Dai Yin, Zhong Yueyi, Xu Fazhi, Gu Siqin, Zhou Huiqin, Wang Jieru, Yin Dongdong, Yin Lei, Shen Xuehuai, Pan Xiaocheng, Liu Xuelan
Anhui Province Key Laboratory of Livestock and Poultry Product Safety Engineering, Livestock and Poultry Epidemic Diseases Research Center of Anhui Province, Institute of Animal Husbandry and Veterinary Science, Anhui Academy of Agricultural Science, Hefei 230031, China.
College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China.
Poult Sci. 2024 Dec;103(12):104452. doi: 10.1016/j.psj.2024.104452. Epub 2024 Oct 30.
Fowl adenovirus serotype 4 (FAdV-4) is the main causative agent of hydropericardium hepatitis syndrome (HHS), which has resulted in huge economic losses to the poultry industry in recent years. Hence, a rapid and simple visual detection method is needed for identification of FAdV-4. In this study, three multienzyme isothermal rapid amplification (MIRA) assays, basic MIRA, MIRA-qPCR and MIRA-LFD were developed for detection of FAdV-4. The amplification primers and reaction conditions were optimized, and the specificity and sensitivity of the assays were evaluated. The MIRA assays were specific for FAdV-4 with no cross-reaction with novel goose astrovirus, H9 subtype avian influenza virus, duck enteritis virus, Muscovy duck reovirus, or duck circovirus. The basic MIRA assay required only one primer pair and the reaction can be completed within 30 min at 36 °C. The MIRA-qPCR and MIRA-LFD assays were completed in 20 min with a minimum detection limit of 1 × 10 copies/μL and 1 × 10 copies/μL, respectively. The results of the MIRA-LFD assay can be observed directly with the naked eye, omitting the need for specialized instruments. The positive rate of three proposed MIRA assays were consistent with that of the conventional PCR assay. The MIRA assays are simple, rapid, and effective diagnostic tools for field detection of FAdV-4.
禽腺病毒4型（FAdV-4）是心包积水肝炎综合征（HHS）的主要病原体，近年来给家禽业造成了巨大经济损失。因此，需要一种快速简便的可视化检测方法来鉴定FAdV-4。在本研究中，开发了三种多酶等温快速扩增（MIRA）检测方法，即基础MIRA、MIRA-qPCR和MIRA-LFD，用于检测FAdV-4。对扩增引物和反应条件进行了优化，并评估了检测方法的特异性和灵敏度。MIRA检测方法对FAdV-4具有特异性，与新型鹅星状病毒、H9亚型禽流感病毒、鸭肠炎病毒、番鸭呼肠孤病毒或鸭圆环病毒无交叉反应。基础MIRA检测方法仅需一对引物，反应可在36℃下30分钟内完成。MIRA-qPCR和MIRA-LFD检测方法分别在20分钟内完成，最低检测限分别为1×10拷贝/μL和1×10拷贝/μL。MIRA-LFD检测方法的结果可直接用肉眼观察，无需专门仪器。三种提出的MIRA检测方法的阳性率与传统PCR检测方法一致。MIRA检测方法是用于现场检测FAdV-4的简单、快速且有效的诊断工具。
