Development and Evaluation of Droplet Digital PCR Assay for the Detection of Reticuloendotheliosis Virus in Attenuated Vaccines

Abstract Background: The use of Reticuloendotheliosis virus (REV) from contaminated live virus vaccine is suspected to be one of the most important causes of massive outbreaks of Reticuloendotheliosis in China. Methods: In this study, we established a droplet digital PCR (ddPCR) detection method for REV and compared its sensitivity to different methods to detect REV contamination in a vaccine. Results: The results indicated that both quantitative PCR and dot-blot methods could detect REV contamination at a dose of 1 TCID50/1,000 feathers, whereas ddPCR could detect REV contamination at a dose of 0.1 TCID50/1,000 feathers, which is 1,000-fold more sensitive than conventional polymerase chain reaction detection (102 TCID50/1000 feathers). ddPCR not only exhibited the highest sensitivity but also proved extremely intuitive, especially to detect REV contamination in vaccines.Conclusions: The ddPCR method established in this study to detect REV contamination in vaccines can effectively detect and quantify low-dose REV contamination. This provides a new method for the rapid detection of REV contamination in various samples, especially vaccines.