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

Abstract

Background: We describe the development and validation of a new quantitative real time PCR (qrt-PCR) method for the enumeration of the toxic benthic dinoflagellate Ostreopsis cf. ovata in marine environment. The benthic Ostreopsis sp. has a world-wide distribution and is associated during high biomass proliferation with the production of potent palytoxin-like compounds affecting human health and environment. Species-specific identification, which is relevant for the complex of different toxins production, by traditional methods of microscopy is difficult due to the high morphological variability, and thus different morphotypes can be easily misinterpreted.

Methodology/findings: The method is based on the SYBR I Green real-time PCR technology and combines the use of a plasmid standard curve with a "gold standard" created with pooled crude extracts from environmental samples collected during a bloom event of Ostreopsis cf. ovata in the Mediterranean Sea. Based on their similar PCR efficiencies (95% and 98%, respectively), the exact rDNA copy number per cell was obtained in cultured and environmental samples. Cell lysates were used as the templates to obtain total recovery of DNA. The analytical sensitivity of the PCR was set at two rDNA copy number and 8.0×10(-4) cell per reaction for plasmid and gold standards, respectively; the sensitivity of the assay was of cells g(-1) fw or 1(-1) in macrophyte and seawater samples, respectively. The reproducibility was determined on the total linear quantification range of both curves confirming the accuracy of the technical set-up in the complete ranges of quantification over time.

Conclusions/significance: We developed a qrt-PCR assay specific, robust and high sample throughput for the absolute quantification of the toxic dinoflagellate Ostreopsis cf. ovata in the environmental samples. This molecular approach may be considered alternative to traditional microscopy and applied for the monitoring of benthic toxic microalgal species in the marine ecosystems.

Figure 1. Dynamic range, sensitivity and specificity of the qrt-PCR assay.

A typical amplification plot (a, d) and the corresponding standard curve (b, e) for the pLSUO and gold standards are shown, respectively. A 204 bp fragment of the LSU gene were amplified in a 10⁶, 10⁵, 10⁴, 10³, 10², 10, 5 and 2 copy dilutions of the pLSUO plasmid (a) and in a 8, 4, 0.8, 0.16, 0.08, 0.016, 0.008, 0.004 and 0.0008 O. cf. ovata cell dilutions (d) of the lysed pool from macroalgae samples. The fluorescence intensity (ΔRn) expressed as logarithmic scale is plotted vs cycle number. Only one replicate is shown. The mean standard curve is obtained by the correlation of Ct values and log₁₀ input plasmid copy (b) or environmental O. cf. ovata cell number (e) measured in 8 independent experiments ± SD, respectively. Melting curve of the the 204 bp amplified fragments of the pLSUO plasmid (c) and environmental O. cf. ovata cells (f) generated by qrt-PCR. To better display the melting curves, only the low input pLSUO plasmid copy number (10, 2 and NTC ) and cell number (0.008 and 0.0008) are shown. As few as two copies of pLSUO plasmid and 0.0008 cell dilutions are clearly detected. The specificity of the 204 bp amplicon was also confirmed by gel electrophoresis and sequencing analysis (data not shown).

Figure 2. Micrograph of Ostreopsis cf. ovata assemblage in Scanning Electron Microscopy (SEM).

Scale bar 50 µm (photo by Romagnoli T.).