Real-time PCR Detection of Dinophysis Species in Irish Coastal Waters

Abstract

Diarrhetic shellfish toxin-producing Dinophysis species occur in Irish coastal waters throughout the year. Dinophysis acuta and Dinophysis acuminata are the most commonly occurring species and are responsible for the majority of closures of Irish mussel farms. This study describes the development of a qualitative real-time poly-merase chain reaction (PCR) assay for identification of D. acuta and D. acuminata in Irish coastal waters. DNA sequence information for the D1-D2 region of the large ribosomal sub-unit (LSU) was obtained, following single-cell PCR of D. acuta and D. acuminata cells isolated from Irish coastal locations. PCR primers and hybridization probes, specific for the detection of D. acuta,were designed for real-time PCR on the LightCycler™. The LightCycler™ software melt curve analysis programme determined that D. acuta was identified by a melt-peak at 61°C , while D. acuminata cells produced a melt peak at 48°C. The limit of detection of the real-time PCR assay was determined to be one to ten plasmid copies of the LSU D1-D2 target region for both species and one to five D. acuminata cells. Lugol's preserved water samples were also tested with the assay. The real-time PCR assay identified Dinophysis species in 100% of samples found to contain Dinophysis species by microscopy and had a greater than 90% correlation with light microscopy for identification of D. acuta and D. acuminata in the samples. The assay can identify and discriminate D. acuta and D. acuminata at low numbers in Irish waters and has the potential to add value to the Irish phytoplankton monitoring programme.

Incorporating molecular tools into routine HAB monitoring programs: Using qPCR to track invasive Prymnesium

“…Owing to factors such as fast processing times and relatively low cost of initial setup and consumables, qPCR provides a relatively inexpensive alternative for HAB species enumeration and monitoring (Galluzzi and Penna, 2010). Multiple qPCR methods have been devised for HAB species, including Pseudo-nitzchia (Fitzpatrick et al, 2010), numerous dinoflagellates (Bowers et al, 2000;Galluzzi et al, 2004;Kavanagh et al, 2010), various cyanobacteria (Koskenniemi et al, 2007;AlTebrineh et al, 2011), and P. parvum (Galluzzi et al, 2008;Manning and La Claire, 2010), but most of these methods have not been incorporated into routine monitoring programs. Since P. parvum invaded and bloomed in Lake Texoma, OK-TX in 2004, we have been monitoring its population dynamics using microscopy-based methods (Hambright et al, 2010).…”

Molecular probes and microarrays for the detection of toxic algae in the genera Dinophysis and Phalacroma (Dinophyta)

“…Hart et al (2007) developed Dinophysis clade-specific primers for amplification of partial LSU rDNA (the D1–D2 region) to avoid cloning. Kavanagh et al (2010) described a quantitative real-time PCR assay with primers and hybridization probes specific for D . acuminata and D .…”

“…Therefore, there is a need for species-specific or strain-specific probes, which can be used to detect only the cells of interest (Anderson 1995). The development of molecular tools, such as antibody probes and oligonucleotide probes for the detection of toxic algae, is steadily underway, but has come into more widespread use only for a limited number of species with probes that can only be used in limited geographical areas (Godhe et al 2007; Miller and Scholin 1996; Simon et al 1997; Scholin et al 1997; Kavanagh et al 2010). …”

Approaches for the detection of harmful algal blooms using oligonucleotide interactions

“…Applications directed at the detection of Alexandrium species such as A. catenella [62][63][64][65][66], A. minutum [26,67], A. fundyense [68,69], A. taylori [64] and A. tamarense [62,63,70] have been thoroughly reported. Similarly, real-time PCR methods were developed for the dinoflagellate species Dinophysis acuta and D. acuminata [71]. Recent outbreaks of Pseudo-nitzschia in the USA have propelled the development of real-time PCR methods for P. australis, P. pungens, P. delicatissima, P. calliantha, P. multistriata [72,73] and P. multiseries [74].…”