Molecular Quantification of Total and Toxigenic <i>Microcystis</i> Using Digital-Droplet-Polymerase-Chain-Reaction-Based Multiplex Assay
The proliferation of harmful cyanobacteria, particularly <i>Microcystis</i>, poses significant risks to drinking and recreational water resources, especially under the influence of climate change. Conventional monitoring methods based on microscopy for harmful cyanobacteria management sy...
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MDPI AG
2025-05-01
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| Series: | Toxins |
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| Online Access: | https://www.mdpi.com/2072-6651/17/5/242 |
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| author | In-Su Kim Hae-Kyung Park |
| author_facet | In-Su Kim Hae-Kyung Park |
| author_sort | In-Su Kim |
| collection | DOAJ |
| description | The proliferation of harmful cyanobacteria, particularly <i>Microcystis</i>, poses significant risks to drinking and recreational water resources, especially under the influence of climate change. Conventional monitoring methods based on microscopy for harmful cyanobacteria management systems are limited in detecting toxigenic genotypes, hindering accurate risk assessment. In this study, we developed a digital droplet PCR (ddPCR)-based method for the simultaneous quantification of total and toxigenic <i>Microcystis</i> in freshwater environments. We targeted the <i>secA</i> gene, specific to the <i>Microcystis</i> genus, and the <i>mcyA</i> gene, associated with microcystin biosynthesis. Custom-designed primers and probes showed high specificity and sensitivity, enabling accurate detection without cross-reactivity. The multiplex ddPCR assay allowed for concurrent quantification of both targets in a single reaction, reducing the analysis time and cost. Application to field samples demonstrated good agreement with microscopic counts and revealed seasonal shifts in toxigenic genotype abundance. Notably, ddPCR detected <i>Microcystis</i> at very low densities—down to 7 cells/mL in the mixed cyanobacterial communities of field samples—even when microscopy failed, highlighting its utility for early bloom detection. This approach provides a reliable and efficient tool for monitoring <i>Microcystis</i> dynamics and assessing toxin production potential, offering significant advantages for the early warning and proactive management of harmful cyanobacterial blooms. |
| format | Article |
| id | doaj-art-c32a066ef14d436bb385a1b44feb7fd4 |
| institution | OA Journals |
| issn | 2072-6651 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Toxins |
| spelling | doaj-art-c32a066ef14d436bb385a1b44feb7fd42025-08-20T01:56:38ZengMDPI AGToxins2072-66512025-05-0117524210.3390/toxins17050242Molecular Quantification of Total and Toxigenic <i>Microcystis</i> Using Digital-Droplet-Polymerase-Chain-Reaction-Based Multiplex AssayIn-Su Kim0Hae-Kyung Park1Nakdong River Environment Research Center, National Institute of Environmental Research, Daegu 43008, Republic of KoreaNakdong River Environment Research Center, National Institute of Environmental Research, Daegu 43008, Republic of KoreaThe proliferation of harmful cyanobacteria, particularly <i>Microcystis</i>, poses significant risks to drinking and recreational water resources, especially under the influence of climate change. Conventional monitoring methods based on microscopy for harmful cyanobacteria management systems are limited in detecting toxigenic genotypes, hindering accurate risk assessment. In this study, we developed a digital droplet PCR (ddPCR)-based method for the simultaneous quantification of total and toxigenic <i>Microcystis</i> in freshwater environments. We targeted the <i>secA</i> gene, specific to the <i>Microcystis</i> genus, and the <i>mcyA</i> gene, associated with microcystin biosynthesis. Custom-designed primers and probes showed high specificity and sensitivity, enabling accurate detection without cross-reactivity. The multiplex ddPCR assay allowed for concurrent quantification of both targets in a single reaction, reducing the analysis time and cost. Application to field samples demonstrated good agreement with microscopic counts and revealed seasonal shifts in toxigenic genotype abundance. Notably, ddPCR detected <i>Microcystis</i> at very low densities—down to 7 cells/mL in the mixed cyanobacterial communities of field samples—even when microscopy failed, highlighting its utility for early bloom detection. This approach provides a reliable and efficient tool for monitoring <i>Microcystis</i> dynamics and assessing toxin production potential, offering significant advantages for the early warning and proactive management of harmful cyanobacterial blooms.https://www.mdpi.com/2072-6651/17/5/242toxigenic <i>Microcystis</i>microcystinsdigital droplet PCRgenus-specific primers<i>mcyA</i><i>secA</i> |
| spellingShingle | In-Su Kim Hae-Kyung Park Molecular Quantification of Total and Toxigenic <i>Microcystis</i> Using Digital-Droplet-Polymerase-Chain-Reaction-Based Multiplex Assay Toxins toxigenic <i>Microcystis</i> microcystins digital droplet PCR genus-specific primers <i>mcyA</i> <i>secA</i> |
| title | Molecular Quantification of Total and Toxigenic <i>Microcystis</i> Using Digital-Droplet-Polymerase-Chain-Reaction-Based Multiplex Assay |
| title_full | Molecular Quantification of Total and Toxigenic <i>Microcystis</i> Using Digital-Droplet-Polymerase-Chain-Reaction-Based Multiplex Assay |
| title_fullStr | Molecular Quantification of Total and Toxigenic <i>Microcystis</i> Using Digital-Droplet-Polymerase-Chain-Reaction-Based Multiplex Assay |
| title_full_unstemmed | Molecular Quantification of Total and Toxigenic <i>Microcystis</i> Using Digital-Droplet-Polymerase-Chain-Reaction-Based Multiplex Assay |
| title_short | Molecular Quantification of Total and Toxigenic <i>Microcystis</i> Using Digital-Droplet-Polymerase-Chain-Reaction-Based Multiplex Assay |
| title_sort | molecular quantification of total and toxigenic i microcystis i using digital droplet polymerase chain reaction based multiplex assay |
| topic | toxigenic <i>Microcystis</i> microcystins digital droplet PCR genus-specific primers <i>mcyA</i> <i>secA</i> |
| url | https://www.mdpi.com/2072-6651/17/5/242 |
| work_keys_str_mv | AT insukim molecularquantificationoftotalandtoxigenicimicrocystisiusingdigitaldropletpolymerasechainreactionbasedmultiplexassay AT haekyungpark molecularquantificationoftotalandtoxigenicimicrocystisiusingdigitaldropletpolymerasechainreactionbasedmultiplexassay |