Cultures and Species identification
For specific identification, two species of Pseudo-nitzschia spp collected in the field and isolated were then cultured in an incubator (light 24 h, 20°C ± 2°C) for more than 6 weeks to 10,000 cells/mL, and DNA was extracted according to the manufacturer’s protocols using the AccuPrep plant genome DNA extraction kit (Bioneer Cooperation, Daejeon, Korea). DNA was amplified using Eukaryotic specific primers (Table 1), DIR and D3Ca, and amplified in MyGenie 32 Thermal Block (Bioneer Cooperation) under the following conditions. The PCR program was 5 min at 95°C, 40 cycles of 30 s at 95°C, 30 s at 47°C, and 60 s at 72°C, and then a final step of 5 min at 72°C. We confirmed by Sanger sequencing that the amplicon was from Pseudo-nitzschia pungens and Pseudo-nitzschia americana.
Table 1
Primers and probes used in this study.
Type
|
Sequence (5′–3′)
|
Authors
|
DIR forward primer
|
ACC CGC TGA ATT TAA GCA TA
|
Scholin et al. (1994)
|
D3Ca reverse primer
|
ACG AAC GAT TTG CAC GTC AG
|
Lenaers et al. (1989)
|
PSE1Forward
|
AGG ATG TGA ATC TGC GCT TT
|
This study
|
PSE1Reverse
|
TCA CCC TCT CAG CTG TCC TT
|
This study
|
Probe
|
TGG CGCC GA ATT GTG GTC TG
|
This study
|
PnGenusFwd
|
CTG TGT AGT GCT TCT TAG AGG
|
Fitzpatrick et al. (2010)
|
PhGenusRev
|
AGG TAG AAC TCG TTG AAT GC
|
Fitzpatrick et al. (2010)
|
Table 2
Filter pore size and materials used in this study.
Pore size (µm)
|
Filter type
|
0.2
|
Cellulose nitrate (Whatman)
|
0.45
|
Mixed cellulose ester (Advantec MFS)
|
1.0
|
Glass fiber (Whatman)
|
2.7
|
Glass fiber (Whatman)
|
2–4
|
Paper (CHMLAB)
|
20–25
|
Paper (CHMLAB)
|
34–42
|
Paper (CHMLAB)
|
In addition, Pseudo-nitzschia multiseries (LIMS-PS-3398), Skeletonema costatum (LIMS-PS-2611), and Alexandrium catenella (LIMS-PS-3427) were provided from LIMS (Library of Marine Samples) and cultured under the above conditions for experiments. Cultured to a minimum of 10,000 cells/mL.
Primer and probe design and in silico test
A new set of primer probes was developed to detect and quantify Pseudo-nitzschia spp. The related sequences were searched using the large subunit rRNA domain (MK682488, MZ838947, MG799158, MK682498, LC483111, KX290874) of the National Biotechnology Information Center (www.ncbi.nlm.nih.gov). Subsequently, aligned with ClustalW (www.genome.jp/tools-bin/clustalw). The primer–probe set of the target species was designed using Primer3 (Whitehead Institute and Howard Hughes Medical Institute, MD) and Oligo Calc (Oligonucleotide Properties Calculator software; Table 1). The probe was double-labeled with fluorescent dyes FAM and BHQ (Bioneer Cooperation) at the 5′ and 3′ ends, respectively. The specificity of the primer-probe is shown in Fig. 1. All data generated or analysed during this study are included in this published article.
Seawater sampling method for eDNA quantitation
To compare the amount of Pseudo-nitzschia spp. cells present, samples were obtained from three stations off the coast of Tongyeong, Korea, in July 2022. Fluid pumps were used to collect 50 cm below the surface of the water each sampling site. Relatively large-pore size (20–25 and 34–42 µm) filters connecting an In-Line Filter Holder (Millipore) to a fluid pump were used to filter the seawater directly on-site and then moved to the laboratory on ice, and the relatively small 0.2, 0.45, 1.0, 2.7, and the 2–4 µm pore size filter take a long time to filter in the field, so transported seawater collected on-site to the laboratory and immediately filtered until the filter clogged. After storage at − 70°C for 24 h, eDNA was extracted from the filter.
eDNA extraction from filters
For filter eDNA extraction, an AccuPrep® plant genomic DNA extraction kit (Bioneer Cooperation) was used. Using sterile scissors and tweezers, each filter was quartered and all four were used for DNA extraction. 600 µL of PL buffer was added, and then 0.4–0.6 mm beads were added. First, the tube was shaken on a vortex for 45 s at maximum speed, incubated at 60°C for 30 min, and then vortexed again. Next, 40 µL of proteinase K and 20 µL of RNase were added to the tube, which was then incubated at 60°C for 1 h. After this, DNA extraction was performed according to the manufacturer’s protocol.
Morphological identification for eDNA quantitative analysis
The seawater collected from the sampling area, 50 cm below the surface of the water using a pump. 1 L was transferred to the polyethylene bottle and fixed with Lugol’s solution. The fixed cells were concentrated 100 times and 1 mL was used for analysis. For qualitative and quantitative analysis, a Sedgwick-Rafter chamber was used under the phase difference microscope (Nikon, Japan, JP/TS100-F). The average value was calculated after three cells counts, and the cell number (cells/mL) was calculated as the existing amount. The sample was magnification of 400–1,000 times for the qualitative analysis.
Quantitative real-time PCR assay
To determine the standard curve for qPCR quantitative analysis, P. pungens cultured in the laboratory were counted for the morphological identification method (12,800 cells/mL). In addition, a 10 mL volume of cell culture was centrifuged at 3,000 rpm for 30 minutes, and the following cell concentrations were obtained through continuous dilution(1.28⋅105–1.28⋅102 cells). P. americana was identified morphologically by Kaczmarska et al. (2005) and grown to 12,800 cells/mL for the experiment, and then continuously diluted as above to obtain a cell concentration of 1.28⋅105–1.28⋅102 cells. And the fluorescence amount of each reaction tube was quantified at each PCR cycle.
The qPCR conditions for quantitative analysis were Thermal Cycler Dice Real-Time System III (TaKaRa, Tokyo, Japan) was used for 5 minutes at 95°C, and then 40 cycles of 95°C for 30 s and 56°C for 30 s. To test the specificity of the Pseudo-nitzschia spp. primer–probe set, To confirm the specificity of Pseudo-nitzschia spp., Skeletonema costatum (LIMS-PS-2611), a diatom provided by LIMS, Alexandrium pacificum (LIMS-PS-0848), a Dinophyta that blooms at the same time as Pseudo-nitzschia spp., Thalassiosira spp., which was isolated and cultured in the field, and f/2 medium were used as negative controls. To compare the accuracy of TaqMan qPCR, the SYBR Green qPCR PnGenus described by Fitzpatrick et al. (2010) was used (Table 1). All qPCR experiments were performed in triplicate for reproducibility testing.