Chen Shuyu, Chen Cuiteng, Zhang Mengyan, Chen YuYi, Zhang Wenyu, Fu Huanru, Huang Yu, Cheng Longfei, Wan Chunhe
Institute of Animal Husbandry and Veterinary Medicine/Fujian Key Laboratory for Avian Diseases Control and Prevention/Fujian Animal Diseases Control Technology Development Centre, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China; School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
Institute of Animal Husbandry and Veterinary Medicine/Fujian Key Laboratory for Avian Diseases Control and Prevention/Fujian Animal Diseases Control Technology Development Centre, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China.
Poult Sci. 2024 Dec;103(12):104426. doi: 10.1016/j.psj.2024.104426. Epub 2024 Oct 18.
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.
禽腺病毒4型（FAdV-4）和鸭腺病毒3型（DAdV-3）是家禽临床疾病的病原体，给中国水禽产业造成了相当大的经济损失。FAdV-4和DAdV-3都被归类于腺病毒科禽腺病毒属。高分辨率熔解（HRM）分析已成为一种有用的病毒基因分型方法，它提供了在qPCR平台上快速开发一种区分技术的可能性，该技术的熔解曲线取决于产物的GC含量。本研究的目的是开发一种用于灵敏检测和区分FAdV-4和DAdV-3的qPCR-HRM分析方法。在此，基于FAdV-4和DAdV-3的100K基因设计了特异性引物，并通过优化反应条件建立了qPCR-HRM分析方法。特异性试验表明，该方法仅能检测FAdV-4和DAdV-3，与其他常见鸭源病毒无交叉反应。敏感性试验表明，FAdV-4和DAdV-3的最低检测限分别为2.84拷贝/μL和2.85拷贝/μL。重复性试验表明，组内和组间分析的变异系数均小于2.5%。调查了FAdV-4和DAdV-3的田间样本分布情况，番鸭中DAdV-3阳性、FAdV-4阳性和混合感染阳性的比例分别为27.78%、16.67%和11.11%。需要进一步研究以更深入了解鸭中FAdV-4和DAdV-3混合感染的发病机制。总之，qPCR-HRM分析为检测和区分FAdV-4和DAdV-3提供了一种准确、灵敏、可靠且经济高效的替代方法。
