Molecular tools for phytoplankton monitoring samples

Abstract

HABs can have severe impacts in fisheries or human health by the consumption of contaminated bivalves. Monitoring assessment (quantitative and qualitative identification) of these organisms, is routinely accomplished by microscopic identification and counting of these organisms. Nonetheless, molecular biology techniques are gaining relevance, once these approaches can easily identify phytoplankton organisms at species level and even cell number quantifications. This work tests 12 methods/kits for genomic DNA extraction and seven DNA polymerases to determine which is the best method for routinely use in a common molecular laboratory, for phytoplankton monitoring samples analyses. From our work, Direct PCR master mix for tissue samples, proved to be the most adequate by its velocity of processivity, practicability, reproducibility, sensitiveness and robustness. However, brands such as Omega Biotek, GRISP, Qiagen and MP Biomedicals also showed good results for conventional DNA extraction as well as all the Taq brands tested (GRISP, GE Healthcare Life Sciences, ThermoFisher Scientific and Promega). Lugol´s solution, with our tested kits did not show negative interference in DNA amplification. The same can be said about mechanical digestion, with no significant differences among kits with or without this homogenization step.

Identification of microalgae cultured in Bold’s Basal medium from freshwater samples, from a high-rise city

“…Methods used to digest the cell wall often require to be adapted from other protocols, to suit the algal genus researched on. There are reports of success with using commercial DNA extraction kits 11 , 12 , phenol–chloroform methods 13 , glass bead disruption, Tris–HCl EDTA-NaCl-SDS extraction 14 , hexadecyl-trimethyl-ammonium bromide (CTAB) 15 , 16 , freeze thaw techniques, tissue homogenizing, grinding 17 and boiling 18 . We modified an existing method 14 to facilitate DNA extraction of our algal isolates.…”