Browsing by Author "Corrales Martínez, Joselyn Griselda"

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Estandarización del método PMAxxTM qPCR para la cuantificación de la carga viable de Salmonella spp. en contenido cecal de origen avícola
(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2022-03) Corrales Martínez, Joselyn Griselda; Calero Cáceres, William Ricardo
Currently, the standardization and validation of quantitative molecular techniques such as qPCR are important for the quality assurance and research sectors. Especially, the standardization of qPCR protocols for the detection of Salmonella spp. is essential, due to the impact it has on the health of the population. The present work aimed to standardize the PMAxxTM-based qPCR method for the detection and quantification of viable cells of S. infantis in poultry cecal content samples. First, staining with PMAxx bacterial viability stain (Trademark) of pure Salmonella culture samples was performed to inhibit the signal of non-viable cells. After DNA extraction with two different kits, a bank of serial dilutions was made, which were used for the construction of standard curves using the Illumina Eco Thermal Cycler (Trademark) Real-Time PCR System. In the analysis of the results, the concentration and volume of dye used significantly inhibited the signal of dead cells. The standard curves obtained with the two DNA extraction kits showed a high linearity greater than 0.99 and a limit of quantification of Ct 31.4. However, the best efficiency was obtained with the QIAamp Fast DNA Stool Mini Kit, which was 84.99 percent. Therefore, the optimization of the method obtained with this kit was used because it had acceptable yield values for the subsequent quantification of S. infantis in samples of poultry cecal contents.