Alejandra Jimenez Mejias

Primases are essential enzymes that initiate DNA replication by synthesizing de novo primers for replicative DNA polymerases, which ensure accurate genome duplication owing to their high specificity, fidelity, and processivity. PrimPols have emerged as versatile DNA-initiating enzymes that are widely distributed across diverse replication systems, from bacteriophages to humans. In this work, a novel PrimPol enzyme, PP271, encoded by bacteriophage vB_EfaS-271, was characterized. PP271 belongs to the archaeal–eukaryotic primase (AEP) superfamily and contains the conserved catalytic core, together with a C-terminal PriCT domain. Enzymatic analysis demonstrated that PP271 functions as a bona fide PrimPol, i.e. a DNA primase capable of synthetizing short DNA primers. PP271 priming activity is strongly stimulated by ADP , which serves as the preferred initiating nucleotide. Additionally, PP271 exhibits relaxed specificity for selecting the priming site, compared to other PrimPols with more restricted template preferences. TruePrime™ amplification technology combines the Thermus thermophilus PrimPol and the highly processive Phi29 DNA polymerase to achieve primer-free DNA amplification. While highly efficient, further improvements in amplification yield, fidelity and product complexity remain possible. Here, we show that DNA primers synthesized by PP271 are efficiently extended by Phi29 DNA polymerase, enabling their coordinated action. Moreover, the short primers generated by PP271 are expected to minimize the contribution of primase-derived errors to the final amplification product while allowing efficient DNA synthesis across a broader range of sequence contexts. Altogether, these findings identify PP271 as a promising alternative for improving primase-dependent DNA amplification. Key points: Bacteriophage EFAS271 encodes PP271, a DNA-dependent DNA primase-polymerase PP271 preferentially uses ADP as the initiating nucleotide during primer synthesis PP271 enables primer-free isothermal DNA amplification with Phi29 DNApol

La principal estrategia para el control de la malaria, según la Organización Mundial de la Salud (OMS), es un diagnóstico rápido y adecuado seguido de un tratamiento efectivo. Los laboratorios de referencia de microbiología tienen entre sus funciones el desarrollo y validación de nuevas metodologías diagnósticas para ayudar al control y erradicación de la malaria en el mundo y en nuestro caso para evitar la reintroducción en nuestro país. En este trabajo se hace una revisión de los métodos más extendidos para el diagnóstico de malaria: i) métodos clásicos, como son la microscopía y los test de diagnóstico rápido, ii) métodos moleculares, como PCR y LAMP, y, iii) por último, los nuevos avances en microscopía automática e inteligencia artificial.