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Portable Accessories For Enriching/ Concentrating Pathogenic Organisms For Agricultural Diagnostics

Jenkins, Daniel
University of Hawaii
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Establish protocols using handheld non-instrumented incubators to enable detection limits of a reference environmental isolate of E. coli equivalent to existing reference methods.Automate a silica-based extraction technique to recover concentrated high quality DNA from large volume environmental samples in the field, using an environmental E. coli model isolate.Transfer new sample preparation technologies including non-instrumented incubators and automated silica-based extraction units to facilitate rapid, sensitive, and distributed agricultural diagnostics in the field.

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Objective 1: For sensitive detection of Salmonella, we have previously used 24 hour enrichment of contaminated milk samples on a disposable glass card followed by a simple silica based DNA extraction on the same card, resulting in detection limits of about 1 CFU/ ml (Kubota, LaBarre et al. 2013). To make incubated enrichment more accessible to food producers and processors in limited space, we have also adapted the principle behind our non-instrumented nucleic acid amplification to make non-instrumented incubators in insulated 850 ml food jars, and demonstrated highly stable temperatures for at least 12 hours (unpublished data, Figure 4). Initial experiments indicate that the devices can be used to successfully enrich E. coli by several orders of magnitude within 8 hours, allowing detection of contaminations that were initially undetectable using our LAMP system.To firmly establish baseline protocols for enrichment periods required to match the sensitivity of reference methods, we will conduct the following experiments:Temperature profiles in handheld incubators will be recorded for varying quantities of boiling water and pre-heating times.Replicates of enrichment media will be inoculated with the pathogen, and incubated in our programmable incubators to simulate enrichment in the portable incubators.Samples of inoculated media will be taken periodically, and appropriate dilutions plated on solid media to enable quantitation of pathogen based on plate counts.Quantitative growth data will be used to determine protocols for portable incubator loading and enrichment times to achieve sufficient enrichment to allow detection limits equivalent to reference laboratory methods including laboratory enrichment steps.

Objective 2: Physical filtration represents the most straightforward approach to concentrate suspensions of pathogen particles, and has been demonstrated in portable systems for assessing bacterial counts in environmental water samples (Leskinen, Kearns et al. 2012). We have used commercial filtration-based spin columns to isolate bacteria from ml quantities of soil drainage (Kubota, Vine et al. 2008; Paret, Kubota et al. 2010), and to isolate bacteria from larger samples we have used glass fiber filters with 5 mm nominal pore size to for processing large (100s of ml) soil slurry samples. The high hydraulic conductivity of the filters enabled samples to be processed quickly (1-2 minutes) using a hand operated vacuum pump in the field, even for samples with very high sediment and other suspended solid loads, and importantly was demonstrated to retain 90% of Ralstonia solanacearum cells (rod shaped bacteria about 1 mm in diameter) from the filtrate (unpublished data; Figure 5). While filtration works well for concentrating bacteria from environmental water samples with small sediment loads, especially when culture based methods are used for downstream detection (Leskinen, Kearns et al. 2012), the co-concentration of inhibitors adhering to other particles in suspension in our experience has limited the effectiveness of simple filtration when used for sample preparation for rapid molecular diagnostic methods.To address this limitation, we propose to adapt conduct a silica based extraction technique on these glass fiber filters to recover purified DNA directly from the filters, suitable for highly-sensitive nucleic acid based detection.Task 1) Process optimization for large volume silica-based nucleic acid extractionCommercial kits for silica-based nucleic acid purification are common and the principles are well understood, but generally these kits are used for relatively small clinical samples. For this task, we will use the well characterized principles to scale the process up, ideally using inexpensive and non-toxic materials/ reagents that can be handled safely and even disposed of in the field. The steps in a silica based extraction process include the following:Sample denaturing with highly concentrated chaotropic salt, lysing cells and denaturing the released negatively charged nucleic acids so they can bind to the negatively charged silica surface through ionic bridging of the chaotropic cation/ zwitterion. Sample washing with alcohol or other solvent incapable of dissolving the ionized nucleic acid molecules, to remove chaotropic reagents and other sample inhibitors. Nucleic acid elution in a suitable buffer such as TE. We will evaluate different conditions for each one of these steps to identify a process meeting the performance criteria to recover > 40% of the nucleic acid in a suspension of a model E. coli isolate, using the lease expensive and hazardous materials available. For the denaturing step on the filter, we will experiment with a) fertilizer grade urea; b) reagent grade urea; c) Guanidinium hydrochloride, and; d) guanidinium isothiocyanate. For the wash step we will experiment with isopropyl and ethyl alcohols at various concentrations, and elute the captured and washed nucleic acids with TE buffer. Recovery of DNA will be quantified through quantitative PCR or quantitative LAMP, and compared to DNA in the original stock culture to evaluate the efficiency of nucleic acid recovery. Prior to the elution step ambient air will be suctioned through the filter to volatilize off residual alcohol which are well known to inhibit polymerase based analytical reactions. Task 2) Automation and scale up of silica based nucleic acid extraction to enable practical application of the silica based extraction technique on high volumes in the field the process needs to be automated using a compact and inexpensive system. We will build and evaluate systems to automate the process in the field using battery powered compressors or vacuum pumps, with quick disconnects to a modular cartridge with a selectable manifold to route the different reagents through the filter and capture ultimately capture the concentrated nucleic acid eluent from the system for further analysis.

Objective 3: For testing the non-instrumented incubators we will work with a commercial aquaponics producer on the island of Hawaii to test their process water to detect contamination with E. coli. Using the characterized growth curves for the organism and quantitative calibrations of the downstream LAMP reaction we'll infer starting titers of the pathogen in the water, with an enrichment period and conditions sufficient to achieve a detection limit of 10 CFU per 100 ml, or approximately an order of magnitude more sensitive than the standards (126 CFU per 100 ml) for agricultural water used to irrigate crops other than sprouts. We will similarly test the silica based extraction devices developed to rapidly test for the presence of E. coli in process water in an aquaponic system. For reference our contamination levels predicted with our methods will be compared to those estimated through the reference method in the FDA Bacterial Analytical Manual. To determine actual detection limits we will artificially inoculate autoclaved process water samples with cultured E. coli at dilute levels to determine. All contaminated samples, whether occurring naturally or through artificial inoculation, will be decontaminated by mixing with bleach to a final concentration of 10% for 30 minutes before being discarded.

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Nat'l. Inst. of Food and Agriculture
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Escherichia coli
Detection Methods