Abstract

DIAZ-FERREIRA, Natalia Jazmín et al. Optimization of a conventional PCR technique for the amplification of the LCR region and the E6 gene of human papillomavirus type 16. Mem. Inst. Investig. Cienc. Salud [online]. 2022, vol.20, n.2, pp.13-19. ISSN 1812-9528.  https://doi.org/10.18004/mem.iics/1812-9528/2022.020.02.13.

Cervical cancer is the fourth most common cancer in women in the world and worldwide HPV 16 is present in approximately 60% of cases. To date, HPV 16 variants are classified into 4 lineages and 16 sublineages, with some variants being associated with lesion severity. Sequencing of the LCR region and the E6 gene is used for variant classification. Therefore, the objective was to optimize two conventional PCRs to detect the LCR region and one PCR for the E6 gene. For this purpose, HPV 16 positive samples, specific primers for the LCR region and the E6 gene were used. The reactions were tested at different alignment temperatures. The concentration of MgCl2, dNTP, and primers was determined following the recommendations of the manufacturer of the DNA polymerase enzyme used. For amplification of the LCR region and the HPV 16 E6 gene, the best results were observed at an annealing temperature of 57°C and 50°C, respectively. The concentration of MgCl2 used in both reactions was 1.5mM, dNTP 0.2mM and primers 0.2uM. The present optimization will be used to sequence the amplified products to determine the variants and subsequently evaluate the functionality and transcriptional activity in order to relate it to cervical pathogenesis.

Keywords : HPV16; PCR; LCR/E6.

        · abstract in Spanish     · text in Spanish     · Spanish ( pdf )