Development and validation of a quantitative PCR assay for the early detection and monitoring of the invasive diatom Didymosphenia geminata |
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| Institution: | 1. Environmental Research Institute, School of Science, Faculty of Science & Engineering, University of Waikato, Private Bag 3105, Hamilton 3240, New Zealand;2. University of Delaware, College of Earth, Ocean, and Environment, Lewes, DE 19958-1242, USA;3. Cawthron Institute, Private Bag 2, Nelson 7042, New Zealand;4. Environmental Risk Analysis Programs, US Department of Agriculture, 4700 River Road, Unit 147, Riverdale, MD 20737, USA;1. Physics Department and Biruni Observatory, College of Sciences, Shiraz University, Shiraz 71454, Iran;2. Research Institute for Astronomy and Astrophysics of Maragha (RIAAM), P.O. Box 55134-441, Maragha, Iran;3. Physics Department, Shahid Beheshti University, Tehran 19839, Iran;1. Department of Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, 2000, South Africa;2. Evolutionary Studies Institute, University of the Witwatersrand, Johannesburg, 2000, South Africa;3. Department of Biodiversity and Conservation Biology, University of the Western Cape, Cape Town, 7535, South Africa;4. Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721, USA;5. School of Animal, Plant and Environmental Science, University of the Witwatersrand, Johannesburg, 2000, South Africa;1. UMR MARBEC, Centre for Marine Biodiversity, Exploitation and Conservation (IRD, Ifremer, Université Montpellier, CNRS), Université Montpellier, CC 093, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France;2. CNRS, UMR 7144, Adaptation et Diversité en Milieu Marin, Equipe EPEP, Station Biologique de Roscoff, 29680 Roscoff, France;3. Sorbonne Universités, Université Pierre et Marie Curie (Paris 6), UMR 7144, Station Biologique de Roscoff, 29680 Roscoff, France;4. UMR MARBEC, Centre for Marine Biodiversity, Exploitation and Conservation (IRD, Ifremer, Université de Montpellier, CNRS), Laboratoire Environnement et Ressources du Languedoc-Roussillon (LER-LR), Station Ifremer, Avenue Jean Monnet, CS 30171, 34203 Sète Cedex, France;1. Department of Environmental Marine Sciences, College of Science and Technology, Hanyang University, Ansan 426-791, Republic of Korea;2. Green Life Science Department, College of Convergence, Sangmyung University, 7 Hongij-dong, Jongno-gu, Seoul 110-743, Republic of Korea;3. Department of Biological Sciences, College of Natural Sciences, Sungkyunkwan University, Suwon 440-746, South Korea;4. Marine Environment Research Division, National Fisheries Research and Development Institute, Busan 619-705, Republic of Korea |
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| Abstract: | Didymosphenia geminata is a large, invasive, freshwater diatom that can produce distinctive and robust mucilaginous stalks. Over the last two decades, there has been a worldwide increase in the distribution and severity of D. geminata blooms. These dense, persistent blooms can have severe impacts on native species and ecosystem functioning. D. geminata is usually identified by microscopic methods that are time consuming, resource intensive, and dependent upon expert taxonomic identification, so the extent of surveillance programs has been limited. As an alternative, we have developed a TaqMan quantitative polymerase chain reaction (QPCR) assay for sensitive and rapid detection and enumeration of D. geminata in environmental samples. Species-specific QPCR primers and probe were designed by aligning the D. geminata 18S ribosomal DNA (rDNA) sequence with closely related diatoms. The QPCR assay was linear (R2 = 1.00) over a detection range of eight orders of magnitude with a lower limit of approximately two D. geminata cells. QPCR analysis of environmental samples employed the comparative cycle threshold (CT)-method with an exogenous plasmid used as an internal reference standard. The assay was evaluated using samples collected during a survey of D. geminata in three rivers in the South Island, New Zealand, and from 13 international locations where D. geminata is known to be present. Positive QPCR amplifications were confirmed as the correct amplification product through direct DNA sequencing. Phylogenetic analysis of 18S rDNA sequences suggests that D. geminata is more closely related to species in the family Cymbellaceae rather than Gomphonemataceae as currently classified. |
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| Keywords: | Detection and quantification Didymosphenia geminata Invasive diatom Montoring and surveillance QPCR |
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