ABSTRACT. This study aimed to develop and validate real-time PCR for the diagnosis of Mycobacterium bovis isolates. Two hundred and seventy-four M. bovis isolates and 156 M. tuberculosis isolates were tested. Both qPCRs amplified all of the 274 M. bovis samples, but none of the 156 M. tuberculosis samples. The qPCR for PE-PGRS 20 had 91% efficiency and a detection limit of 0.32 ng (sensitivity and specificity for qPCR "Mbovis.100" were 99.64 and 100%, respectively). The qPCR for RD4 had 100% efficiency, and a detection limit of 4 pg (diagnostic sensitivity and specificity were 100 and 100%. The qPCR tests were performed using 4 extraction sets, 3 qPCR kits, and with a range of equipment; yet, all combinations produced similar results in a diagnostic test, demonstrating the robustness of this method. The techniques proved to be efficient, robust, sensitive, and specific for the diagnosis of M. bovis.

Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p < 0.001, CI = 0.84 – 1.0). The diagnostic sensitivity and specificity were 100% (CI = 95.94% – 100%) and 100% (CI = 93.98% – 100%). This qPCR was developed with the goal of diagnosing the bacillus M. tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method.

A multiplex PCR technique for detection of Brucella spp. in samples of bacterial suspension was validated as a complementary tool in the diagnosis of the disease. This technique allows the characterization of the agent without performing

No abstract

BackgroundThe allergic test of mallein is one of the most frequently used tests, together with the Complement Fixation Test (CFT), for the diagnosis of glanders in endemic areas. Mallein, a purified protein derivative (PPD), is produced similarly to PPD tuberculin and the end product is a primarily proteic antigen, which is only poorly purified. The immuno-allergic activity of mallein is believed to be due to a high molecular weight group of proteins present in the antigen. To improve the quality of the antigen, in terms of sensitivity and specificity, a new method of mallein production was developed, in which purification was accomplished by ultrafiltration in a Tangential Flow Filtration system (TFF).ResultsThe TFF methodology efficiently separated the high and low molecular weight protein groups of mallein. The five TFF-purified malleins, produced from Burkholderia mallei strains isolated from clinical cases of glanders in Brazil, proved to be more potent than standard mallein in the induction of an allergic reaction in sensitized animals. Regarding specificity, two of the purified malleins were equivalent to the standard and three were less specific.ConclusionSome of the TFF-purified malleins showed considerable potential to be used as an auxiliary test in the diagnosis of glanders.