Source: UNIV OF HAWAII submitted to
PORTABLE ACCESSORIES FOR ENRICHING/ CONCENTRATING PATHOGENIC ORGANISMS FOR AGRICULTURAL DIAGNOSTICS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1004671
Grant No.
(N/A)
Project No.
HAW05027-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Apr 24, 2015
Project End Date
Sep 30, 2019
Grant Year
(N/A)
Project Director
Jenkins, DA, M.
Recipient Organization
UNIV OF HAWAII
3190 MAILE WAY
HONOLULU,HI 96822
Performing Department
Molecular Biosciences & Bioengineering
Non Technical Summary
Effective containment and control of disease organisms on plants, animals, and food is predicated on widespread surveillance and rapid detection. Technologies for detection have improved drastically in recent years, especially in their speed, portability, and ability to resolve very closely related organisms that may have significantly different virulence and other biological characteristics influencing persistence and dispersal. Unfortunately, advances in sample preparation have not kept pace with new portable detection systems to enable them to have detection limits comparable to more tedious and expensive reference laboratory methods. The objective of this project is to develop truly portable and inexpensive sample preparation modules to rapidly isolate and concentrate trace quantities of pathogenic organisms from large (i.e. 100s of ml and larger) samples of agricultural and environmental samples. Development of these systems would enable truly distributed diagnostics and surveillance programs to rapidly identify and manage disease outbreaks in the field.
Animal Health Component
0%
Research Effort Categories
Basic
25%
Applied
25%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
4027299202040%
2127299104030%
7127299104030%
Goals / Objectives
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.
Project Methods
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 extractionTo 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.

Progress 04/24/15 to 09/30/19

Outputs
Target Audience:Regulatory agencies and officials overseeing trade in agricultural and biological materials. Agricultural producers and processors. Basic experimental biologists. Analytical Chemists. Farmers, and agricultural diagnostic service providers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?-Application of industry standard engineering CAD software (SolidWorks) -Design and application of molecular diagnostic techniques like LAMP. -Design, servicing, maintenance, and application of equipment and techniques including plasma treatment, machine vision, spectroscopy. -Classic microbiological techniques including sterile culture and plate counting. -Collaboration and communication with industry professionals for commercial product manufacture. -Software development including for Android devices, embedded system control, and digital communications between devices. -Experimental design and data analysis, including use of machine learning tools. How have the results been disseminated to communities of interest?We have reported results in professional meetings, as well as to allied agencies and organizations in planning meetings for future research and development. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Over the course of the project we accomplished all of the major goals we identified, and we were also able to make advances in other related areas. We successfully built and characterized handheld non-instrumented incubators that were able to maintain near optimal temperatures (37 C) for culture based enrichment of common enteric pathogens such as Salmonella and E. coli, for periods in excess of 12 hours, enabling detection of contamination levels down to near 1 CFU / g on common food materials following one cycle of enrichment using routine molecular detection techniques. We also leveraged a NIFA funded project to develop handheld cartridges to separate bacterial contaminants from large samples of environmental water samples using electroflotation, demonstrating ~100 fold concentration of bacteria in samples in a 15 minute process, significantly improving ability to detect trace amounts of bacteria that could contaminate food or drinking or recreational waters. For objective 3 (technology transfer) we are working with local agencies (Hawaii Department of Health) and organizations (Surfrider Foundation) to implement use of some of these technologies for routine testing of recreational waters for indicators of fecal contamination. We are also continuing to work on chemical additives to improve flocculation and recovery of bacteria from our electroflotation cartridge, and integration with silica based gene extraction in a fully automated two-step process. We have also pursued related objectives related to this project, most notably development of alternative molecular probe technologies and data analysis to distinguish between single nucleotide polymorphisms (i.e. to differentiate pathogen isolates with different virulence, antibiotic resistance, or other important traits), and advanced new approaches to implement isothermal molecular diagnostics for improved quality and quantity of actionable data with minimal user manipulation of samples (i.e. single ready to use kits that include control reactions to ensure integrity of results, and potentially expanded diagnostic panels to simultaneously determine presence of multiple pathogens of interest that might present similar symptoms but which have significantly different control strategies or consequences to management).

