomic copies/cell [16]; therefore, our findings are significant for food-borne pathogen detection, as we have demonstrated the ability to detect <10 cells in a sample.


Optical Imaging of Paramagnetic Bead-DNA Aggregation Inhibition Allows for Low Copy Number Detection of Infectious Pathogens.

DuVall JA, Borba JC, Shafagati N, Luzader D, Shukla N, Li J, Kehn-Hall K, Kendall MM, Feldman SH, Landers JP - PLoS ONE (2015)

Lower limit of Escherichia coli O157:H7 detection via PiBA.Limit of detection was determined to be conservatively 20 copies (2 out of 3 analyses were positive using 2 genomic copies of template DNA). Each starting template amount was tested three times, n = 3. The LOD was determined using Escherichia coli O157:H7 strain EDL933 isolated from ground beef.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4466016&req=5

pone.0129830.g004: Lower limit of Escherichia coli O157:H7 detection via PiBA.Limit of detection was determined to be conservatively 20 copies (2 out of 3 analyses were positive using 2 genomic copies of template DNA). Each starting template amount was tested three times, n = 3. The LOD was determined using Escherichia coli O157:H7 strain EDL933 isolated from ground beef.
Mentions: Having shown (at least with the targets tested) that PiBA had the desired specificity for infectious pathogen detection, the sensitivity of the assay was evaluated using the primer set specific for E. coli O157:H7 stx2 gene sequences (S1 Table). Solutions prepared through serial dilution provided starting template concentrations that varied from less than 1 to a high of 2000 genomic copies (per amplification reaction). Fig 4 (corresponding to S6 Fig) shows that the aggregation inhibition with template concentrations from 2000 copies down to 20 copies was unequivocal. At a template concentration of 2 copies, significant aggregation inhibition was observed in 2 of 3 analyses. Further dilution to less than 1 copy per reaction (0.2 copies) produced no observable aggregation inhibition. Thus, we conclude that the limit of detection lies around 20 starting copies of genomic DNA. Research has shown that E. coli contains approximately 4 genomic copies/cell [16]; therefore, our findings are significant for food-borne pathogen detection, as we have demonstrated the ability to detect <10 cells in a sample.

Bottom Line: The fragments generated via LAMP fail to induce chaotrope-mediated bead aggregation; however, due to their ability to passivate the bead surface, they effectively inhibit bead aggregation by longer 'trigger' DNA.We also show the concentration dependence of aggregation inhibition, suggesting the potential for quantification of target nucleic acid in clinical and environmental samples.Lastly, we demonstrate the ability to rapidly detect infectious pathogens by utilizing a cell phone and custom-written application (App), making this novel detection modality fully portable for point-of-care use.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Virginia, Charlottesville, VA, United States of America.

ABSTRACT
DNA-paramagnetic silica bead aggregation in a rotating magnetic field facilitates the quantification of DNA with femtogram sensitivity, but yields no sequence-specific information. Here we provide an original description of aggregation inhibition for the detection of DNA and RNA in a sequence-specific manner following loop-mediated isothermal amplification (LAMP). The fragments generated via LAMP fail to induce chaotrope-mediated bead aggregation; however, due to their ability to passivate the bead surface, they effectively inhibit bead aggregation by longer 'trigger' DNA. We demonstrate the utility of aggregation inhibition as a method for the detection of bacterial and viral pathogens with sensitivity that approaches single copies of the target. We successfully use this methodology for the detection of notable food-borne pathogens Escherichia coli O157:H7 and Salmonella enterica, as well as Rift Valley fever virus, a weaponizable virus of national security concern. We also show the concentration dependence of aggregation inhibition, suggesting the potential for quantification of target nucleic acid in clinical and environmental samples. Lastly, we demonstrate the ability to rapidly detect infectious pathogens by utilizing a cell phone and custom-written application (App), making this novel detection modality fully portable for point-of-care use.

No MeSH data available.


Related in: MedlinePlus