Comparison and Evaluation of Real-Time Taqman PCR for Detection and Quantification of Ebolavirus

Given that ebolavirus causes severe and frequently lethal disease, its rapid and accurate detection using available and validated methods is essential for controlling infection. Real-time reverse-transcription PCR (RT-PCR) has proven to be an invaluable tool for ebolaviruses diagnostics. Many assays with different targets have been developed, but they have not been externally compared or validated, and limits of detection are not uniformly reported. Here we compared and evaluated the sensitivity, reproducibility and specificity of 23 in-house assays under the same conditions. Our results showed that these assays were highly gene- and species- specific when evaluated using in vitro RNA transcripts and viral RNA, and the potential limits of detection were uniformly reported ranging from 10(2) to 10(6) in vitro synthesized RNA transcripts copies per mu L and 1-100 TCID50/mL. The comparison of these assays indicated that those targeting the more conservative NP gene could be the better option for EVD case definition and quantitative measurement because of its higher sensitivity for the same species. Our analysis could contribute to the standardization of ebolavirus detection and quantification assays, which can offer a better understanding of the meaning of results across laboratories and time points, as well as make them easy to implement, especially under outbreak conditions.

Automated summary

The study compared and evaluated the sensitivity, reproducibility, and specificity of 23 in-house assays for ebolavirus detection, finding that targeting the more conservative NP gene may offer better sensitivity for EVD case definition and quantitative measurement. This analysis could contribute to the standardization of ebolavirus detection and quantification assays, providing a better understanding of results and making implementation easier, particularly during outbreaks.