Detection and differentiation of fowl adenovirus serotype 4 and duck adenovirus 3 using high resolution melting curve assay

• Published in: Poultry science Vol. 103; no. 12; p. 104426
• Main Authors: Chen, Shuyu, Chen, Cuiteng, Zhang, Mengyan, Chen, YuYi, Zhang, Wenyu, Fu, Huanru, Huang, Yu, Cheng, Longfei, Wan, Chunhe
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.12.2024; Elsevier
• Subjects: Adenoviridae Infections - veterinary; Adenoviridae Infections - virology; Animals; Aviadenovirus; Aviadenovirus - classification; Aviadenovirus - genetics; Aviadenovirus - isolation & purification; China; cost effectiveness; cross reaction; DAdV-3; Differentiation; Ducks - virology; Epidemiological investigation; FAdV-4; family; genotyping; genus; industry; mixed infection; Muscovy; pathogenesis; poultry; Poultry Diseases - diagnosis; Poultry Diseases - virology; qPCR-HRM assay; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - veterinary; Sensitivity and Specificity; Serogroup; serotypes; viruses; waterfowl; China; FAdV-4; DAdV-3; Epidemiological investigation; qPCR-HRM assay; Differentiation
• ISSN: 0032-5791; 1525-3171; 1525-3171
• DOI: 10.1016/j.psj.2024.104426

Fowl adenovirus type 4 (FAdV-4) and duck adenovirus type 3 (DAdV-3) are the causative agents of clinical diseases in poultry and have caused considerable economic losses to the waterfowl industry in China. Both FAdV-4 and DAdV-3 are classified into the genus Aviadenovirus under the family Adenoviridae. The high-resolution melting (HRM) assay has become a useful method for virus genotyping, which offers the possibility of rapidly developing a differentiation technique in which the melting profile depends on the GC content of the product in the qPCR platform. The aim of this study was to develop a qPCR-HRM assay for sensitive FAdV-4 and DAdV-3 detection and differentiation. Here, specific primers were designed on the basis of the 100 K genes of FAdV-4 and DAdV-3, and a qPCR-HRM assay was established through optimization of the reaction conditions. A specificity test revealed that this method could detect only FAdV-4 and DAdV-3, with no cross-reaction with other common duck-derived viruses. A sensitivity test revealed that the lowest detection limits of FAdV-4 and DAdV-3 were 2.84 copies/µL and 2.85 copies/µL, respectively. A repeatability test demonstrated that the coefficient of variation was less than 2.5 % in both the intragroup and the intergroup analyses. Field sample distributions of FAdV-4 and DAdV-3 were investigated, and the percentages of DAdV-3-positive, FAdV-4-positive and coinfection-positive in Muscovy ducks were 27.78 %, 16.67 % and 11.11 %, respectively. Further studies are needed to provide more insight into the pathogenesis of FAdV-4 and DAdV-3 coinfection in ducks. In conclusion, the qPCR-HRM assay provides an accurate, sensitive, reliable and cost-effective alternative method for detecting and distinguishing FAdV-4 and DAdV-3.