Species-Specific Detection and Quantification of Toxic Marine Dinoflagellates Alexandrium tamarense and A. catenella byReal-Time PCR Assay
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A Real-time polymerase chain reaction (PCR) assay was designed and evaluated for rapid detection and quantification of the toxic dinoflagellates Alexandrium catenella and A. tamarense, which cause paralytic shellfish poisoning. Two sets of PCR primers and fluorogenic probes targeting these two species were derived from the sequence of 28S ribosomal DNA. PCR specificity was examined in closely related Alexandrium spp. and many other microalgae. A. catenella–specific primers and probe detected the PCR amplification only from A. catenella strains, and nonspecific signals were not detected from any microalgae. Also, A. tamarense–specific primers and probe also detected the targeted species, suggesting the strict species specificity of each PCR. This assay could detect one cell of each species, showing its high sensitivity. Moreover, using the developed standard curves, A. tamarense and A. catenella could be quantified in agreement with the quantification by optical microscopy. The performance characteristics of species specificity, sensitivity, and rapidity suggest that this method is applicable to the monitoring of the toxic A. tamarense and A. catenella.
Keywordstoxic dinoflagellate Alexandrium paralytic shellfish poisoning (PSP) real-time PCR species-specific identification quantification
We thank Drs. M. Yamaguchi, T. Uchida (Fisheries Research Agency of Japan), S. Yoshimatsu (Kagawa Fisheries Research Institute), M. Ishida (Aichi Fisheries Research Institute), I. Imai (Kyoto University), M. Yoshida (Nagasaki University), and B. Reguera (Spanish Institute of Oceanography) for providing algal strains. We also thank Mr. Baba (Yamaguchi Prefectural Fisheries Experimental Station), Mr. Tamori (Oita Institute of Marine and Fisheries Science), and Dr. M. Yamaguchi for sampling natural seawater. This work was supported in part by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan, and a grant-in-aid for scientific research (13556033) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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