Qu Guanggang, Li Yun, Zhao Zhongwei, Miao Lizhong, Wei Feng, Tang Na, Xu Qingqing, Nair Venugopal, Yao Yongxiu, Shen Zhiqiang
Binzhou Animal Science and Veterinary Medicine Academy and UK-China Centre of Excellence for Research on Avian Diseases, Binzhou, China.
College of Veterinary Medicine, Shandong Agricultural University, Tai'an, China.
Front Vet Sci. 2022 Jul 7;9:847194. doi: 10.3389/fvets.2022.847194. eCollection 2022.
Avian leukosis caused by avian leukosis virus (ALV), belonging to the genus of the family , is associated with benign and malignant tumors in hemopoietic cells in poultry. Although several methods have been developed for ALV detection, most of them are not suitable for rapid on-site testing due to instrument limitations, professional operators, or the low sensitivity of the method. Herein, we described the real-time recombinase polymerase amplification (RPA) assay for rapid detection of ALV subgroup J (ALV-J). The major viral structural glycoprotein gp85, highly specific for the subgroup, was used as the molecular target for the real-time RPA assay. The results were obtained at 38°C within 20 min, with the detection sensitivity of 10 copies/μl of standard plasmid pMD18-T-gp85 as the template per reaction. Real-time RPA was capable of ALV-J-specific detection without cross-reaction with other non-targeted avian pathogens. Of the 62 clinical samples tested, the ALV-positive rates of real-time RPA, PCR, and real-time PCR were 66.13% (41/62), 59.68% (37/62), and 67.74% (42/62), respectively. The diagnostic agreement between real-time RPA and real-time PCR was 98.39% (61/62), and the kappa value was 0.9636. The developed real-time ALV-J assay seems promising for rapid and sensitive detection of ALV-J in diagnostic laboratories. It is suitable for on-site detection, especially in a poor resource environment, thus facilitating the prevention and control of ALV-J.
禽白血病病毒（ALV）引起的禽白血病属于该科属，与家禽造血细胞中的良性和恶性肿瘤有关。尽管已经开发了几种用于检测ALV的方法，但由于仪器限制、专业操作人员或方法灵敏度低，大多数方法不适用于快速现场检测。在此，我们描述了用于快速检测J亚群禽白血病病毒（ALV-J）的实时重组酶聚合酶扩增（RPA）检测方法。将对该亚群具有高度特异性的主要病毒结构糖蛋白gp85用作实时RPA检测的分子靶标。在38°C下20分钟内获得结果，每个反应以标准质粒pMD18-T-gp85作为模板的检测灵敏度为10拷贝/μl。实时RPA能够特异性检测ALV-J，与其他非靶向禽病原体无交叉反应。在测试的62份临床样本中，实时RPA、PCR和实时PCR的ALV阳性率分别为66.13%（41/62）、59.68%（37/62）和67.74%（42/62）。实时RPA与实时PCR之间的诊断一致性为98.39%（61/62），kappa值为0.9636。所开发的实时ALV-J检测方法似乎有望在诊断实验室中快速、灵敏地检测ALV-J。它适用于现场检测，特别是在资源匮乏的环境中，从而有助于ALV-J的防控。
