Validation of the multiplex PCR for identification of Brucella spp.

Abstract

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

Validation of real-time PCR technique for detection of Mycobacterium bovis and Brucella abortus in bovine raw milk

“…Samples of homogenized tank milk from a herd certified free of brucellosis and tuberculosis were used. For contamination of the samples, one colony forming unit (CFU) of reference strains M. bovis AN5 CRNC 01 [16] and B. abortus 2380 [17] from LANAGRO/MG bacterial collection were homogenized in 150 μL and added in 1350 μL of raw milk. From this tube (equivalent to 7.8372 × 10 9 CFU/mL), serial dilutions were performed up to 10 −12 .…”

“…The following program was used: pre-cycle 50°C for 2 min, 95°C for 15 min, and PCR at 95°C for 15 s, 60°C for 60 s for 50 cycles. The standard samples of M. bovis AN5 CRNC 01 [16] and B. abortus 2380 [17] were used throughout the qPCR standardization and validation processes as positive controls. In all rounds of qPCR, negative controls (all reagents mixed without adding templates) were added to verify the contamination of the reagents used in the extraction of the DNA and of the Master Mix components used in the qPCR.…”

“…The parameters used for the validation performed in this study were based on those described in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2019 [3] Corynebacterium pseudotuberculosis CRNC 09/02, Rhodococcus equi CRNC09/01, and Nocardia asteroides CRNC10/01, as described by Sales [15]. To test the specificity of the Bru.is6501.128 and Bru.ERID.92 primers, strains of B abortus, B. abortus S19, B. abortus biovar 1, B. abortus biovar 3, B. melitensis, B. suis, Trueperella pyogenes, Rhodococcus equi, Salmonella enterica, Staphylococcus aureus, and Escherichia coli were used, as described by Orzil et al [17]. All the strains used for the analytical specificity tests were inactivated and belonged to the LANAGRO/ MG bacterial collection.…”

A quantum dot fluorescent microsphere based immunochromatographic strip for detection of brucellosis

“…However, these methods both use the whole cell and whole smooth lipopolysaccharides (S-LPS) as the antigen to detect Brucella serum antibodies, which can cause false positives and cross-reactivity with other Gram-negative bacteria such as Yersinia entercolitica [7,8,9]. Molecular biology methods, such as polymerase chain reaction (PCR) [10], real time PCR (qPCR) and multiplex PCR [11], provide qualitative and quantitative results with good accuracy and sensitivity. However, these methods require expensive instruments and professional operators, are time consuming and they produce aerosol pollution [12].…”

A sandwich immunoassay for brucellosis diagnosis based on immune magnetic beads and quantum dots

“…However, these methods usually use the whole cell or smooth lipopolysaccharides (S-LPS) as the antigen to detect Bru- cella antibodies in the patient's serum, which can lead to false positives and cross-reactivity with other Gram-negative bacteria such as Yersinia entercolitica O:9 [9][10][11]. Molecular biology detection technology has also been used for the diagnosis of brucellosis, such as Polymerase Chain Reaction (PCR) [12], real time PCR and multiplex PCR [13], etc., and they also demonstrate good accuracy and sensitivity. These methods are expensive for routine lab analysis and require trained personnel.…”