Optimization of Brucella abortus Protocols for Downstream Molecular Applications.

ABSTRACT

We compared the performances of various DNA extraction kits for their ability to recover Brucella abortus strain 19 inoculated into Brucella-free bovine tissues. Tissues were homogenized in a FastPrep bead homogenizer and extracted in triplicate by using one of five kits (Qiagen DNeasy, GE Illustra, Omega Bio-tek E.Z.N.A., Quanta Extracta, and IBI Science DNA Tissue kit). Whole blood was also taken from animals prior to chemical euthanasia, aliquoted, and then fractioned into buffy coat, red blood cells, and plasma. DNA was extracted from whole blood, buffy coat, and plasma by using four kits (Qiagen DNeasy, Omega Bio-tek E.Z.N.A., IBI Science DNA Blood kit, and 5PRIME PerfectPure). Previously reported primers targeting strain 19 were used to amplify extracted DNA and identify the optimal extraction kit. Real-time PCR was performed, and kits were compared for statistical differences by using quantification cycles as an outcome measure. Omega Bio-tek E.Z.N.A. was superior (P < 0.0068) in its lower quantification cycle values across all tissue kits. The IBI Science DNA Blood kit was superior to Qiagen DNeasy, 5PRIME PerfectPure, and Quanta Extracta (P < 0.0001, P = 0.0004, and P = 0.0013, respectively) but was not different from Omega Bio-tek E.Z.N.A. (P = 1.0). In summary, the optimal extraction kit for B. abortus strain 19 for tissues is Omega Bio-tek E.Z.N.A., and that for blood and its fractions is the IBI Science Mini Genomic DNA kit. Eluted DNA was also concentrated by using the Zymo Research DNA Clean & Concentrator-25 kit. Concentrated eluted DNA with the target was superior (P = <0.0001) to unconcentrated eluted DNA.