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IDENTIFICATION OF PFIESTERIA PISCICIDA (DINOPHYCEAE) AND PFIESTERIA-LIKE ORGANISMS USING INTERNAL TRANSCRIBED SPACER-SPECIFIC PCR ASSAYS1

Authors

  • R. Wayne Litaker,

    Corresponding author
    1. Center for Coastal Fisheries and Habitat Research, National Ocean Service, National Oceanic Atmospheric Administration,
      101 Pivers Island Rd., Beaufort, North Carolina 28516-9722, USA
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  • Mark W. Vandersea,

    1. Center for Coastal Fisheries and Habitat Research, National Ocean Service, National Oceanic Atmospheric Administration,
      101 Pivers Island Rd., Beaufort, North Carolina 28516-9722, USA
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  • Steven R. Kibler,

    1. Center for Coastal Fisheries and Habitat Research, National Ocean Service, National Oceanic Atmospheric Administration,
      101 Pivers Island Rd., Beaufort, North Carolina 28516-9722, USA
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  • Kimberly S. Reece,

    1. Virginia Institute of Marine Science, The College of William and Mary, P.O. Box 1346, Gloucester Point,
      Virginia 23062-1346, USA
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  • Nancy A. Stokes,

    1. Virginia Institute of Marine Science, The College of William and Mary, P.O. Box 1346, Gloucester Point,
      Virginia 23062-1346, USA
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  • Karen A. Steidinger,

    1. Florida Fish and Wildlife Conservation Commission, Florida Marine Research Institute, 100 Eighth Avenue SE, St. Petersburg,
      Florida 33701-5020, USA
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  • Dave F. Millie,

    1. Florida Fish and Wildlife Conservation Commission, Florida Marine Research Institute, 100 Eighth Avenue SE, St. Petersburg,
      Florida 33701-5020, USA
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  • Brian J. Bendis,

    1. Florida Fish and Wildlife Conservation Commission, Florida Marine Research Institute, 100 Eighth Avenue SE, St. Petersburg,
      Florida 33701-5020, USA
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  • Ryan J. Pigg,

    1. Florida Fish and Wildlife Conservation Commission, Florida Marine Research Institute, 100 Eighth Avenue SE, St. Petersburg,
      Florida 33701-5020, USA
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  • Patricia A. Tester

    1. Center for Coastal Fisheries and Habitat Research, National Ocean Service, National Oceanic Atmospheric Administration,
      101 Pivers Island Rd., Beaufort, North Carolina 28516-9722, USA
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  • 1

    Received 23 July 2002. Accepted 31 March 2003.

  • The U.S. Government has the right to retain a nonexclusive royalty-free license in and to any copyright covering this article.

Author for correspondence: e-mail Wayne.Litaker@noaa.gov

Abstract

The putative harmful algal bloom dinoflagellate, Pfiesteria piscicida (Steidinger et Burkholder), frequently co-occurs with other morphologically similar species collectively known as Pfiesteria-like organisms (PLOs). This study specifically evaluated whether unique sequences in the internal transcribed spacer (ITS) regions, ITS1 and ITS2, could be used to develop PCR assays capable of detecting PLOs in natural assemblages. ITS regions were selected because they are more variable than the flanking small subunit or large subunit rRNA genes and more likely to contain species-specific sequences. Sequencing of the ITS regions revealed unique oligonucleotide primer binding sites for Pfiesteria piscicida, Pfiesteria shumwayae (Glasgow et Burkholder), Florida “Lucy” species, two cryptoperidiniopsoid species, “H/V14” and “PLO21,” and the estuarine mixotroph, Karlodinium micrum (Leadbetter et Dodge). These PCR assays had a minimum sensitivity of 100 cells in a 100-mL sample (1 cell·mL−1) and were successfully used to detect PLOs in the St. Johns River system in Florida, USA. DNA purification and aspects of PCR assay development, PCR optimization, PCR assay controls, and collection of field samples are discussed.

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