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J Phycol. 2012 Aug;48(4):902-15. doi: 10.1111/j.1529-8817.2012.01146.x. Epub 2012 May 10.

DEVELOPMENT OF SEMI-QUANTITATIVE PCR ASSAYS FOR THE DETECTION AND ENUMERATION OF GAMBIERDISCUS SPECIES (GONYAULACALES, DINOPHYCEAE)(1).

Author information

1
NOS/NOAA, Center for Coastal Fisheries and Habitat Research, 101 Pivers Island Road, Beaufort, North Carolina 28516, USAMarine Conservation Molecular Facility, Duke University Marine Laboratory, Nicholas School of the Environment, 135 Marine Lab Road, Beaufort, North Carolina 28516, USADepartment of Botany, United States National Herbarium, National Museum of Natural History, Smithsonian Institution, 4210 Silver Hill Road, Suitland, Maryland, 20746, USATropical Marine Science Institute, 14 Kent Ridge Road, National University of Singapore, Singapore City 119223, SingaporeAquatic Ecosystem Health, Department of Environmental and resource Management, GPO Box 2454, Brisbane, Quennsland 4001, AustraliaLaboratoire Des Micro-Algues Toxiques, Institut Louis Malardé, BP 30 98713 Papeete, TahitiNOS/NOAA, Center for Coastal Fisheries and Habitat Research, 101 Pivers Island Road, Beaufort, North Carolina 28516, USA.

Abstract

Ciguatera fish poisoning (CFP) is a serious health problem in tropical regions and is caused by the bioaccumulation of lipophilic toxins produced by dinoflagellates in the genus Gambierdiscus. Gambierdiscus species are morphologically similar and are difficult to distinguish from one another even when using scanning electron microscopy. Improved identification and detection methods that are sensitive and rapid are needed to identify toxic species and investigate potential distribution and abundance patterns in relation to incidences of CFP. This study presents the first species-specific, semi-quantitative polymerase chain reaction (qPCR) assays that can be used to address these questions. These assays are specific for five Gambierdiscus species and one undescribed ribotype. The assays utilized a SYBR green format and targeted unique sequences found within the SSU, ITS, and the D1/D3 LSU ribosomal domains. Standard curves were constructed using known concentrations of cultured cells and 10-fold serial dilutions of rDNA PCR amplicons containing the target sequence for each specific assay. Assay sensitivity and accuracy were tested using DNA extracts purified from known concentrations of multiple Gambierdiscus species. The qPCR assays were used to assess Gambierdiscus species diversity and abundance in samples collected from nearshore areas adjacent to Ft. Pierce and Jupiter, Florida USA. The results indicated that the practical limit of detection for each assay was 10 cells per sample. Most interestingly, the qPCR analysis revealed that as many as four species of Gambierdiscus were present in a single macrophyte sample.

KEYWORDS:

Gambierdiscus belizeanus; Gambierdiscus caribaeus; Gambierdiscus carolinianus; Gambierdiscus carpenteri; Gambierdiscus ribotype 2; Gambierdiscus ruetzleri; SYBR green; ciguatera; qPCR

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