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Applied and Environmental Microbiology, November 2002, p. 5394-5407, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5394-5407.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Characterization of the rRNA Locus of Pfiesteria piscicida and Development of Standard and Quantitative PCR-Based Detection Assays Targeted to the Nontranscribed Spacer

Keiko Saito, Tomás Drgon,{dagger} José A. F. Robledo, Danara N. Krupatkina, and Gerardo R. Vasta*

Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202

Received 21 May 2002/ Accepted 16 August 2002

Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 µg of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.

* Corresponding author. Mailing address: Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 East Pratt St., Baltimore, MD 21202. Phone: (410) 234-8826. Fax: (410) 234-8896. E-mail: vasta{at}umbi.umd.edu.

{dagger} Present address: National Institute on Drug Abuse, Baltimore, MD 21224.

Applied and Environmental Microbiology, November 2002, p. 5394-5407, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5394-5407.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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