49B-16 |
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M. S. RAMELLA1, M. A. Kroth2, C. Tagliari2, E. S. Sant'Anna2, and A. C. M. Arisi2. (1) Food Science and Technology Department, Federal University of Santa Catarina, Av. Admar Gonzaga 1346 - Itacorubi, Florianópolis, SC, 88034001, Brazil, (2) Brazil Lophyus gastrophysus has important commercial value in Brazil and its production is almost completely exported to Europe. In 2001, its exportation revenues represented approximately US$ 21,7 million dollars. Due to increase in international trade in fish and its products, authentication has become necessary. RAPD (random amplified polymorphic DNA) has been used for fish identification since it is reliable and low cost. Because of its simplicity and speed in execution, the RAPD procedure is very effective. Despite these advantages, RAPD reactions show high variability among species, therefore RAPD optimization consumes long time. In this study, we described the optimization of RAPD protocol for identification of L. gastrophysus. Different conditions (annealing temperatures, MgCl2 concentrations, DNA quantity) were tested in order to find reproducible and adequate profiles. Genomic DNA was isolated from froozen muscle of eight individual according to the protocol of Asahida et al. (1996), after minor modifications. DNA concentration and purity were determined by spectrophotometry. Tests with annealing temperature (36 oC and 40 oC), DNA quantity (12.5 ng and 25.0 ng) and MgCl2 concentrations (2.0 mM and 2.5 mM) were performed with four 10-mer primers. The amplifications were resolved on agarose gel electrophoresis and photodocumentated. Optimal conditions for RAPD analysis were determined by the highest number of fragments and accurate bands. Amplifications performed with primers A01, A02, A03 and A16 generate the best RAPD profiles when the conditions were annealing temperature of 36 oC, 25.0 ng of DNA quantity and 2.5 mM MgCl2. Exact identification of the species and origin of marine products is necessary, particularly for foreign trade. We determine optimal conditions for RAPD analysis to identify L. gastrophysusL. gastrophysus. It could be used as an accurate, rapid tool to expose commercial fraud.
Session 49B, Aquatic Food Products: General
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