Detecção de Brucella abortus em tecidos bovinos utilizando ensaios de PCR e qPCR¹

Abstract

The aim of the study was to evaluate the technical polymerase chain reaction (PCR) and Real-Time PCR (qPCR) to detect Brucella abortus from bovine tissues with suggestive lesions of brucellosis. For this, 21 fragments of bovine tissues collected at abattoirs of Mato Grosso do Sul were processed and subjected to microbiological culture and extraction of genomic DNA to perform the PCR reactions and qPCR. Eight samples of microbiological culture showed bacterial growth and five samples were confirmed as B. abortus by PCR. DNA of Brucella (IS711 primers) was detected in 13 (61.9%) directly from tissue samples and 17 (81%) from tissue homogenate samples. With the species-specific set of primers BruAb2_0168F and BruAb2_0168R, 14 (66%) tissue samples and 18 (85.7%) tissue homogenate samples were positive. Six positive samples in the species-specific PCR were sequenced and the best hit in the BLASTn analysis was B. abortus. By qPCR, 21 (100%) tissue samples and 19 (90.5%) tissue homogenate samples were positive for B. abortus. Ten samples of DNA from bovine blood from an accredited-free herd were used as negative control in PCR and qPCR analysis using the primers BruAb2_0168F and BruAb2_0168R, and no one amplified by PCR, whereas two samples were amplified by qPCR (20%). In conclusion, both techniques detect the presence of B. abortus directly from tissues and homogenized, but the qPCR showed high sensitivity. The results indicate that qPCR can represent an alternative tool for faster and more accurate detection of B. abortus directly from tissues, and use in health surveillance programs by presenting satisfactory sensitivity and specificity.

Detecção de Brucella abortus em linfonodos de búfalas (Bubalus bubalis) em diferentes fases da gestação

“…em fluidos e tecidos (Sola et al 2014). Essa ferramenta, de grande valia, vem se destacando no diagnóstico da brucelose por apresentar altos níveis de sensibilidade e especificidade (Hinić et al 2009, Caitano et al 2014.…”

Detecção de Brucella sp. em leite informal comercializado no estado de Alagoas, Brasil: um breve alerta à saúde pública

“…Entretanto, resultados duvidosos podem ser gerados quando ainda não houve soroconversão ou através do uso extensivo de vacinas, o que incrementaria a resposta imune nas infecções em curso (Vanzini et al, 2001;Nielsen, 2002;Chand et al, 2005). Por conta dessas limitações, o uso de técnicas moleculares, como a reação em cadeia da polimerase (PCR), é adotado cada vez mais, sobretudo por sua rapidez, baixo risco biológico e incremento da sensibilidade (Yu e Nielsen, 2010;Amoroso et al, 2011;Caitano et al, 2014;Alamian et al, 2017).…”

Brucellosis in water buffaloes

“…The direct methods of diagnosis of brucellosis can be performed by bacterial isolation and detection of nucleic acids by molecular biology. A test that has shown promising results for brucellosis diagnosis is qPCR because of its high levels of sensitivity and specificity (Hinić et al 2009, Caitano et al 2014. Moreover, it has been a promising alternative for the direct diagnosis of slow growing organisms because of its ability to detect small amounts of DNA in the sample regardless of its viability (Erlich et al 1991, Al Dahouk et al 2003.…”