The Development of a DNA Based Specific Assay for Pfiesteria piscicida in Water and Sediments

EPA Grant Number: R827084
Title: The Development of a DNA Based Specific Assay for Pfiesteria piscicida in Water and Sediments
Investigators: Oldach, David , Rublee, Parke
Institution: University of Maryland - Baltimore , University of North Carolina at Greensboro
EPA Project Officer: Packard, Benjamin H
Project Period: October 8, 1998 through October 7, 2001 (Extended to August 31, 2002)
Project Amount: $709,537
RFA: Ecology and Oceanography of Harmful Algal Blooms (1998) RFA Text |  Recipients Lists
Research Category: Water Quality , Harmful Algal Blooms , Water , Ecosystems


The recent association of Pfiesteria-like dinoflagellates with fish kill events and adverse human health effects has highlighted the need for research aimed at predicting, mitigating, and preventing such occurrences. However, the unique life cycle of Pfiesteria like dinoflagellates, the absence of axenic culture for their study, and the biohazard associated with attempted culture of toxin-producing organisms have hampered progress in these endeavors. To address some of these inherent difficulties, we have adapted molecular methods for the detection of known and novel dinoflagellate species. In Phase-1 of the proposed project the investigators will use a molecular ecological approach to determine dinoflagellate diversity in Maryland, North Carolina, and South Carolina estuarine sites and, using SSU (18s rDNA) gene sequences, analyze phylogenetic relationships among those organisms. Qualitative changes in species diversity will be measured utilizing ?dinoflagellate specific polymerase chain reaction (PCR) primers (18s rRNA), heteroduplex mobility assay (HMA) to gauge diversity within the amplified pool of dinoflagellate cDNA, and heteroduplex tracking assay (HTA) to identify characterized and novel dinoflagellate gene sequences within the sampled community. Changes in dinoflagellate community structure in selected waterways over time will be correlated with estuarine physicochemical and biological parameters. The investigators will assay specifically for Pfiesteria piscicida and correlate the presence or absence of this species with environmental parameters and effects. The proposed approach, while having demonstrated capability to detect specific species such as P. piscicida, will also be used to identify novel dinoflagellate gene sequences (i.e., species) associated with environmental events such as fish kills. A library of estuarine dinoflagellate 18s rDNA gene sequences will be constructed, and correlated/validated with direct sequencing of cultures generated by collaborating laboratories. This strategy will also provide an assessment of the clonality of dinoflagellate cultures, enhancing efforts by culture facilities to generate clonal dinoflagellate cultures for detailed genetic and biochemical analyses. Utilizing sequence data already available and novel sequence data generated through the above analyses, species specific quantitative PCR detection using kinetic thermal cycling assays will be developed. The kinetic thermal cycling assay system will utilize fluorogenic probes in the 5?exonuclease assay to determine target sequence copy numbers in assayed samples. In Phase I of the proposed study, this assay will be laboratory based. In Phase II, the assay will be adapted to newly developed battery powered miniaturized analytical thermal cycling instruments (MATCI) with field testing capability. Significant effort will be exerted in developing DNA extraction methods for both water and sediment that can support such field analyses. Utilizing this technology, a quantitative PCR assay for Pfiesteria piscicida will be developed, and field tested both in Maryland and North Carolina.


These studies will be carried out in parallel with longitudinal cohort studies of individuals with occupational exposure to the same waterways (thus, at potential risk for Pfiesteria-related illness). In pilot study epidemiological analyses, results of qualitative PCR assays for the presence of planktonic Pfiesteria piscicida will be correlated with observed health effects in exposed individuals (as well as with environmental events such as fish kills). The quantitative PCR assay will be field tested in the context of the ongoing Maryland cohort study in an effort to help define ?significant exposures to Pfiesteria piscicida.

Expected Results:

1) Development of PCR/heteroduplex mobility based assays permitting rapid assessment of dinoflagellate culture clonality. 2) Identification of characteristic HMA patterns (molecular fingerprints) associated with specific estuarine heterotrophic dinoflagellate dinoflagellates. 3) SSU sequence determination for Pfiesteria piscicida and Pfiesteria-like dinoflagellates (HMA directed sequence discovery). 4) Expansion of an estuarine dinoflagellate SSU sequence matrix, permitting: 5) Development of species specific PCR primer/probe combinations for environmental monitoring. 6) Development of species specific quantitative PCR utilizing fluorogenic probe hybridization strategies (5?-3? exonuclease assay). 7) Deployment of quantitative assays in a portable real-time, PCR device that can be used in-situ for assessment of harmful algal blooms. 8) Correlation of environmental monitoring for Pfiesteria piscicida and related dinoflagellates with results of an ongoing Pfiesteria-related illness cohort study among Maryland Chesapeake Bay watermen.

Publications and Presentations:

Publications have been submitted on this project: View all 90 publications for this project

Journal Articles:

Journal Articles have been submitted on this project: View all 17 journal articles for this project

Supplemental Keywords:

Pfiesteria piscicida, toxin-producing dinoflagellates, harmful algal blooms, phytoplankton, phycology, real-time PCR, MATCI (miniaturized analytic thermal cycling instruments), heteroduplex mobility assays, SSU sequences., RFA, Scientific Discipline, Geographic Area, Water, Waste, Ecosystem Protection/Environmental Exposure & Risk, Contaminated Sediments, State, Oceanography, Environmental Microbiology, algal blooms, Ecological Risk Assessment, Ecology and Ecosystems, marine ecosystem, bloom dynamics, dinoflagellates, DNA based molecular diagnostics, fish kills, contaminated sediment, phytoplankton, polymerase chain reaction, Maryland (MD), pfiesteria, South Carolina (SC), North Carolina (NC), ECOHAB, gene sequences

Progress and Final Reports:

  • 1999
  • 2000 Progress Report
  • 2001 Progress Report
  • Final Report