New Approach using the real-time PCR method for estimation of the toxic marine dinoflagellate Ostreopsis cf. ovata in marine environment

摘要

Background: We describe the development and validation of a new quantitative real time PCR (qrt-PCR) method for the\udenumeration of the toxic benthic dinoflagellate Ostreopsis cf. ovata in marine environment. The benthic Ostreopsis sp. has a\udworld-wide distribution and is associated during high biomass proliferation with the production of potent palytoxin-like\udcompounds affecting human health and environment. Species-specific identification, which is relevant for the complex of\uddifferent toxins production, by traditional methods of microscopy is difficult due to the high morphological variability, and\udthus different morphotypes can be easily misinterpreted.\udMethodology/Findings: The method is based on the SYBR I Green real-time PCR technology and combines the use of a\udplasmid standard curve with a ‘‘gold standard’’ created with pooled crude extracts from environmental samples collected\udduring a bloom event of Ostreopsis cf. ovata in the Mediterranean Sea. Based on their similar PCR efficiencies (95% and 98%,\udrespectively), the exact rDNA copy number per cell was obtained in cultured and environmental samples. Cell lysates were\udused as the templates to obtain total recovery of DNA. The analytical sensitivity of the PCR was set at two rDNA copy\udnumber and 8.061024 cell per reaction for plasmid and gold standards, respectively; the sensitivity of the assay was of cells\udg21 fw or 121 in macrophyte and seawater samples, respectively. The reproducibility was determined on the total linear\udquantification range of both curves confirming the accuracy of the technical set-up in the complete ranges of quantification\udover time.\udConclusions/Significance: We developed a qrt-PCR assay specific, robust and high sample throughput for the absolute\udquantification of the toxic dinoflagellate Ostreopsis cf. ovata in the environmental samples. This molecular approach may be\udconsidered alternative to traditional microscopy and applied for the monitoring of benthic toxic microalgal species in the\udmarine ecosystems.