Usefulness of an in vitro-transcribed RNA control for the detection and quantification of Yellow fever virus through real-time reverse transcription-polymerase chain reaction

ABSTRACT: Introduction: Unvaccinated individuals in endemic areas with proven enzootic transmission of Yellow fever virus are at risk of infection due to a dramatic shift in the epidemiology of the disease over recent years. For this reason, epidemiological surveillance and laboratory confirmation of cases have become mandatory. Objective: To develop and test a control RNA for YFV detection through real-time RT-PCR. Methods: A 437-bp insert containing the T7 promoter and the target sequences for two different in-house protocols was designed in the context of the pUC57 vector and obtained through gene synthesis. After T7-driven in vitro transcription, standard curves were developed for Log10 serial dilutions of the YFV control RNA with 8 replicates. Results: A dynamic range of quantification of 10 orders of magnitude was observed with a limit of detection of 6.3 GCE/µL (95% CI, 2.6 to 139.4 GCE/µL). Conclusion: The plasmid construct is available for YFV molecular test validation on clinical, entomological, and epizootic samples. Keywords: In vitro transcription; Molecular detection; Plasmid DNA; RT-qPCR; Yellow fever virus.

• Autores: Navas Navas, María Cristina; Laiton Donato, Katherine; Quintero Cortés, Paula; Franco Salazar, Juan P.; Peláez Carvajal, Dioselina; Junglen, Sandra; Parra Henao, Gabriel; Usme Ciro, Jose A.
• Tipo de recurso: Article of investigation
• Fecha de publicación: 2024
• Institución: Universidad de Antioquia
• Repositorio: Repositorio UdeA
• Idioma: eng
• OAI Identifier: oai:bibliotecadigital.udea.edu.co:10495/39595
• Acceso en línea: https://hdl.handle.net/10495/39595; https://www.sciencedirect.com/science/article/pii/S2666991923000167
• Palabra clave: Reacción en Cadena de la Polimerasa de Transcriptasa Inversa; Reverse Transcriptase Polymerase Chain Reaction; Transcripción Reversa; Reverse Transcription; Fiebre Amarilla; Yellow Fever; Virus de la Fiebre Amarilla; Yellow fever virus; Diagnóstico; Diagnosis; Epidemiología; Epidemiology; ARN; RNA; https://id.nlm.nih.gov/mesh/D020133; https://id.nlm.nih.gov/mesh/D048348; https://id.nlm.nih.gov/mesh/D015004; https://id.nlm.nih.gov/mesh/D015005; https://id.nlm.nih.gov/mesh/D003933; https://id.nlm.nih.gov/mesh/D004813; https://id.nlm.nih.gov/mesh/D012313
• Rights: openAccess
• License: https://creativecommons.org/licenses/by-nc-nd/4.0/