Quantitative PCR Coupled with Melt Curve Analysis for Detection of Selected Pseudo-nitzschia spp. (Bacillariophyceae) from the Northwestern Mediterranean Sea

Abstract

The frequency and intensity of Pseudo-nitzschia spp. blooms along the coast of Catalonia have been increasing over the past 20 years. As species from this genus that are documented as toxigenic have been found in local waters, with both toxic and nontoxic species cooccurring in the same bloom, there is a need to develop management tools for discriminating the difference. Currently, differentiation of toxic and nontoxic species requires time-consuming electron microscopy to distinguish taxonomic features that would allow identification as to species, and cryptic species can still remain misidentified. In this study, cells of Pseudo-nitzschia from clonal cultures isolated from seawater were characterized to their species identity using scanning electron microscopy, and subsamples of each culture were used to create an internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 ribosomal DNA database for development of species-specific quantitative PCR (qPCR) assays. Once developed, these qPCR assays were applied to field samples collected over a 2-year period in Alfaques Bay in the northwestern Mediterranean Sea to evaluate the possibility of a comprehensive surveillance for all Pseudo-nitzschia spp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. Total Pseudo-nitzschia cell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eight Pseudo-nitzschia species-specific qPCR assays using duplicate samples. Species-specific qPCR followed by melt curve analysis allowed differentiation of amplicons and identification of false positives, and results correlated well with the total Pseudo-nitzschia cell counts from optical microscopy.

Toxigenic algae and associated phycotoxins in two coastal embayments in the Ebro Delta (NW Mediterranean)

“…by light microscopy and presence of the toxin domoic acid (DA). These samples were then processed (following Andree et al, 2011;Fawley and Fawley, 2004) by targeting the species Pseudo-nitzschia arenysensis (=Pseudo-nitzschia delicatissima, strain Ra3), Pseudo-nitzschia brasiliana, Pseudo-nitzschia calliantha, Pseudo-nitzschia delicatissima (strain Ra2), Pseudo-nitzschia fraudulenta, Pseudo-nitzschia galaxiae, Pseudo-nitzschia multistriata, and Pseudo-nitzschia pungens. For DNA extraction, cell pellets were thawed and suspended in 200 mL lysis buffer (1 M NaCl, 70 mM Tris, 30 mM EDTA, pH 8.0), then each was transferred to a 2 mL cryo-tube containing approximately 50 mg of 0.5 mm-diameter zirconium glass beads (BioSpec, Bartlesville, Oklahoma, USA).…”

“…Extraction of the genomic DNA continued from this supernatant using a GeneClean Kit (MP Biomedicals, LLC, LLC, Santa Ana, California, USA), following the procedure of Fawley and Fawley (2004). A hemispecific assay, containing one genus-specific 5.8S primer and one species-specific ITS-1 or ITS-2 primer, was used for identification of taxa (Andree et al, 2011). Duplicate amplifications were performed on an ABI 7300 real-time PCR system (ABI, Carlsbad, California, USA) in 20 mL volumes extended for 45 cycles with a standard two-step protocol at 94 8C for 30 s followed by primer annealing/extension at 65 8C for 30 s. SYBR Green I dye in reaction mixtures was used for amplification detection and melt curve analysis.…”

“…Duplicate amplifications were performed on an ABI 7300 real-time PCR system (ABI, Carlsbad, California, USA) in 20 mL volumes extended for 45 cycles with a standard two-step protocol at 94 8C for 30 s followed by primer annealing/extension at 65 8C for 30 s. SYBR Green I dye in reaction mixtures was used for amplification detection and melt curve analysis. The thermal profile for melt curve determination started with an incubation of 1 min at 60 8C with a gradual increase in temperature (1 8C/15 s) to 95 8C, during which time changes in fluorescence were monitored (Andree et al, 2011).…”

“…Samples were only considered positive when the shape and temperature (Tm) of the melt curve matched the control. Although curve shape may vary slightly due to point mutations within the amplified region (Andree et al, 2011), significant deviations from the control samples were considered as negative for that specific internal transcribed spacer (ITS) PCR assay.…”

“…For example, such techniques aided in the discrimination of Pseudo-nitzschia populations and cultured isolates at the species level over time (Andree et al, 2011;Quijano-Scheggia et al, 2008). Nevertheless, analyses of field samples were based mainly on integrated samples throughout the water column or on a crude distinction between surface and bottom samples.…”