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.

Late Holocene changes in cyanobacterial community structure in maritime Antarctic lakes

“…(16ST58). This is in agreement with other studies (Coolen et al 2008;Savichtcheva et al 2011) in which Synechococcus sequences were detected along the entire sediment cores. The members of this OTU thrive in various habitats, including marine environments (Baltic Sea, AF330254) (97.8 % similarity) and freshwater lakes (100 % similarity) (Lake Superior, USA; DQ526409) (Fig.…”

“…Fossil or ancient DNA (aDNA) is increasingly becoming part of the paleoecological tool-box (Savichtcheva et al 2011) and has enabled, for example, refined reconstructions of past climate changes, inferred past changes in microbial and multicellular community structure in lakes, terrestrial habitats and the marine environment (Coolen et al 2004;Anderson-Carpenter et al 2011;Boere et al 2011), and study of the colonization-extinction dynamics in particular taxonomic groups (Mergeay et al 2007). The use of aDNA in paleoecology is, however, impeded by its stability and preservation potential in sediments, which depend on interacting factors such as microbial activity, adsorption of DNA to substrates, salinity, oxygen concentration, temperature and pressure (Boere et al 2011).…”

Establishment and field applications of real-time PCR methods for the quantification of potential MIB-producing cyanobacteria in aquatic systems

“…Considering the volume of samples collected, the limit of quantification (LOQ) of these qPCRs should be at the level of 10 3 copies L −1 in field, and this LOQ was verified in the practical monitoring of the three waters. Unlike the qPCR assays targeting geosmin-producing Streptomyces (Auffret et al 2011) and Anabaena (Su et al 2013;Tsao et al 2014) based on SYBR Green, the qPCR methods developed in this study introduced TaqMan technique, which has a high specificity (Savichtcheva et al 2011;Srivastava et al 2013), to quantify odor-producing organisms. To our knowledge, this study is the first report of qPCR assays to detect and quantify potential MIB-producing cyanobacteria.…”

Nutritional stress induces exchange of cell material and energetic coupling between bacterial species

“…H 2 S concentration was determined as described in Giuliani et al 51 Real-time qPCR. After extraction of genomic DNA from D. vulgaris and C. acetobutylicum using the Wizard Genomic DNA Purification Kit (Promega), DNA purity and concentration were determined and the number of copies was determined by qPCR as described by Savichtcheva et al 52 The primers used for the qPCR (dsrA and endoG) are listed in Supplementary Reverse transcription PCR. Reverse transcription PCR was performed according to Fiévet et al 53 for estimating the level of expression of genes coding pyruvate ferredoxin oxidoreductase and lactate dehydrogenase.…”