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)

Abstract

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.