Молекулярная биология: открытый доступ

Evaluation of Bacterial DNA Extraction Methods on Marine Samples Integrating a Process Control

Abstract

Erwan Bourdonnais, Thomas Brauge, Sabine Debuiche, Cédric Le Bris and Graziella Midelet*

To investigate the microbial community in the marine environment by molecular approaches, it is important to extract DNA in sufficient quantity and purity. The presence of inhibitors in the samples can lead to false negative results or a loss of information, but can be highlighted by a process control in the experiments. We compared seven bacterial DNA extraction methods on marine samples: fish skin, gills and guts, mollusk meat, phytoplankton and zooplankton. A process control (Listeria monocytogenes) was added in half of the samples. The performance of the DNA extraction methods were compared to produce the more pure and concentrated DNA for qPCR amplification targeting the bacterial tufgene and the process control hlyAgene. The purity and concentration of DNA were determined by spectrophotometry assays. The results showed that the highest purity and concentration of DNA were obtained using the PowerBiofilm and PureLink Microbiome kits. The qPCR data confirmed these kits produced better bacterial DNA purity and concentration with higher amplification efficiency. In some samples, the presence of inhibitors was detected by qPCR targeting the hlyAgene, showing that the samples were heterogeneous contaminated with inhibitors. The DNA extracts are suitable for genetic downstream applications in the marine environment.