Capture probe conjugated to paramagnetic nanoparticles for purification of Alexandrium species (Dinophyceae) DNA from environmental samples.
OBJECTIVE
To develop a rapid, cost-effective and selective Alexandrium DNA extraction procedure from environmental samples in order to provide good-quality template for the downstream PCR-based detection assay.


RESULTS
In this study, we tested a DNA extraction method based on silica-coated, superparamagnetic nanoparticles conjugated to a DNA-capture sequence (probe) complementary to a specific region of 5.8S rDNA of the genus Alexandrium. Cultured Alexandrium catenella cells were used as the harmful algal bloom species for the DNA extraction. Then, a PCR assay was performed with primers specific for the genus Alexandrium to assess the specificity and sensitivity of the nucleic acid extraction method. This method was applied to both cultured and field samples, reaching in both cases a detection limit of one A. catenella cell.


CONCLUSIONS
The results suggest that the use of probe-conjugated paramagnetic nanoparticles could be effective for the specific purification of microalgal DNA in cultured or environmental samples, ensuring sensitivity and specificity of the subsequent PCR assays.


CONCLUSIONS
The DNA extraction method optimized in this study represents a progress towards the rapid and efficient direct detection of Alexandrium cells in seawater monitoring. In fact, this method requires no other equipment than a magnet and a hybridization oven and, in principle, can be adapted to different toxic microalgal species and can be automated, allowing the processing of a high number of samples.
Galluzzi
L
L
Center of Biotechnology, University of Urbino, Fano (PU), Italy. l.galluzzi@uniurb.it
Bertozzini
E
E
del Campo
A
A
Penna
A
A
Bruce
I J
IJ
Magnani
M
M
D000818:Animals; D017931:DNA Primers; D048028:DNA, Algal / Q000032:analysis*; D004141:Dinoflagellida / Q000235:genetics*; D004784:Environmental Monitoring / Q000379:methods*; D005068:Eutrophication; D015336:Molecular Probe Techniques; D049329:Nanostructures; D009693:Nucleic Acid Hybridization / Q000379:methods; D010839:Phytoplankton; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012623:Seawater; D012680:Sensitivity and Specificity; D014871:Water Microbiology
D017931:DNA Primers; D048028:DNA, Algal