Diseño de primers del gen meca para detección de Staphylococcus aureus resistente a meticilina por Amplificación de Polimerasa y Recombinasa, Perú 2022.
No Thumbnail Available
Date
2023
Journal Title
Journal ISSN
Volume Title
Publisher
Universidad Nacional de Trujillo
Abstract
El presente trabajo de investigación tuvo como objetivo diseñar primers in silico del gen mecA para la detección de cepas peruanas de S. aureus resistente a la meticilina (SARM) por Amplificación de Polimerasa y Recombinasa (RPA),
validando y verificando el cumplimiento de características físico químicas deseables para dichos primers, su especificidad y capacidad de amplificación in silico de tal manera que en el futuro se pueda comprobar su amplificación y
capacidad de diagnóstico a nivel de laboratorio. Se obtuvieron 32 secuencias del gen mecA de la base de datos RefSeq del Centro Nacional de Información Biotecnológica (NCBI) procedentes de Perú, consolidando la información en una
metadata. Se diseñaron los primers usando los programas bioinformáticos Primer- BLAST y Primer3Plus. Se seleccionaron los dos mejores pares de primers para RPA: el par Nº1 (mecA-RPA-F1 y mecA-RPA-R1) y el par Nº2 (mecA-RPA-F2 y mecA-RPA-R2) al evaluar los parámetros termodinámicos y estructuras secundarias con los programas Oligoanalyzer Tool y Beacon Designer Free Edition. Se verificó la capacidad de amplificar in silico de los primers del gen mecA de S. aureus usando los programas In silico PCR amplificación y PCR Test - Secuences Manipulation Suite (SMS). Se complementó evaluando la especificidad de los primers mediante el software BLAST-N que mostró la capacidad de los primers
propuestos para identificar las cepas peruanas de Staphylococcus aureus (con 100% de identidad), las cuales representaron el 62% del total de especies identificadas, mientras que el 38% fueron otras especies de Staphylococcus.
The aim of this research was to design in silico primers of the mecA gene that would allow detecting Peruvian strains of methicillin-resistant Staphylococcus aureus (MRSA) by Polymerase and Recombinase Amplification (RPA), verifying and validating that the desirable physical chemical characteristics of these primers were accomplished. Moreover, their specificity and in silico amplification capacity of were evaluated. Thus, letting pendent the evaluation of the amplification and diagnostic capacity that should be done at the laboratory level in the future. Thirtytwo mecA gene sequences from Peru were obtained from the RefSeq database of the National Center for Biotechnology Information (NCBI), integrating the information in a metadata. Primers were designed using the bioinformatics programs Primer-BLAST and Primer3Plus. The two best primer pairs for RPA were selected: pair Nº1 (mecA-RPA-F1 and mecA-RPA-R1) and pair Nº2 (mecA-RPAF2 and mecA-RPA-R2) by evaluating the thermodynamic parameters and secondary structures with the Oligoanalyzer Tool and Beacon Designer Free Edition programs. The in silico amplification capability of the Staphylococcus aureus mecA gene primers was verified using the In silico PCR amplification and PCR Test - Sequences Manipulation Suite (SMS) programs. This assessment was complemented by evaluating the specificity of the primers using the BLAST-N software that showed the ability of the proposed primers to identify the Peruvian strains of Staphylococcus aureus (with 100% identity), which represented the 62% of the total number of identified species, while the 38% were other Staphylococcus species.
The aim of this research was to design in silico primers of the mecA gene that would allow detecting Peruvian strains of methicillin-resistant Staphylococcus aureus (MRSA) by Polymerase and Recombinase Amplification (RPA), verifying and validating that the desirable physical chemical characteristics of these primers were accomplished. Moreover, their specificity and in silico amplification capacity of were evaluated. Thus, letting pendent the evaluation of the amplification and diagnostic capacity that should be done at the laboratory level in the future. Thirtytwo mecA gene sequences from Peru were obtained from the RefSeq database of the National Center for Biotechnology Information (NCBI), integrating the information in a metadata. Primers were designed using the bioinformatics programs Primer-BLAST and Primer3Plus. The two best primer pairs for RPA were selected: pair Nº1 (mecA-RPA-F1 and mecA-RPA-R1) and pair Nº2 (mecA-RPAF2 and mecA-RPA-R2) by evaluating the thermodynamic parameters and secondary structures with the Oligoanalyzer Tool and Beacon Designer Free Edition programs. The in silico amplification capability of the Staphylococcus aureus mecA gene primers was verified using the In silico PCR amplification and PCR Test - Sequences Manipulation Suite (SMS) programs. This assessment was complemented by evaluating the specificity of the primers using the BLAST-N software that showed the ability of the proposed primers to identify the Peruvian strains of Staphylococcus aureus (with 100% identity), which represented the 62% of the total number of identified species, while the 38% were other Staphylococcus species.
Description
Keywords
Staphylococcus aureus, Antibióticos, Amplificación de Polimerasa y Recombinasa (RPA)