Tropical and subtropical agroecosystems
versão On-line ISSN 1870-0462
GONZALEZ-DE LA CRUZ, J. Ulises et al. Protocol for extraction of bacterial metagenome DNA to prawn Macrobrachium carcinus L. Trop. subtrop. agroecosyt [online]. 2011, vol.14, n.3, pp. 875-883. ISSN 1870-0462.
In this work we adapted a protocol for the extraction of metagenomic DNA (ADNmg) bacteria in the digestive system (intestines, stomach and hepatopáncreas) of Macrobrachium carcinus L., with reference to the method of extracting bacterial DNA from soils and sediments (Rojas-Herrera et al., 2008). This methodology consisted of enzymatic, physics, mechanics and chemistry after a series of tests was abolished enzymatic lysis. However, the success ADNmg extraction was influenced mainly by the preparation of the samples, in particular the hepatopáncreas, where it was necessary to remove the fat by thermal shock temperature and phase separation by centrifugation with the sample frozen. The effectiveness of isolated DNA fragmentation was verified by gel electrophoresis in denaturing gradient (DGGE) after amplification with universal primers. In general, it had a low diversity (19 phylotypes) between the different organs analyzed of 13.5 ± 1 (intestines) to 11.7 ± 0.96 (stomach). The Shannon-Weaver index (2.45), Simpsons (10.88) and equity (0972) obtained from the digitization of the image of the gel, suggested that the phylotypes that form the gut microflora M. carcinus, is distributed unevenly between the different organs analyzed.
Palavras-chave : M. carcinus; metagenomic DNA; bacterial diversity; DGGE.