ABSTRACT
A commercial kit intended for Taq polymerase inhibitor removal was tested to detect Shiga-toxigenic Escherichia coli (STEC) by polymersase chain reaction (PCR) directly from cattle fecal samples. Forty-five samples were analysed for the presence of stx genes. Results were compared to those obtained by two other methods: amplification of DNA purified by a non-commercial procedure (heat lysis protocol), and amplification of DNA from samples cultured in solid media, commonly used in our lab. Identical numbers of positive samples (33/45, 73 %) were obtained with the QIAamp DNA stool purification kit and the culturing procedure, suggesting an adequate removal of inhibitors that interfere in PCR amplification from the feces. Besides, the number of positive samples detected using DNA purified by the non-commercial protocol was lower, 25/39 (64%) than that achieved by using the kit. In conclusion, the use of the QIAamp DNA stool purification kit provided a rapid stx gene detection by PCR in bovine fecal samples.
Subject(s)
Cattle/microbiology , Escherichia coli/isolation & purification , Feces/microbiology , Polymerase Chain Reaction/methods , Reagent Kits, Diagnostic , Shiga Toxins/analysis , Animals , Enzyme Inhibitors/pharmacology , Escherichia coli/metabolism , Food Contamination/prevention & control , Rectum/microbiology , Sensitivity and Specificity , Taq Polymerase/antagonists & inhibitorsABSTRACT
A commercial kit intended for Taq polymerase inhibitor removal was tested to detect Shiga-toxigenic Escherichia coli (STEC) by polymersase chain reaction (PCR) directly from cattle fecal samples. Forty-five samples were analysed for the presence of stx genes. Results were compared to those obtained by two other methods: amplification of DNA purified by a non-commercial procedure (heat lysis protocol), and amplification of DNA from samples cultured in solid media, commonly used in our lab. Identical numbers of positive samples (33/45, 73 %) were obtained with the QIAamp DNA stool purification kit and the culturing procedure, suggesting an adequate removal of inhibitors that interfere in PCR amplification from the feces. Besides, the number of positive samples detected using DNA purified by the non-commercial protocol was lower, 25/39 (64%) than that achieved by using the kit. In conclusion, the use of the QIAamp DNA stool purification kit provided a rapid stx gene detection by PCR in bovine fecal samples.
Un kit comercial diseñado para la eliminación de inhibidores de la polimerasa Taq fue ensayado para la detección de STEC por PCR en muestras fecales de bovinos. Cuarenta y cinco muestras fueron evaluadas por la presencia de genes stx. Los resultados fueron comparados con aquéllos obtenidos por otros dos métodos: amplificación de ADN purificado por un procedimiento no comercial (protocolo de lisis por calor), y amplificación de ADN de muestras cultivadas en medio sólido, comúnmente usado en nuestro laboratorio. El mismo número de muestras positivas (33/45, 73 %), fueron obtenidas con el QIAamp DNA stool purification kit y el procedimiento de cultivo, sugiriendo una eliminación adecuada de inhibidores que interfieren con la amplificación en materia fecal. Por otro lado, el número de muestras positivas detectadas usando ADN purificado por el protocolo no comercial fue menor, 25/39 (64%). En conclusión, el uso del kit QIAamp DNA stool purification permitió una detección rápida de genes stx por PCR en muestras fecales bovinas.
Subject(s)
Animals , Cattle/microbiology , Escherichia coli/isolation & purification , Feces/microbiology , Polymerase Chain Reaction/methods , Reagent Kits, Diagnostic , Shiga Toxins/analysis , Enzyme Inhibitors/pharmacology , Escherichia coli/metabolism , Food Contamination/prevention & control , Rectum/microbiology , Sensitivity and Specificity , Taq Polymerase/antagonists & inhibitorsABSTRACT
A commercial kit intended for Taq polymerase inhibitor removal was tested to detect Shiga-toxigenic Escherichia coli (STEC) by polymersase chain reaction (PCR) directly from cattle fecal samples. Forty-five samples were analysed for the presence of stx genes. Results were compared to those obtained by two other methods: amplification of DNA purified by a non-commercial procedure (heat lysis protocol), and amplification of DNA from samples cultured in solid media, commonly used in our lab. Identical numbers of positive samples (33/45, 73
) were obtained with the QIAamp DNA stool purification kit and the culturing procedure, suggesting an adequate removal of inhibitors that interfere in PCR amplification from the feces. Besides, the number of positive samples detected using DNA purified by the non-commercial protocol was lower, 25/39 (64
) than that achieved by using the kit. In conclusion, the use of the QIAamp DNA stool purification kit provided a rapid stx gene detection by PCR in bovine fecal samples.
ABSTRACT
Different experimental approaches were evaluated for their ability to detect stx genes by PCR and identify Shiga toxin-producing Escherichia coli (STEC) in bovine fecal samples. One hundred and sixty fecal samples from steers in Argentina were processed by protocols that involved: (1) enrichment of fecal samples and DNA extraction using a commercially available kit (Protocol A); (2) plating on selective media after enrichment of the fecal sample followed by heat-lysis DNA extraction from the confluent growth zone (Protocol B); (3) analysis of individual colonies isolated from direct fecal culture on MacConkey agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (Protocol C), used as Gold Standard. PCR performed on bacteria from the confluent growth zone (Protocol B) proved to be the most sensitive methodology. In addition, enrichment for greater than 6h, enhanced sensitivity. Among eight STEC isolates, four were O8:H19 and four were stx2/eae-negative. An STEC isolate was characterized as O26:H11 with a stx1/eae/EHEC-hlyA genotype, often associated with human disease. Finally, no STEC O157 strains were isolated using these methods.
Subject(s)
Cattle Diseases/microbiology , Escherichia coli Infections/veterinary , Escherichia coli/metabolism , Polymerase Chain Reaction/veterinary , Shiga Toxin 1/biosynthesis , Shiga Toxin 2/biosynthesis , Animals , Argentina , Cattle , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Drug Resistance, Bacterial , Escherichia coli/genetics , Escherichia coli/isolation & purification , Escherichia coli Infections/diagnosis , Escherichia coli Infections/microbiology , Feces/microbiology , Male , O Antigens/blood , Polymerase Chain Reaction/methods , Sensitivity and Specificity , Shiga Toxin 1/genetics , Shiga Toxin 2/genetics , VirulenceABSTRACT
Chorizo is a raw sausage, which is manufactured with beef, pork meat and pork fat, additives and spices. In Argentina, the expenditure of chorizo is through butchery and supermarkets where the product can be found packaged in both polyethylene films and vacuum sealed pouches. In the latter type of packaging an appearance problem has been detected in relation to drip loss. The aim of the work was to solve such problem through the incorporation of soy protein isolate (SPI). The sensory, microbiological and chemical stability of the product and its drip loss during a storage period of 14 days were studied. By adding a 2.5% SPI, the drip loss was prevented without introducing any change in the flavour, aroma and juiciness characteristics of the product. These sensory attributes were kept stable during the storage period studied. Chemical composition, oxidative and microbiological stability were not affected by the addition of SPI during the storage period, being similar for added and non-added SPI samples. Finally, SPI can be used in chorizos to improve their overall appearance during refrigerated storage while the product quality characteristics are not altered.