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      Comparación de la técnica de Inmunoseparación Magnética y el Método Convencional para el aislamiento de Salmonella spp. en leche pasteurizada contaminada artificialmente Translated title: Comparison of Magnetic Immunoseparation technique and the conventional method for isolation of Salmonella spp. from artificially contaminated pasteurized milk

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          Abstract

          La leche pasteurizada es un excelente sustrato para Salmonella por lo que requiere un minucioso control de calidad que garantice la inocuidad para su consumo. El objetivo de la presente investigación fue comparar las técnicas de Inmunoseparación Magnética (ISM) y el Método Convencional (MC) para la detección de Salmonella spp. en leche pasteurizada contaminada artificialmente. Se trabajó con seis grupos de muestras de leche pasteurizada inoculadas con 50 cel/mL de Salmonella spp. más1x 10³ cel/mL de un “coctel” de Enterobacterias (Escherichia coli, Klebsiella oxytoca, Shigella boydii y Enterobacter cloacae). Se evaluó el crecimiento de Salmonella mediante el MC (COVENIN 1291-2004) e ISM, utilizando diferentes protocolos de esta última, variando el factor de dilución de la muestra (sin dilución y 10³) y el número de lavados (3 y 5). Resultados: Se logró recuperar Salmonella spp. mediante ISM a partir del caldo de enriquecimiento selectivo y pre-enriquecimiento del MC. La ISM redujo a tres días el tiempo de obtención de resultados con respecto al MC. Aplicando ISM directamente en leche pasteurizada inoculada, no se logró recuperación bacteriana. Se demostró que el factor dilución de la muestra no influyó en los resultados de la ISM, mientras que el número de lavados durante la técnica sí fue determinante. El MC logró recuperar Salmonella spp en 100% (n=30) de las muestras. Conclusión: La aplicación de la ISM para el aislamiento de Salmonella spp. en muestras de leche pasteurizada reduce significativamente el tiempo de obtención de resultados con respecto al MC.

          Translated abstract

          Pasteurized milk is an excellent substrate for Salmonella and therefore requires a thorough quality control to ensure its safety for human consumption. The aim of this study was to compare the techniques of Magnetic Immunoseparation (MIS) and the conventional method (CM) for the detection of Salmonella spp. in artificially contaminated pasteurized milk. Six groups of pasteurized milk samples were artificially contaminated with 50 cells/mL of Salmonella spp. plus 1 x 10³ cells/mL of a "cocktail" of Enterobacteriaceae (Escherichia coli, Klebsiella oxytoca, Shigella boydii and Enterobacter cloacae). Salmonella growth by MC (COVENIN 1291-2004) and MIS was evaluated using different protocols of the latter, varying the dilution factor (without dilution and 10³) and the number of washes (3 and 5). Results: We succeeded in recovering Salmonella spp. by MIS from selective enrichment broth and pre-enrichment of CM. The MIS reduced the time for obtaining results down to three days with respect to CM. Bacterial recovery was not achieved by applying MIS directly into inoculated pasteurized milk. It was shown that the sample dilution factor did not influence MIS results, while the number of washings during the procedure greatly influenced them. The CM was able to recover Salmonella spp. in 100% (n=30) of the samples. Conclusion: The application of the MIS for the isolation of Salmonella spp. in samples of pasteurized milk significantly reduces the time for obtaining results with respect to CM.

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          Most cited references26

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          Multidrug-resistant Salmonella Typhimurium Infection from Milk Contaminated after Pasteurization

          An outbreak of multidrug-resistant Salmonella enterica serotype Typhimurium infections occurred in Pennsylvania and New Jersey. A case-control study implicated pasteurized milk from a dairy, and an inspection indicated the potential for contamination after pasteurization. Dairy cattle are the likely reservoir, and milk may be an important vehicle of Salmonella transmission to humans.
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            Rapid detection of Salmonella in milk by electrochemical magneto-immunosensing.

            A very simple and rapid method for the detection of Salmonella in milk is reported. In this approach, the bacteria are captured and preconcentrated from milk samples with magnetic beads through an immunological reaction. A second polyclonal antibody labeled with peroxidase is used as serological confirmation with electrochemical detection based on a magneto-electrode. The 'IMS/m-GEC electrochemical immunosensing' approach shows a limit of detection of 5 x 10(3) and 7.5 x 10(3)CFU mL(-1) in LB and in milk diluted 1/10 in LB broth, respectively, in 50 min without any pretreatment. If the skimmed-milk is preenriched for 6h, the method is able to detect as low as 1.4 CFU mL(-1), while if it is preenriched for 8h, as low as 0.108 x CFU mL(-1) (2.7 x CFU in 25 g of milk, in 5 samples of 5 mL) are detected accordingly with the legislation. Moreover, the method is able to clearly distinguish between food pathogenic bacteria such as Salmonella and Escherichia coli. The features of this approach are discussed and compared with classical culture methods.
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              The detection of Salmonella using a combined immunomagnetic separation and ELISA end-detection procedure.

              The aim of this study was to develop a rapid immunoassay to detect Salmonella bacteria. Skimmed milk powder (SMP) in buffered peptone water was inoculated with six Salmonella strains (Salm. typhimurium, Salm. virchow, Salm. enteritidis, Salm. give, Salm. ealing and Salm. arizonae) at three inoculum levels (about 2-200 cfu 25 g(-1) SMP) and incubated (37 degrees C) overnight. Heat-treated salmonella cells were immobilized on paramagnetic particles and detected within 3 h using the Salmonella genus-specific monoclonal antibody M105 in a microtitre plate based assay. The rapid Salmonella detection method combining immunomagnetic separation and ELISA had a total isolation and detection time of less than 24 h, which is significantly shorter than the conventional techniques requiring 72-96 h. The technique had a sensitivity limit of 10(5)-10(6) cfu ml(-1).
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                Author and article information

                Journal
                s
                Salus
                Salus
                Universidad de Carabobo (Valencia, Carabobo, Venezuela )
                1316-7138
                December 2011
                : 15
                : 3
                : 19-23
                Affiliations
                [01] orgnameUniversidad de Carabobo orgdiv1Facultad de Ciencias de la Salud orgdiv2Escuela de Ciencias Biomédicas y Tecnológicas
                [02] Carabobo orgnameLaboratorio Clínico Cesar Sánchez Font Venezuela
                [04] orgnameUniversidad de Carabobo orgdiv1Facultad de Ciencias de la Salud orgdiv2Departamento de Microbiología Venezuela
                [03] orgnameUniversidad de Carabobo orgdiv1Facultad de Ciencias de la Salud orgdiv2Escuela de Bioanálisis Venezuela
                Article
                S1316-71382011000300007 S1316-7138(11)01500307
                f1285adb-2955-4f62-b5ed-3a9f8d2b3031

                http://creativecommons.org/licenses/by/4.0/

                History
                : February 2011
                : July 2011
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 19, Pages: 5
                Product

                SciELO Venezuela

                Self URI: Texto completo solamente en formato PDF (ES)
                Categories
                Artículos

                inmunoseparación magnética,Salmonella spp,magnetic immunoseparation,Leche,Milk

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