Development and validation of a quantitative PCR assay for the early detection and monitoring of the invasive diatom Didymosphenia geminata

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.