CICEET Progress Report for the period 9/16/05 Through 3/15/06

Project Title: A Multichannel Handheld Sensor for Microbial Contaminants
Principal Investigator(s): John H. Paul
Additional Investigator(s): David P. Fries
Project Start Date: 10/01/06

Figures

Figure 1

Tasks to meet objectives
These objectives actually encompass the first 14 months of the project. Our first task was to organize and integrate the engineering and molecular biological components of the project. The second task was to initiate experimentation on a field RNA extraction kit. A third task was to determine how the RNA extraction technology performed in our existing handheld (one channel) analyzer. A fourth task was initiate the design and construction of the multichannel analyzer.

Progress on Tasks
In terms of the organization and integration of the molecular biology with the engineering, the first task was to hire an engineer for this project. Pragnesh Bhansali was hired to develop the multichannel analyzer. The molecular biology personnel (Erica Casper and Stacey Patterson) did a NASBA assay with Pragnesh so he could see the details of the process and incorporate this into design of the multichannel analyzer. Experiments to simplify the lab RNA extraction protocol (ie. remove filtration towers and microcentrifugation steps from the protocol) were performed. Some of these were analyzed on the EasyQ analyzer and some were analyzed using the single channel handheld analyzer. Progress has been made on the design of the analyzer, but fabrication has not yet begun.

Difficulties
One difficulty was the delay in receiving funding from NOAA/CICEET. A second setback was that Pragnesh blew out his knee playing soccer and required surgery. There was a delay in the surgery because of health insurance issues. He is now sound as a pound and proceeding with the project.

Project Objectives for Next Reporting Period

Objectives
1. To develop a multichannel handheld analyzer to detect microbial contaminants based upon Nucleic Acid Sequence Based Amplification (NASBA), 2. Develop a simple method for field nucleic acid extraction from environmental samples

Tasks to Meet Objectives
Our first task is to initiate fabrication of the multichannel handheld analyzer. It is our experience that hardware development often takes longer than anticipated. Our second task is to transfer the molecular biology technology to a new employee as both Erica Casper and Stacey Patterson are leaving the project. A third task is to have more adapters made for the field extraction kit. A fourth task would be to see how we can simplify the NASBA assay for field application.

Work Plan for Next Reporting Period
Once the design is agreed upon, we will begin fabrication of the sample block and integration of the detectors and heaters with the sample block. A new individual will be trained on the NASBA end of the project to continue extraction technology development and provide support for field testing in the second year.

Anticipated Success in Meeting Project Objectives
We anticipate success in meeting our objectives.

Overall Project Timeline Update
At this early juncture in the project period we are on schedule, and have no reason to update the timeline.

Preliminary Data
We have collected some preliminary data using the field extraction protocol. Samples of K. brevis cultures and field samples were extracted simultaneously by the field extraction protocol and the lab method. Results were expressed in terms of cells based upon cell culture standards run simultaneously. An excellent agreement was found between the two methods. The results are in Figure 1.

Dissemination
Publications: None yet

Workshops: None yet

Conferences: None yet

Manuals, Protocols: None yet

Outreach Activities: None yet

Contact with End Users: We have discussed the project with David Heil, the Director of the SEAS program in Florida, briefly at a conference in St. Petersburg. We have been in email contact as well.

Patent, Copyright, Invention Disclosure Activity: None yet We have previously submitted a disclosure on the K. brevis NASBA Assay, and a patent application has been filed.

End User Advisor Feedback
Name: David Heil, Ph.D., M.P.H.
Organization: Bureau of Aquaculture Environmental Services
Location: 1203 Governors Square Boulevard, 5th Floor, Tallahassee, FL 32301
Phone number: (850) 488-5471
Email: heild@doacs.state.fl.us
1) At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
Estuarine monitoring of red tide concentrations in Molluscan shellfish growing waters. May allow for up to the minute information on presence/absence/concentrations of red tide in estuarine environments. Once a red tide bloom is established there is less need for actual K. brevis water sampling (subsequent overnight shipping). May allows for faster response time to close shellfish harvesting areas. May determine more quickly when red tide is no longer in the water. Each of our five field offices would have the ability to aggressively monitor red tide in the water. Additionally, those engaged in beach monitoring for bathers could use the device from shore and would have to rely less on HAB flags and other sources for information.

2) What, if anything, has changed about this project’s potential applicability since the last reporting period (not applicable to the first Progress Report)?
None. If successful, this would be a major advancement in red tide management/analysis for Molluscan shellfish growing waters.

3) Do you see any key challenges that the researchers may want to address or keep in mind?
Fabrication must produce an easily portable and durable sensor to withstand rigors of potential everyday use in saltwater environment. Ideally it would be capable of measurements throughout the water column (0 to 20 feet). Additionally, United States Food and Drug Administration (FDA) criteria for K. brevis are measured in cells/Liter. Therefore, conversion by the instrument (or inclusion of a conversion table for the user) from the NASBA result to an FDA definable result would be beneficial. Does the estuarine salinity gradient affect the NASBA results? (i.e. as K. brevis cells rupture around 25ppt or lower)?

4) Does this report offer you enough information to adequately address the above questions?
Yes. If cultures of the “new” Karenia species become available, they should also be evaluated.

5) Other feedback?
None.