CICEET Progress Report for the period 9/16/3 through 3/15/03

Project Title: Assay and Sensor Development to Identify, Detect, and Quantify Microbial Contaminants
Principal Investigator(s): Jack Fell, Kelly Goodwin, Peter Ortner

Accomplishments
Scheduled Tasks:
The following tasks were scheduled for activity and/or completion during the designated 6-month reporting period:

1. Further testing of the Bfra probe on microplates.
2. Continue Rookery Bay NERR sample analysis using the K. brevis microplate assay.
3. Testing and optimization of sewage-indicating microplate assays.
4. Testing and optimization of new Luminex probes for bacterial indicators of sewage.
5. Design and test a Luminex probe for the fecal bacterium Enterococcus faecalis.
6. Evaluate detection limit of Luminex assays.

Progress on Tasks
We have made substantial progress on the tasks outlined above. Progress is described in the “Preliminary Data” section.

Difficulties Encountered
We had difficulty obtaining Karenia mikimotoi DNA and live culture.

Anticipated Success in Meeting Project Objectives in Scheduled Project Period
We anticipate being able to meet project objectives.

Preliminary Data
Microplate Assay
Detection of Toxic Dinoflagellates
The microplate assay provided identification of several toxic dinoflagellates, including Karenia brevis, the organism responsible for red tide in the Rookery Bay NERR. The assay was evaluated with 110 environmental samples and was consistent with species-specific PCR, sequencing, and microscopic observation. Target sensitivity for this study was a negative result consistent with a report of “not present” provided by a regional monitoring program. With HPLC purified probes, this objective was met in every case tested. Assay sensitivity allowed detection of K. brevis when it was “present” in the water (<1000 cells/L), as defined by the Florida Marine Research Institute (FMRI).

Detection of Sewage-Indicating Bacteria
The assay uses a matrix of targets rather than a single indicator species to identify sewage contamination. The hybridization technique is rapid, taking apx. 100 minutes to complete. DNA probes were immobilized for a variety of sewage indicating bacteria. The “ColinSitu” probe specifically detected E. coli, the “ENTI” probe detected a variety of Enterobacteriaceae species, “Bdis” specifically detected Bacteroides distasonis, and “Bfra” detected other Bacteroides from the Bacteroides fragilis group (BFG). Hybridization was achieved at one temperature with DNA amplified from a single set of primers. Bdis did not detect Prevotella ruminicola, a Bacteroides found in ruminants. Bdis is a promising probe to help distinguish between human and nonhuman sewage contamination in coastal waters.

Luminex Assay
Detection of Sewage-Indicating Bacteria
The Luminex system uses microspheres equipped with species-specific probes to capture target DNA. This DNA hybridization assay detected microbial indicators of fecal contamination. Group-specific primers were used to amplify DNA extracted from cultures and environmental samples (seawater and river water). Multiplexing of the group-specific primers and probes allowed many bacterial species to be identified with a single assay; using only a single PCR run and requiring little sample DNA (Figure 1). Bacterial targets in the assay included Escherichia coli, Enterococci faecalis, Bacteroides distasonis, the Bacteroides fragilis group, and total coliforms. The assay successfully detected all species and groups with no cross reactivity. Targets were successfully detected at a sensitivity level of 500 fg of genomic DNA (pre-PCR), which is equivalent to approximately 100 cells (Figure 2 and Figure 3).

Tasks and activities for the next reporting period

Tasks for the next reporting period

1. Test Luminex and microplate assays against traditional culture-based methods for detection of fecal bacteria.
2. Sequence bacteria from environmental samples to compare culture based and molecular based methods.
3. Use Luminex for harmful algae detection.

Work plan to accomplish tasks
Testing Luminex and microplate assay against traditional culture-based methods requires obtaining the ingredients to make a variety of selective media. EPA protocols will be used to culture bacteria from environmental samples for comparison to molecular identification methods. A high-throughput sequencer will be used to sequence colonies grown on the media. Harmful algae detection will require designing and testing probes for a variety of harmful algae.

Concerns or difficulties
None.

Expenditures
Expenditures were in the expected range.