Development and validation of a field deployable quantitative real-time polymerase chain reaction assay (qRT-PCR) for Ebolavirus

Authors

• DM Figueroa
• E Kuisma
• MJ Matson
• T Bushmaker
• SN Seifert
• S Olson
• RJ Fischer
• VJ Munster

Abstract

Besides humans, Ebolavirus (EBOV) has been found to affect an array of primates, including gorillas. This poses a risk as people who encounter the deceased primates can become infected. Direct diagnosis of whether EBOV is the cause of mortality is instrumental in mitigating the dangers a dead gorilla poses. Field diagnosis for high consequence pathogens, such as EBOV, is challenging as sample preservation and travel to a laboratory can delay results. Here, we developed a point-of-encounter (PoE) system, utilizing the Biomeme (BM) field qRT-PCR system. First, we validated the compatibility of the BM syringe-based extraction kit with our standard inactivation methods for EBOV. The BM kit extracted 3.04x108 genome copies/mL compared to 1.04x109 genome copies/mL by the Qiagen Viral RNA kit. Next, we compared the BM against Roche 480 qRT-PCR reagents with the Smart Cycler instrument. On average, the amplification efficiencies of the assays differed by less than 3% (± 23 STD) across two EBOV variants. No cross reactivity was observed with other species of Ebolavirus: Reston Ebolavirus, Sudan Ebolavirus, Tai Forest Ebolavirus and Bundibugyo Ebolavirus. The BM system was further validated by testing swabs from an EBOV infected non-human primate at day 0, 4 and 7 from 4 locations on the carcass. All swabs were positive, consistent with previous carcass sampling experiments. The instrument has also been tested in the field, in Montana, USA and the Republic of Congo and presented no problems. We conclude that the BM with the EBOV assay is field compatible, producing results in 1.5 hours in remote and resource poor locations.

Scientific Focus Area: Microbiology and Infectious Diseases

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