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: McLamore, E., Datta, S.P.A., Morgan, V., Cavallaro, N., Kiker, G., Jenkins, D.M., Rong, Y., Gomes, C., Claussen, J., Vanegas, D., and Alocilja, E. 2019. SNAPS: Sensor Analytics Point Solutions for detection and decision support systems. Sensors 19(22): 4935.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Jenkins, D.M., Lee, B.E., Jun, S., Reyes-de-Corcuera, J., and McLamore, E.S. 2019. ABE-Stat, a fully open-source and versatile wireless potentiostat project including electrochemical impedance spectroscopy. Journal of the Electrochemical Society 166(9): B3056-B3065.


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Regulatory agencies and officials overseeing trade in agricultural and biological materials. Agricultural producers and processors. Basic experimental biologists. Analytical Chemists. Farmers, and agricultural diagnostic service providers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Application of industry standard engineering CAD software (SolidWorks) Design and application of molecular diagnostic techniques like LAMP. Design, servicing, maintenance, and application of equipment and techniques including plasma treatment, machine vision, spectroscopy. Classic microbiological techniques including sterile culture and plate counting. Collaboration and communication with industry professionals for commercial product manufacture. Software development including for Android devices, embedded system control, and digital communications between devices. Experimental design and data analysis. How have the results been disseminated to communities of interest?We have reported results in professional meetings. What do you plan to do during the next reporting period to accomplish the goals?This is the last year of our project, and we are focused on submitting applications for follow up funding to commercialize the technologies developed in this research, as well as to develop ancillary technologies to improve their value. This includes integration of silica based extraction of DNA / RNA onto our automated electroflotation platform and improved design of the assembly, as well as improved design of a hardware design for conducting isothermal gene-based diagnostics in the field using LAMP, and new LAMP based molecular probes for identifying SNPs in pathogenic and other organisms (i.e. for quickly identifying traits such as virulence and antibiotic resistance).

Impacts
What was accomplished under these goals? We finalized comprehensive laboratory based validation of handheld (non-instrumented) incubators, and have continued to incrementally improve performance of electroflotation based sample preparation technologies to accelerate and facilitate sensitive detection of pathogenic organisms in the field, and prepared for submission of a variety of grants to support more detailed characterization of the technologies for widespread use and commercialization (applications which have since been submitted to USDA-SBIR, as well as Water Resources Research Center at the University of Hawaii), and plans to integrate a silica based DNA extraction technology onto our automated electroflotation platform (which we have since begun testing in the new project year).

