Implementación y evaluación del ensayo amplificación isotérmica mediada por un lazo (lamp)para detección de brucella abortus en muestras biológicas de bovinos

Abstract

Bovine brucellosis is an infectious-transmissible disease caused by Brucella abortus, which is characterized by mainly affecting the reproductive tract of adult females. This disease causes great impacts in the economic sector and represents a great problem in public Health. In the present work we proposed to implement a LAMP assay using genetic material from the B. abortus RB51 and S19 vaccine strains inoculated and extracted from bovine milk (1 X 106 - 1 X 100 CFU/ml milk) and lymph node (10 X 106-10 X 100 UFC/g tissue) samples, and then evaluate the analytical sensitivity using different detection methods. Obtaining DNA from B. abortus susceptible to being amplified in the milk samples, establishing a level of analytical sensitivity by agarose-TAE gel electrophoresis stained with Ethidium bromide of 10 CFU/ml milk, and the same sensitivity by use SYBR green for fluorescence detection, with this dye under natural light was observed a change of color from orange to green as well as an increase of fluorescence in the positive samples. As regards tissues inoculated with the vaccine strains, great variability was observed in the amplification of the total DNA. Therefore, it did not obtain a reliable assay when applying the LAMP method for the detection of B. abortus presuming the presence of inhibitors of endogenous origin in the tissues of lymph nodes.