Abstract
Diagnosis of brucellosis is made by culture and serological tests. Microbiological isolation is considered the gold standard; however, it requires a minimum of viable bacteria in the sample and an incubation period of up to 4 weeks. The time limitation led to the evaluation of a new molecular biology method based on real-time PCR for rapid and more sensitive detection of Brucella. To evaluate a molecular biology test for the detection of Brucella spp. in humans. The design of primers and probes was carried out using the Beacon Designer v8.1 (BD8) program, using the sequence of the 31 kDa outer membrane protein (BSCP31) for genus, and for the detection of specie the alkB gene regions and BMI1162 for B. abortus and B. melitensis, respectively. An internal reaction control (IPC) was added, with the purpose of positive validation of samples. The PCR test was evaluated using 64 samples of blood from individuals with suspected brucellosis. These samples were also processed by serology with Rose Bengal, standard agglutination and agglutination tests in the presence of 2-mercaptoethanol. Statistical analysis was performed with a 2×2 contingency table using EPIDAT 3.0 software. The designed oligonucleotides were successfully tested, a detection limit of 5 fg was obtained, which is equivalent to 1 CFU in the serum sample. The diagnostic sensitivity and specificitywere 97.2 and 96% respectively. In this work the identification of Brucella spp. at the level of genus and species was performed by a highly sensitive real-time PCR test. This test considerably is reducing the identification time for Brucella species whereby we can recommend it as a support tool for the timely diagnosis of human brucellosis.
