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 - 05/05/23
Graphical abstract |
Highlights |
• | A positive control plasmid DNA for molecular diagnostics of YFV was generated. |
• | T7-driven transcription allowed the generation of a YFV positive control RNA. |
• | The control RNA was successfully used for the detection and quantification of YFV through real-time RT-PCR. |
• | The YFV construct is available for use with two recommended YFV detection assays. |
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.
Le texte complet de cet article est disponible en PDF.Keywords : Yellow fever virus, RT-qPCR, Molecular detection, In vitro transcription, Plasmid DNA
Abbreviations : GCE, NHP, NTC, PAHO, YFV, RFLP, RT-qPCR
Plan
Vol 53 - N° 3
Article 104654- avril 2023 Retour au numéro
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