Evaluation of <Emphasis Type="Italic">eryC</Emphasis> as a Molecular Marker for the Quantitative Detection of <Emphasis Type="Italic">Brucella</Emphasis> Spp. by Real-Time PCR in Food Samples |
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Authors: | David Rodríguez-Lázaro Lorena López-Enríquez Alain A Ocampo-Sosa Pilar Muñoz José María Blasco Clara Marín Marta Hernández |
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Affiliation: | 1.Microbiology Division, Department of Biotechnology and Food Science,University of Burgos,Burgos,Spain;2.Laboratory of Molecular Biology and Microbiology,Instituto Tecnológico Agrario de Castilla y León (ITACyL),Valladolid,Spain;3.Microbiology Unit,University Hospital “Marqués de Valdecilla”,Santander,Spain;4.Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITAA),Gobierno de Aragón,Zaragoza,Spain;5.Departamento de Ingeniería Agrícola y Forestal, Tecnología de los Alimentos, E.T.S. Ingenierías Agrarias,Universidad de Valladolid,Palencia,Spain |
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Abstract: | We evaluated the capacity of the Brucella sp. eryC gene as a diagnostic marker for brucellosis by quantitative real-time PCR. eryC gene encodes the enzyme d-erythrulose-1-phosphate dehydrogenase that plays an important role in the erythritol metabolism and is related with the Brucella survival in the intracellular environment of the macrophage. The assay includes an internal amplification control (IAC) in order to avoid false negative results. It was 100% specific, with an analytical sensitivity of 1 genome equivalent (GE) in 43% of the reactions, being the quantification highly linear (R 2 > 0.9953) and efficient (PCR efficiency >0.8820) over a 6-log dynamic range, down to 10 GE. Finally, the applicability of this assay was evaluated with artificially contaminated biological matrices implicated in the transmission of this bacterium such as sheep raw milk and pig blood. The eryC-IAC real-time PCR assay allowed detection of as few as ten Brucella cells per 25 ml of sheep raw milk or per 1 ml of pig blood. In conclusion, we present an alternative for the detection of Brucella genus and therefore facilitate the establishment of preventive and prophylactic measures in food and farm environments. |
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