Publications

  • Type: Other Status: Published Year Published: 2017 Citation: Jenkins, D.M. 2017. Mobile Tools for Biosecurity and Natural Resource Management: From Agricultural Diagnostics to Aerial Control of Invasive Species. Invited presentation to the Zhejiang Academy of Agricultural Sciences and Zhejiang University, November 11, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Jenkins, D.M. 2018. Evaluation of X-Ray Irradiation as a Non-Chemical Method of Control and Management of Coconut Rhinoceros Beetle. Presentation 1801554 at the 2018 Annual International Meeting of the American Society of Agricultural and Biological Engineers, Detroit, MI, July 31, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Jenkins, D.M. 2018. Evaluation of Lighting Preference to Enhance Trap Catch of Asian Citrus Psyllid and Coconut Rhinoceros Beetle. Presentation 1801553 at the 2018 Annual International Meeting of the American Society of Agricultural and Biological Engineers, Detroit, MI, July 31, 2018.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Diaz, L., Li, Y., Kubota, R., and Jenkins, D.M. 2018. Enrichment of Escherischia coli O157:H7 and Salmonella Typhimurium using a portable incubator increases the detection sensitivity of loop mediated amplification (LAMP). Food Protection Trends (in press).
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Larrea-Sarmiento, A., Dhakal, U., Boluk, G., Fatdal, L., Alvarez, A., Strayer, A., Paret, M., Jones, J., Jenkins, D.M., and Arif, M. 2018. Development of a genome-informed loop mediated isothermal amplification assay for rapid and specific detection of Xanthomonas euvesicatoria. Scientific Reports 8:14298.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Diaz, L., Jenkins, D.M., Li, Y., McNealy, T., Walter, N., and Kubota, R. 2018. Electroflotation of Escherichia coli improves detection rates by Loop-mediated isothermal amplification. Transactions of the American Society of Agricultural and Biological Engineers 61(4): 1209-1220.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Jenkins, D.M. and Kurasaki, R. 2018. ABE-VIEW: Android interface for wireless data acquisition and control. Sensors 18(8): 2647.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience: Regulatory agencies and officials overseeing trade in agricultural and biological materials, and food safety Agricultural producers and processors Food retailers. Basic experimental biologists Analytical Chemists Farmers, and agricultural diagnostic service providers Extension agents working on food safety Changes/Problems:No major changes in approach were taken this project year from those already taken or anticipated in previous years. What opportunities for training and professional development has the project provided?-Application of industry standard engineering CAD software (SolidWorks) -Design and application of molecular diagnostic techniques like LAMP. -Design, servicing, maintenance, and application of equipment and techniques including plasma treatment, machine vision, spectroscopy, -Classic microbiological techniques including sterile culture and plate counting. -Collaboration and communication with industry professionals for commercial product manufacture. -Software development including for Android devices, embedded system control, and digital communications between devices. -Experimental design and data analysis. How have the results been disseminated to communities of interest?Results have been communicated to colleagues through presentation at professional meetings. In the reported project year we submitted a manuscript for peer-review publication, and have collected data to support submission of numerous other publications. Preparation of these manuscripts andoutreach / commercialization of the new technologies will be the primary focus for the on-going project year. What do you plan to do during the next reporting period to accomplish the goals?Now that we have demonstrated the proof of principle of the technologies we have been developing, our primary objectives in the on-going project year are as follows: 1) Prepare and submit manuscripts for peer review describing the advances made in our technologies. 2) Conduct additional experiments to demonstrate scale up of electroflotation for greater sensitivity of detection of highly dispersed pathogens in the environment, on foods, and in process / wash water in food processing facilities. 3) Continue working with partners to improve performance, add new methods, and commercialize applications for potentiostat and other rapid detection technologies.

