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. 2013 Mar 13;8(3):e57627.
doi: 10.1371/journal.pone.0057627. Print 2013.

Quantitative PCR method for enumeration of cells of cryptic species of the toxic marine dinoflagellate Ostreopsis spp. in coastal waters of Japan

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Quantitative PCR method for enumeration of cells of cryptic species of the toxic marine dinoflagellate Ostreopsis spp. in coastal waters of Japan

Naohito Hariganeya et al. PLoS One. .

Abstract

Monitoring of harmful algal bloom (HAB) species in coastal waters is important for assessment of environmental impacts associated with HABs. Co-occurrence of multiple cryptic species such as toxic dinoflagellate Ostreopsis species make reliable microscopic identification difficult, so the employment of molecular tools is often necessary. Here we developed new qPCR method by which cells of cryptic species can be enumerated based on actual gene number of target species. The qPCR assay targets the LSU rDNA of Ostreopsis spp. from Japan. First, we constructed standard curves with a linearized plasmid containing the target rDNA. We then determined the number of rDNA copies per cell of target species from a single cell isolated from environmental samples using the qPCR assay. Differences in the DNA recovery efficiency was calculated by adding exogenous plasmid to a portion of the sample lysate before and after DNA extraction followed by qPCR. Then, the number of cells of each species was calculated by division of the total number of rDNA copies of each species in the samples by the number of rDNA copies per cell. To test our procedure, we determined the total number of rDNA copies using environmental samples containing no target cells but spiked with cultured cells of several species of Ostreopsis. The numbers estimated by the qPCR method closely approximated total numbers of cells added. Finally, the numbers of cells of target species in environmental samples containing cryptic species were enumerated by the qPCR method and the total numbers also closely approximated the microscopy cell counts. We developed a qPCR method that provides accurate enumeration of each cryptic species in environments. This method is expected to be a powerful tool for monitoring the various HAB species that occur as cryptic species in coastal waters.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Scheme of the cell quantification method by qPCR used in this study.
Normalization with DNA recovery efficiency (RE) by addition of exogenous plasmid DNA was considered in the qPCR method.
Figure 2
Figure 2. Standard curves of circular and linear plasmid with or without the addition of an environmental sample.
Standard curves of pGEM-3z assay generated by circular and linearized pGEM-3Z plasmid with or without the addition of an environmental sample were generated. Error bars represent standard deviation of triplicate PCR reactions. Black circle: circular pGEM-3Z with an environmental sample. Open circle: circular pGEM-3Z. Black triangle: linear pGEM-3Z with the environmental sample. Open triangle: linearized pGEM-3Z.
Figure 3
Figure 3. Dynamic range and sensitivity of the qPCR assay.
Standard curves were generated by plasmids containing the 28S rDNA D8/D10 sequences of various species of Ostreopsis. Each standard curve was generated by the plasmid containing the target sequence of 28S rDNA D8/D10 using O. cf. ovata primer and probe set (A), Ostreopsis sp. 1 primer and probe set (B), Ostreopsis sp. 5 primer and probe set (C) Ostreopsis sp. 6 primer and probe set (D). Error bars represent standard deviation of triplicate PCR reactions.
Figure 4
Figure 4. Standard curves of linear plasmid spiked with or without an environmental sample.
Standard curves of linear target plasmid containing the 28S rDNA spiked with DNA from the environmental sample (Maizuru100904) using various Ostreopsis species-specific primer and probe sets (A: O. cf. ovata; B: Ostreopsis sp. 1; C: Ostreopsis sp. 5; D: Ostreopsis sp. 6) were constructed with a 10-fold dilution series of the plasmid. Error bars represent standard deviation of triplicate PCR reactions. Black circle: plasmid containing the 28S rDNA spiked with DNA from the environmental sample. Open circle: plasmid containing the 28S rDNA without DNA from the environmental sample.
Figure 5
Figure 5. Standard curves of Ostreopsis genomic DNA and control plasmid.
Cells of the four Ostreopsis species were harvested from cultures, sonicated and spiked with 4.0×109 copies of linearized pGEM-3Z plasmid, respectively. Then, the genomic DNA were extracted. Ten-fold dilution series of the genomic DNA were prepared and then spiked with 2 µl of DNA solution extracted from the environmental sample (Maizuru100904). Using the dilution series as templates, qPCR was conducted with Ostreopsis species-specific primer and probe set (Black circle) and pGEM primer and probe set (Open circle). Error bars represent standard deviation of triplicate PCR reactions. Standard curves of O. cf. ovata (A), Ostreopsis sp. 1 (B), Ostreopsis sp. 5 (C) and Ostreopsis sp.6 (D).
Figure 6
Figure 6. Validation of normalization with DNA recovery efficiency.
Comparison of the ‘tentative’ number of 28S rDNA copies determined by qPCR (White column), ‘normalized’ number of 28S rDNA copies determined by qPCR normalized with DNA recovery efficiency (Grey column) and ‘actual’ number of copies (1.50×106 and 2.00×106 molecules) of added plasmid containing the 28S rDNA D8/D10 sequences of O. cf. ovata and Ostreopsis sp. 5 (Black column). Values are mean ± SD. Asterisk show significant difference between qPCR results without normalization and that with normalization by DNA recovery efficiency (p<0.05).
Figure 7
Figure 7. Cell quantification of each species in mixed samples of cultured cells of various species.
Cell quantification of each Ostreopsis species in three mixed culture samples (A–C) spiked with the environmental sample (Maizuru100904) by qPCR considering normalization with RE. A: qPCR quantification result of four species in mixed culture sample (Sample A) containing 900±200 cells of cultured O. cf. ovata, 1,500±350 cells of Ostreopsis sp. 1, 1,500±230 cells of Ostreopsis sp. 5 and 1,260±110 cells of Ostreopsis sp. 6. B: qPCR result in Sample B containing 90,000±5,000 cells of cultured O. cf. ovata, 100±10 cells of Ostreopsis sp. 1, 80±10 cells of Ostreopsis sp. 5 and 50±5 cells of Ostreopsis sp. 6. C: qPCR result in Sample C containing O. cf. ovata, 80,000±4,000 cells of Ostreopsis sp. 1, 70±10 cells of Ostreopsis sp. 5 and 50±10 cells of Ostreopsis sp. 6. White columns: cell number determined by qPCR method considering normalization with RE, Black columns: actual number of cells spiked in environmental sample. Numbers on x-axis represented Log10 cell number. Values are mean ± SD.
Figure 8
Figure 8. Cell enumeration of Ostreopsis species in environmental samples.
Number of cells of four species of Ostreopsis were determined by qPCR considering normalization with RE in environmental samples collected from various locals of Japanese coastal waters listed in Table 5. Cell number of O. cf. ovata (red), Ostreopsis sp. 1 (blue), Ostreopsis sp. 5 (green), Ostreopsis sp. 6 (purple) as determined by qPCR with RE. Total cell number of Ostreopsis species counted by microscopy (white). Numbers on y-axis represented Log10 cells g−1 fresh weight algae. Values are mean ± SD.

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