Ebola Virus Epidemiology and Evolution in Nigeria

Folarin, Onikepe A.; Ehichioya, Deborah; Schaffner, Stephen F.; Winnicki, Sarah M.; Wohl, Shirlee; Eromon, Philomena; West, Kendra L.; Gladden-Young, Adrianne; Oyejide, Nicholas E.; Matranga, Christian B.; Deme, Awa Bineta; James, Ayorinde; Tomkins-Tinch, Christopher; Onyewurunwa, Kenneth; Ladner, Jason T.; Palacios, Gustavo; Nosamiefan, Iguosadolo; Andersen, Kristian G.; Omilabu, Sunday; Park, Daniel J.; Yozwiak, Nathan L.; Nasidi, Abdusallam; Garry, Robert F.; Tomori, Oyewale; Sabeti, Pardis C.; Happi, Christian T.

EBOV-specific diagnostic tests were performed on the suspected EBOV samples at RUN with RT-PCR using the SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity DNA Polymerase (Life Technologies). The 25-µL assay mix included 5 µL of RNA, KGH primer set [2] at a 250 nmol/L final concentration (forward, GTC GTT CCA ACA ATC GAG CG; reverse, CGT CCC GTA GCT TTR GCC AT), 12.5 µL of ×2 Reaction Mix and 0.5 µL of SuperScript III RT/Platinum Taq High Fidelity Enzyme Mix. The cycling conditions were 60°C for 20 minutes and 94°C for 5 minutes, followed by 35 cycles of 94°C, 58°C, and 68°C for 15 seconds each, with a final extension at 68°C for 2 minutes. RT-PCR was performed on an Eppendorf Mastercycler thermocycler. The samples were analyzed on 1.5% agarose gel, and visual results were recorded.