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Войти в исследования Infectious diseases now · 2023
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.
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Laiton-Donato Katherine, Quintero-Cortés Paula, Franco-Salazar Juan P, Peláez-Carvajal Dioselina, Navas Maria-Cristina, Junglen Sandra, Parra-Henao Gabriel, Usme-Ciro Jose A
Аннотация
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.
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.