Validación de técnicas de PCR cuantitativa (qPCR) para la cuantificación de genes de resistencia a antibióticos

Abstract

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.