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

<div><p>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 <i>Escherichia coli</i> O157:H7 and <i>Salmonella enterica</i>, 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.</p></div

Strain-specific PiBA detection of <i>E</i>. <i>coli</i>.

<p>A) PiBA detection of Enterohemorrhagic <i>E</i>. <i>coli</i> O157:H7 (EHEC) using primers specific to rfbE gene, shown in blue. Enteroaggregative <i>E</i>. <i>coli</i> O42 (EAEC) detection using primers specific to aggR gene, shown in green. B) Detection of <i>E</i>. <i>coli</i> O157 DNA extracted from human stool. 86–24 and TW14359 are O157 strains isolated from outbreaks in 1985 and 2006, respectively. EAEC O42 was used here as an off-target (-) Control.</p

Lower limit of <i>Escherichia coli</i> O157:H7 detection via PiBA.

<p>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 <i>Escherichia coli</i> O157:H7 strain EDL933 isolated from ground beef.</p

Detecting the presence of DNA specific to the human thyroid peroxidase (TPOX) gene and Rift Valley fever virus (RVFV) RNA.

<p>A) PiBA for detection of human-specific DNA from whole blood. Primers were designed to target the TPOX gene. B) RVFV detection from infected (viral RNA in a background of host RNA) samples. #1–4 represent blinded viral samples. C) Results of blinded analysis of samples. D) Results suggesting that “% Inhibition of Aggregation” is concentration dependent and varies with time of amplification.</p

Determining the statistical and empirical (visual) thresholds for genetic analysis via PiBA.

<p>A) Optical images of (+/-) Controls. B) Graph of “% Aggregation”, error bars represent standard deviation, n = 3. C) Probability distribution functions (PDF) calculated for (+/-) Controls. Threshold set as [μ-3σ] for (+) Control. X axis shows “% Maximum Inhibition of Aggregation”, (-) Control set as “0%”. D) Graphical representation of PDF. “Probability of Positive Result” represents likelihood of value to cross the calculated statistical threshold. Empirical threshold serves as visual representation of positive/negative results. For all data represented, the empirical threshold is set at 50%. Values above this threshold are considered positive and values below are considered negative.</p

PiBA for specific food-borne pathogen detection.

<p>A) PiBA using primers specific to <i>Escherichia coli</i>. B) PiBA using primers specific to <i>Salmonella enterica</i>. Here, <i>Listeria monocytogenes</i> was used as an off-target (-) Control.</p