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Development and evaluation of a droplet digital PCR assay for the detection of fowl adenovirus serotypes 4 and 10 in attenuated vaccines

Dong, Guiwei, Meng, Fanfeng, Zhang, Yubiao, Cui, Zhizhong, Lidan, Hou, Chang, Shuang, Zhao, Peng
Journal of virological methods 2019 v.265 pp. 59-65
Adenoviridae, Newcastle disease virus, chickens, droplets, feathers, genomics, live vaccines, quantitative polymerase chain reaction, rapid methods, serotypes, China
In recent years, there has been an increase in reported cases of fowl adenovirus serotype 4 (FAdV-4) in chickens in China. The use of live attenuated vaccines contaminated with FAdV-4 has been proved to be one of the important causes of massive outbreaks of hydropericardium syndrome. To detect the contamination with FAdV-4 in attenuated vaccines more promptly and accurately, a droplet digital PCR (ddPCR) assay was developed for the rapid detection of FAdV-4 and FAdV-10. The ability of this assay to detect FAdV-4 contamination in attenuated Newcastle disease virus vaccines was assessed in comparison to a quantitative real-time PCR (qPCR) and a conventional PCR assay. The findings indicated that the ddPCR assay could detect FAdV-4 contamination at 0.1 EID50/1,000 feathers, while the qPCR could detect FAdV-4 contamination at 1 EID50/1,000 feathers with identical genomic targets, which was 1,000-fold more sensitive than conventional PCR detection with a sensitivity of 102 EID50/1,000 feathers. The ddPCR assay also showed high specificity for FAdV-4/10 and no positive signals were detected for other FAdVs. Consequently, the intuitive and rapid results were especially suitable for the detection of FAdV-4 contamination in vaccines. In this study, a ddPCR assay was developed to effectively detect and quantify low-dose FAdV-4 contamination, providing a new method for rapid detection of FAdV-4 contamination in various samples, especially vaccines.