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Quantitative PCR Coupled with Melting Curve Analysis for Detection of Microalgae

AuthorsAndree, Karl B.; Fernández-Tejedor, Margarita; Elandaloussi, Laurence M.; Quijano-Scheggia, Sonia ; Sampedro, Nagore ; Garcés, Esther ; Camp, Jordi ; Diogène, Jorge
Issue DateNov-2010
PublisherHellenic Centre for Marine Research
Citation14th International Conference on Harmful Algae. Abstract book: 26 (2010)
AbstractAlong the coast of Catalonia large blooms of Pseudo-nitzschia spp. have been increasing steadily in frequency and magnitude during the past twenty years of surveillance and are of serious concern due to their toxic potential. Among species from this genus found locally, P. calliantha, P. delicatissima and P. multistriata have been documented as toxic elsewhere. However rapid assessment, by morphometry, of species identity through optical microscopy is not possible for members of this genus due to the small size of the cells and the presence of cryptic species. Therefore differentiation of potentially toxic and non-toxic species is also not possible. We describe herein a methodology initiated to attempt a remedy to the inability to adequately differentiate species of the genus Pseudo nitzschia, and present the results from field testing of this technique. Clonal cultures of locally isolated Pseudo-nitzschia species were characterized morphologically using scanning electron microscopy and samples of each culture were used in creating an ITS-5.8S ribosomal DNA sequence database for designing primers specific for the predominant species encountered in Catalan coastal waters. Melting curve analysis from the resulting qPCR assays allowed differentiation of amplicons and identification of false positives in seawater samples. This methodology was applied to seawater samples collected during a two year period from Alfaques Bay in the south of Catalonia to assess the changing population structure of Pseudo-nitzschia blooms. Results obtained from this qPCR methodology, which included eight different species, showed good overall statistical correlation with the optical microscopy method of enumeration at the genus level
Description14th International Conference on Harmful Algae, 1-5 November 2010, Creta, Greece
Publisher version (URL)http://www.issha.org/Welcome-to-ISSHA/Conferences/ICHA-Conferences/Past-conferences
Appears in Collections:(ICM) Comunicaciones congresos
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