Impacts
What was accomplished under these goals? In this project year we demonstrated practical application of non-instrumented incubators to enable detection of trace contaminations of food-borne pathogens in the field. This technology consists of a handheld cartridge to which a small quantity of boiling water is added. Latent energy is stored at the melting temperature of a phase change material in the cartridge, allowing the desired temperature to be maintained over a period of 8+ hours by storing the cartridge in a small thermos. We have demonstrated growth curves for several strains of Salmonella and E. coli under the observed conditions in the cartridges, and also demonstrated that the cartridges could be used to enrich 1 CFU/ml of these strains inoculated in foods like milk, spinach, and ground beef to detectable levels within 8 hours. This technology is a very affordable and discrete option to enable testing of food for bacterial contaminations in food processing plants, in the environment, and other locations with very rudimentary labs and portable diagnostic tools which are commercially available. We have also optimized electroflotation media and conditions in a portable electroflotation cartridge developed in previous years to enable greater than 3 order of magnitude (1000x) improvement in detection limit of food-borne pathogens (Salmonella and E. coli) in environmental scale (hundreds of milliliter) water samples, with a 20 minute automated process. This represents a very significant improvement in ability to rapidly detect trace contaminations of these pathogens and effectively implement control strategies to prevent contamination of foods, and to prevent distribution of contaminated foods and logistical difficulties of recalls. The technology may be scaled up for even more sensitive detection of trace contaminations dispersed across large environmental scales. To support other research by collaborators in other states, we have also developed an open-source,potentiostat device. The device was designed to be affordable, handheld, and wireless (Bluetooth and WiFi) to facilitate adoption for diagnostic technologies in the field or on-line in processing environments, but with performance to support high quality, sensitive measurements. The hardware is capable of any standard voltammetric or amperometric technique (i.e. cyclic voltammetry and differential pulse voltammetry), and to the knowledge of the authors it is the smallest and definitely the most affordable instrument capable of conducting electrochemical impedance spectroscopy (EIS). We are currently working on a new iteration of the prototype to enable EIS between 100 Hz and 1 kHz (by incorporation of an external slower clock for the built in network analyzer, and/or use of a faster Analog to Digital Converter for to extend the frequency range at the lower end of the spectrum. We are also working on upgrades to the firmwarefirmware to improve signal to noise ratio (i.e. by disabling the WiFi during analytical steps, or taking a consensus approach to measurement to reject spurious noise. The hardware is interfaced to a customized Android app available freely on Android Play (https://play.google.com/store/apps/details?id=com.diagenetix.abestat&hl=en). As a service to the profession to facilitate custom hardware development and testing by other groups, we have also developed a published Android app that can be used to easily control and collect data wirelessly from Bluetooth enabled hardware (https://play.google.com/store/apps/details?id=com.uhmbe.DAQCTRL&hl=en).This app allows a user to connect to a remote hardware device through a Bluetooth modem. Once connected the app notifies the remote device of the connection so it can populate and configure the interface through coded commands. Available elements for the interface include 16 generic data fields which can be plotted on an interactive graph in real time, 8 configurable buttons, 4 radio groups that can each be configured with 4 radio buttons, and 8 controls to send numerical input to the remote device. User interaction with the elements in the app result in coded information sent back to the remote device (i.e. so it can recognize button presses / selections, and receive numerical input). Numerical and textual data sent to the application can be saved to a comma delimited (.csv) file, and shared by e-mail.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Diaz, L., D.M. Jenkins, R. Kubota, N. Walter, L. Yong, and T. McNealy. 2017. Electroflotation of Escherichia coli Improves Detection Rates by Loop-mediated Isothermal Amplification. Transactions of American Society for Agricultural and Biological Engineers.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Diaz, L., D.M. Jenkins, Y. Li, T. McNealy, and T. Tzeng. 2017. Electroflotation of Escheriscia coli improves detection rates by Loop Mediated Isothermal Amplification. Presentation/ Paper 170164 at 2017 International Meeting of American Society of Agricultural and Biological Engineers, Spokane, WA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Jenkins, D.M., J.L. Diaz, Y. Li, R. Kubota, and J. Obatake. 2017. Handheld non-instrumented incubator for field-based pathogen enrichment. Presentation / paper 1701434 at 2017 International Meeting of American Society of Agricultural and Biological Engineers, Spokane, WA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Jenkins, D.M., R. Kubota, K. Berghorn, L. Diaz. 2017. Clinical and Food-safety applications of point-of-care gene-based diagnostics. Presentation 1701430 at 2017 International Meeting of American Society of Agricultural and Biological Engineers, Spokane, WA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Jenkins, D.M. and J. Reyes-de-Corcuera. 2017. Handheld, open-source potentiostat for high-performance electrochemical analysis in the field. Presentation 1701478 at 2017 International Meeting of American Society of Agricultural and Biological Engineers, Spokane, WA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Jenkins, D.M. and J. Reyes-de-Corcuera. 2017. Open-source Android app for facilitating customized data acquisition, visualization, and control. Presentation 1701479 at 2017 International Meeting of American Society of Agricultural and Biological Engineers, Spokane, WA.


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Regulatory agencies and officials overseeing trade in agricultural and biological materials. Agricultural producers and processors. Basic experimental biologists. Analytical Chemists. Farmers, and agricultural diagnostic service providers. Changes/Problems:Our previous hypothesis that we could make highly affordable electrode arrays by screen printing corrosion resistant materials onto standard metallic printed circuit boards proved to be incorrect, at least using the materials that we experimented as adhesion to the underlying metal substrate was not completely reliable over long durations of electrolysis. This required us to redesign the system using more traditional corrosion resistant materials. The new designs are only just not being completed, and we anticipate diving into the basic experimental phase of the research over the next few months. What opportunities for training and professional development has the project provided?An undergraduate student was provided training in microcontroller programming, basic instrumentation including recording temperature profiles, and programming a customized system to replicate these profiles for use in growth kinetics experiments. She was also trained in standard microbiological plating techniques for cell enumeration/ quantification. A graduate student was provided training in customized circuit design and assembly, writing structured code to control devices and communicate data between devices, and a variety of fabrication and screen printing techniques required to design and build a electroflotation based cartridge to rapidly capture trace quantities of pathogenic bacteria from a large slurry (100s of ml or more). A post-doctoral research was given the opportunity to train these students with basic molecular biology techniques for system validation (i.e., determination of improvements in detection limits based on newly developed methods/ treatments). How have the results been disseminated to communities of interest?Results have been communicated at a professional meeting (2016 Annual International Meeting of the American Society of Agricultural and Biological Engineers), as well as at the project directors meeting for the USDA-AFRI Food Safety program. What do you plan to do during the next reporting period to accomplish the goals?During the current project year, we have developed a new electrode array system using corrosion-resistant "dimensionally stable anode" materials (conductive mixed metal oxide layer coated on a titanium substrate) to ensure that we can replicate high quality results. With this system we intend to evaluate different electroflotation conditions (both for media and the electrical waveforms) for rapid and efficient recovery of pathogens from dilute suspensions, and quantify improvements in detection limits for representative molecular diagnostics. We also plan to complete the quantification of growth kinetics for several pathogens (several strains of E coli and Salmonella) in our handheld non-instrumented incubators to determine requisite incubation periods to achieve adequate levels of sensitivity in these same diagnostics.

