Palabras clave
Muestras de Saliva
RT-qPCR múltiples
Solución Coadyuvante
Estructura Secundaria del ARN
Cómo citar
Resumen
Introducción: El desempeño de la RT-qPCR para detectar SARS-CoV-2 varía porque los SNP en los sitios de cebadores/sondas y las estructuras secundarias del ARN (en particular, los bucles de tallo en las UTR 5′/3′) pueden obstaculizar la hibridación y la elongación de la polimerasa, alterando el Ct y la fluorescencia. Una solución desnaturalizante (DS; compuesta por cloruro de tetrametilamonio y dimetilsulfóxido) puede alterar estas estructuras y mejorar la detección. Objetivo: Evaluar el efecto de la SD en la eficiencia de la RT-qPCR para la detección de SARS-CoV-2 en muestras de saliva individuales o agrupadas de pacientes diagnosticados con COVID-19. Metodología: Se determinó in silico el patrón de hibridación entre los cebadores y sondas autorizados por el CDC (Centers for Disease Control and Prevention) respecto a la región consenso del gen N de SARS-CoV-2, además de generar su estructura secundaria para definir las posiciones variables. La validación de las RT-qPCR individuales y agrupadas con la SD se realizó con ARN total en 20 de 40 muestras de saliva positivas a SARS-CoV-2. Resultados: La región consenso del gen N1 de las variantes de SARS-CoV-2: Alfa, Beta, Delta, Gamma, GH490R, Lambda, Mu y Ómicron del año 2021, presentó más variaciones respecto a la región N2, no obstante, se localizaron dentro de las burbujas adyacentes a un tallo y en la región espaciadora. Las RT-qPCR individuales para las regiones N1 y N2 con SD no mostraron Cts estadísticamente significativos, sin embargo, estos disminuyeron, con una diferencia notable en la señal de fluorescencia, para la región N1 en las reacciones de RT-qPCR agrupadas. Conclusión: El procedimiento confirma la ventaja de la inclusión de la SD en la RT-qPCR para detectar SARS-CoV-2 en muestras de saliva.
Citas
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Esta obra está bajo una licencia internacional Creative Commons Atribución 4.0.
Derechos de autor 2025 Lina Maria Vera Cala, Nathaniel A. Navarro-Baron, Yordy S. Cangrejo-Useda, Luis A. Pardo-Díaz, Cristian E. Cadena-Caballero, Carlos J. Barrios Hernandez, Francisco Martinez-Perez