Gel documentation equipment

Once the optimization was done, diagnostic specificity and sensitivity were determined with a reaction mixture consisting of 1.6 μM inner primers (FIP and BIP), 0.2 μM outer primers (F3 and B3) and 0.8 μM loop primers (FLP and RLP) at a final concentration of the same reagents described above and the selected target DNA. The diagnostic sensitivity of the technique was determined by performing serial dilutions of the DNA extracted from a blood sample with 2% parasitemia using dilutions in a range of 10⁻²-10⁻⁹. Additionally, the diagnostic specificity of the LAMP technique was performed using DNA from B. bovis, Anaplasma marginale, A. phagocytophilum, A. centrale, Trypanosoma theileri, Bos taurus, Homo sapiens, Rhipicephalus microplus and Neospora caninum. The results were determined visually according to the procedures reported by Goto et al. (13 (link)) using agarose gels stained with GelRed (Sigma-Aldrich, Burlington, MA, United States) and analyzed using a gel documentation equipment (Bio-Rad. Hercules, California, United States).

To compare the sensitivity of the LAMP developed in this study with the nested PCR (nPCR), which was considered the gold standard for the molecular diagnosis of B. bigemina, the primers, procedures, and programs reported by the World Organization for Animal Health, (23 ) and by Figueroa et al. (24 (link)) were used as references. The analysis of the results was done visually (13 (link)) and on an agarose gel stained with GelRed using a gel documentation equipment (Bio-Rad. Hercules, California, United States).