A PCR-based assay to facilitate early detection of Diplosoma listerianum in Atlantic Canada

Abstract

The recent detection of the invasive colonial tunicate Diplosoma listerianum in Havre- Aubert, Magdalen Islands (Quebec, Canada) in 2008, prompted the development of a molecular assay as a method to detect and monitor for the potential invasion of this species in Prince Edward Island. The aim of this study was to design a highly sensitive, species-specific Polymerase Chain Reaction (PCR) assay capable of detecting D. listerianum with a high efficacy in local water samples. To accomplish this, oligonucleotide primer sets were designed from the 18S rDNA gene of D. listerianum. Primer sets were evaluated for specificity using the GenBank database, followed by a series of spiked water sample trials involving various tunicate species. Assay efficacy was tested and then evaluated by conducting spiked water sample trials using D. listerianum samples from two different geographic locations (Japan and Canada). Primer sets that were shown to be species specific were then tested for their analytical sensitivity and environmental efficacy by spiking local water samples with various amounts of D. listerianum tissue. The primer set DlistF1/DlistR1 was found to be species specific and yielded no false positive results when tested with tissue from the four invasive tunicate species currently present on Prince Edward Island (PEI) (Styela clava, Botryllus schlosseri, Botrylloides violaceus, and Ciona intestinalis). This assay was also capable of detecting D. listerianum DNA from two different populations, demonstrating its potential for use in other geographic locations, which may possess different haplotypes of the species. As the results of this study demonstrate, the DlistF1/DlistR1 assay has a high analytical sensitivity, detecting DNA from as little as 1 zooid in a water sample, and was not inhibited when tested with water samples collected from various bays across both PEI and the Magdalen Islands. The DlistF1/DlistR1 molecular assay provides a monitoring tool for shellfish aquaculture regions and can be used to facilitate early detection of this species. This level of early detection is beneficial to facilitate the implementation of mitigation programs in time to prevent D. listerianum from reaching nuisance levels.