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Optimization of a DNA extraction protocol for hemolyzed and coagulated bovine blood for use in molecular detection of Anaplasma spp.

Abstract:

Anaplasma spp. bacteria cause anaplasmosis, a disease which negatively affects livestock production worldwide. Molecular detection by PCR requires efficient extraction of DNA from whole blood, which in turn depends on blood sample quality. Failures in sampling procedures and/or sample storage can lead to hemolysis and blood clotting, which can hamper diagnosis. An established DNA extraction protocol using Chelex® 100 resin was modified to optimize detection of Anaplasma spp. in hemolyzed and coagulated bovine blood samples, as well as reduce its cost. The optimized protocol extracted highly pure DNA effective in PCR analysis. Efficiency of the optimized protocol was compared with two commercial DNA extraction kits. When used in PCR detection of Anaplasma spp., the concordance values for all three were high (Cohen's Kappa = 0.72). The optimized protocol is effective at extracting DNA from complex blood samples and is much less costly than commercial methods, a clear advantage when operating under limited budgets.