Impacts
What was accomplished under these goals? During this project year, we completely redesigned the control system for controlling the conditions for electroflotation, and developed an entirely new electroflotation cartridge based on electrodearrays printed on a customized printed circuit board. We experimented with a variety of screen printing materials including customized formulations of carbon paste and conductive (carbon-filled) silicone to protect the underlying electrode arrays from corrosion, to use as a stable experimental platform to test different electroflotation conditions for the ability to recover trace amounts of pathogenic bacteria from suspension (to facilitate highly sensitive detection using rapid field based diagnostics). We also developed standard protocols for safely enumerating cells from the treatment to quantify device performance. In this project year we also started quantifying the growth kinetics of E.coli in our non-instrumented heaters (developed to enable rapid pathogen enrichment in the field, to facilitate rapid and sensitive pathogen detection), replicating the temperature profiles we recorded in these devices in a programmable incubator we designed. The objective of these experiments is to determine the required enrichment times under standard conditions in these handheld incubators to achieve desired detection limits for pathogens of interest, in order to meet performance requirements for regulatory significance (i.e., to ensure reliable testing results when testing for food-borne pathogens).

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Diaz, L.M., McNealy, T., Li, Y., Kubota, R., and Jenkins, D.M. 2016. Recovery of concentrated microbial pathogens using a portable electroflotation system. Presentation 162461482, Annual International Meeting of the American Society of Agricultural and Biological Engineers. Orlando, FL, July 18, 2016.


Progress 04/24/15 to 09/30/15

Outputs
Target Audience:Regulatory agencies and officials overseeing trade in agricultural and biological materials. Agricultural producers and processors. Basic experimental biologists. Analytical Chemists. Farmers, and agricultural diagnostic service providers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Support provided under this project enabled the training of a graduate student, particularly in interfacing and hardware development in developing the different elements of the electroflotation system. How have the results been disseminated to communities of interest?Results have been disseminated in publications, as well as at professional conferences (ASABE 2015, APS 2015) What do you plan to do during the next reporting period to accomplish the goals?Work in the next project period will focus onrefining andvalidating sample preparation technologies for portable diagnostics, setting up enrichment and capture experiments with pathogenic bacteria using adequate safety controls.

Impacts
What was accomplished under these goals? Our primary accomplishments during the project year include the development of two new experimental platforms to facilitate sample preparation in the field, including a portable non-instrumented incubator for sample enrichment, and an electroflotation based system to capture bacterial and other microbial contaminants dispersed in large aqueous samples. We have also refined and improved new molecular tools for detection of individual genes in a reaction, and conducted some preliminary kinetics experiments on different reaction conditions with different primer sets, enzymes, and other conditions to guide improved primer design in the future.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Kubota, R. and Jenkins, D.M. 2015. Real-time multiplex applications of Loop Mediated AMPlification by Assimilating Probes. International Journal of Molecular Sciences. 16(3), 4786-4799
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Keremane, M.L., Ramadugu, C., Rodriguez, E., Kubota, R., Shibata, S., Hall, D.G., Roose, M.L., Jenkins, D.M., and Lee, R.F. 2015. A rapid field detection system for citrus huanglongbing associated 'Candidatus Liberibacter asiaticus' from the psyllid vector, Diaphorina citri Kuwayama and its implications in disease management. Crop Protection 68:41-48