NS5 Region Cloning Protocol, for the Construction of an Accurate Standard Curve in the Detection and Diagnosis of the Yellow Fever Virus.

The yellow fever virus (YF), belonging to the Flaviviridae family, has an RNA genome of 10,862 nucleotides and continues to cause significant epidemics in Africa and South America despite an effective vaccine. Timely laboratory identification is essential for emergency immunization campaigns, but delays often lead to unconfirmed outbreaks. The presence of other diseases with similar symptoms complicates differential diagnosis, particularly in endemic regions, and the recent resurgence of YF activity has renewed interest in this arboviral disease. Genomic DNA (gDNA) and plasmids with cloned target sequences serve as standards in quantitative PCR, requiring specific calculations to determine their masses relative to target nucleic acid copy numbers.