Identificación de BRUCELLA SPP. mediante la técnica de reacción en cadena de la polimerasa para la detección de brucelosis

Abstract

Brucellosis is a zoonotic disease caused by an intracellular Gram-negative bacterium called Brucella spp. It causes brucellosis, a disease that affects humans and animals such as cows, pigs, dogs, cats, sheep, among others. This disease is transmitted through direct contact with infected animals or through the consumption of their meat or milk derivatives. For this reason, rapid and early detection of bacteria is essential to avoid an epidemic. Among the main detection methods, we have the end-point polymerase chain reaction (PCR), characterized by its high specificity and sensitivity. In this research work, a protocol for end-point PCR analysis was developed. The practical work was carried out at the Multidisciplinary Research Center of the National Institute for Public Health Research – INSPI – “Dr. Leopoldo Izquieta Peréz”. With this protocol, 30 bovine blood samples collected from the province of Manabí were analyzed. The end-point PCR amplification products were visualized by 2% agarose gel electrophoresis. As a first positive control, a commercial vaccine strain 19 was used, which had a concentration of 11 ng/uL, obtaining an amplification for Brucella melitensis (151 bp). In addition, a second positive control was used from a Brucella culture concentrate, property of INSPI, with a concentration of 243 ng/uL; as well as a third positive control, the commercial vaccine Bovilis RB-51, with a concentration of 19 ng/uL. The latter presented amplification products for two species: B. abortus (248 bp) and B. melitensis (151 bp). The semi-quantitative identification of the protocol sensitivity was performed through serial duplicate dilutions of the second positive control. The minimum sensitivity was identified as 0.03 ng/uL with observable fragments in agarose gel of 248 bp and 151 bp. Meanwhile, the bovine samples did not present amplification for any of the identified causal agents of brucellosis.