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DEVELOPMENT OF SEMI-QUANTITATIVE PCR ASSAYS FOR THE DETECTION AND ENUMERATION OF GAMBIERDISCUS SPECIES (GONYAULACALES, DINOPHYCEAE)1
Article first published online: 10 MAY 2012
© 2012 Phycological Society of America
Journal of Phycology
Volume 48, Issue 4, pages 902–915, August 2012
How to Cite
Vandersea, M. W., Kibler, S. R., Holland, W. C., Tester, P. A., Schultz, T. F., Faust, M. A., Holmes, M. J., Chinain, M. and Wayne Litaker, R. (2012), DEVELOPMENT OF SEMI-QUANTITATIVE PCR ASSAYS FOR THE DETECTION AND ENUMERATION OF GAMBIERDISCUS SPECIES (GONYAULACALES, DINOPHYCEAE). Journal of Phycology, 48: 902–915. doi: 10.1111/j.1529-8817.2012.01146.x
Received 10 February 2011. Accepted 12 March 2012.
- Issue published online: 1 AUG 2012
- Article first published online: 10 MAY 2012
- Accepted manuscript online: 20 MAR 2012 08:07AM EST
- Gambierdiscus belizeanus;
- Gambierdiscus caribaeus;
- Gambierdiscus carolinianus;
- Gambierdiscus carpenteri;
- Gambierdiscus ruetzleri;
- Gambierdiscus ribotype 2;
- SYBR green
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.