Optimized Protocol for Cyanobacterial 16S rRNA Analysis in Danube Delta Lakes

Abstract

Molecular biology protocols have been more and more accessible to researchers for ecological investigations, however, these protocols always require optimization steps for the analysis of specific types of samples. The purpose of this study was to optimize a molecular protocol for the analysis of cyanobacterial 16S rRNA in Danube Delta shallows lakes. In this regard, several commercial DNA extraction kits were tested in comparison with potassium ethyl xanthogenate extraction method on different matrices. The obtained DNA was further used for 16S rRNA PCR optimization. Finally, an optimized protocol is proposed for the molecular analysis of cyanobacteria group in freshwater samples. The best DNA extraction method was the potassium xanthogenate extraction from dried cyanobacterial biomass. A dynamic in total genomic eDNA was observed, reflecting the seasonal difference in phytoplankton biomass from the studied lakes. The PCR protocol optimized by us can be successfully applied for the identification of a broad range of cyanobacterial genetic markers.

Using FlowCam and molecular techniques to assess the diversity of Cyanobacteria species in water used for food production

“…Isolation and cultivation. The isolation and cultivation of cyanobacteria were laboratory prepared using a modified BG11 medium as per Gumbo et al 43 , Moza and Postolache 29 , and Valadez-Cano et al 30 . Cyanobacteria were grown in a liquid medium (BG11).…”

“…A better understanding of treatment requirements can be achieved through monitoring of cell counts or biovolume estimates in source waters when integrated with toxin information. Advanced digital flow cytometry (FlowCAM) and Polymerized Chain Reaction PCR, amongst other methods, have been widely used to detect, identify and classify cyanobacterial species in drinking water 10,[28][29][30] . Digital flow cytometry FlowCAM (Fluid Imaging Technologies, Scarborough, Maine U.S.A) has been used for cell counting of cyanoHABs since being used by Sieracki et al 31 .…”