Ciencia e Ingeniería en Alimentos y Biotecnología

URI permanente para esta comunidadhttp://repositorio.uta.edu.ec/handle/123456789/412

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2019 - 201912020 - 20231

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Tipo de acceso: Acceso abiertoTema: search.filters.subject.PCR

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  • Miniatura
    Item type:Ítem,
    Diseño y estandarización de un protocolo de diagnóstico molecular por RT-PCR para la genotipificación de las variantes de VPH predominantes en Ecuador (Genotipos:16, 18, 45, 58, 31)
    (Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2023-09)
    Limachi Coello, Kevin Patricio
    ;
    Núñez Villacís, Lorena de los Ángeles
    Human Papillomavirus (HPV) is the most common sexually transmitted pathogen worldwide. Nowadays, there are more than 200 HPV genotypes classified into low-risk (LR-HPV) and high-risk HPV strains (HR-HPV). The HR-HPV group is precursor of cervical cancer, which represents the fourth most deadly type of cancer worldwide and the second leading cause of death of women in Ecuador. Early and specific diagnosis leads to an improvement in the quality of life, low mortality rates and help to choose an efficient treatment. This study was based on the development of a Real Time PCR (RT-PCR) protocol for the genotyping of HR-HPV variants found in Ecuador (16, 18, 45, 58 and 31). Which were chosen through bibliographic analysis of clinical-population studies and research carried out in Ecuador in the last 8 years. A bioinformatic analysis of HPV genomes was developed, which result in a set of 10 primers with the capacity to identify the mentioned genotypes by SYBR Green technology. In addition to a thermal cycling protocol for RT-PCR amplification standardized experimentally in an in-house test for Laboratory of Medical Specialties Ochoa & Ochoa. Finally, we get a protocol able to recognize the HPV genotypes 16, 18, 45, 58 and 31 with a minimal synthetic DNA amount of 0.05 picograms per microliter. This study could be used as a genotyping prototype to be evaluated in human clinical samples.
  • Miniatura
    Item type:Ítem,
    Validación de técnicas de PCR cuantitativa (qPCR) para la cuantificación de genes de resistencia a antibióticos
    (Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Ingeniería Bioquímica, 2019-12)
    Valle Ramos, María Judith
    ;
    Calero Cáceres, William Ricardo
    The increment in the usage of genomic techniques applied to diagnosis and research demands to have validated techniques, in order to obtain reliable and reproducible results during the experimental tests. This work aimed to validate real-time PCR (qPCR) techniques, using both TaqMan and Sybr Green methods. The applied equipment was 7500 Fast Real-Time PCR System thermal cycler. Pure standards of the different recombinant plasmids (blaTEM, sul1, qnrS, ermB, and 16S rDNA) were used. Additionally, a synthetic plasmid standard was designed for tetW gene. The results indicate that the standard curves presented high efficiencies (between 97.4 to 109. 8 percent), as well as linear correlations greater than 0.99 and limits of quantification between Ct 34.4 to 34.8. Therefore, it was demonstrated that the qPCR techniques have acceptable performances since the values obtained are within the optimal ranges. These validated qPCR techniques will be used as a control tool to evaluate the dissemination of antibiotic resistance genes from environmental samples, obtaining truthful data in a short time.