title: Quantitative PCR enumeration of total/toxic Planktothrix rubescens and total cyanobacteria in preserved DNA isolated from lake sediments.
abstract: The variability of spatial distribution and the determinism of cyanobacterial blooms, as well as their impact at the lake scale, are still not understood, partly due to the lack of long-term climatic and environmental monitoring data. The paucity of these data can be alleviated by the use of proxy data from high-resolution sampling of sediments. Coupling paleolimnological and molecular tools and using biomarkers such as preserved DNA are promising approaches, although they have not been performed often enough so far. In our study, a quantitative PCR (qPCR) technique was applied to enumerate total cyanobacterial and total and toxic Planktothrix communities in preserved DNA derived from sediments of three lakes located in the French Alps (Lake Geneva, Lake Bourget, and Lake Annecy), containing a wide range of cyanobacterial species. Preserved DNA from lake sediments was analyzed to assess its quality, quantity, and integrity, with further application for qPCR. We applied the qPCR assay to enumerate the total cyanobacterial community, and multiplex qPCR assays were applied to quantify total and microcystin-producing Planktothrix populations in a single reaction tube. These methods were optimized, calibrated, and applied to sediment samples, and the specificity and reproducibility of qPCR enumeration were tested. Accurate estimation of potential inhibition within sediment samples was performed to assess the sensitivity of such enumeration by qPCR. Some precautions needed for interpreting qPCR results in the context of paleolimnological approaches are discussed. We concluded that the qPCR assay can be used successfully for the analysis of lake sediments when DNA is well preserved in order to assess the presence and dominance of cyanobacterial and Planktothrix communities.
authors: INRA-UMR 42 CARRTEL, Centre Alpin de Recherche sur les Re´seaux Trophiques des Ecosyste`mes Limniques, 74203 Thonon-les-Bains Cedex, France.
authors: Olga
authors: O
authors: Savichtcheva
authors: Didier
authors: D
authors: Debroas
authors: Rainer
authors: R
authors: Kurmayer
authors: Clement
authors: C
authors: Villar
authors: Jean Philippe
authors: JP
authors: Jenny
authors: Fabien
authors: F
authors: Arnaud
authors: Marie Elodie
authors: ME
authors: Perga
authors: Isabelle
authors: I
authors: Domaizon
mesh_terms: D058491:Bacterial Load; D000458:Cyanobacteria / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005618:Fresh Water / Q000382:microbiology*; D019015:Geologic Sediments / Q000382:microbiology*; D052998:Microcystins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D052998:Microcystins; C078588:microcystin