National Pathogen Collection Center for Aquatic Animals, Shanghai Ocean University, Shanghai, China.
Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai Ocean University, Shanghai, China.
J Fish Dis. 2024 Jun;47(6):e13930. doi: 10.1111/jfd.13930. Epub 2024 Feb 13.
Large yellow croaker (Larimichthys crocea) is a vital marine-cultured species in China. Large yellow croaker iridovirus (LYCIV) can cause a high mortality rate in L. crocea. Rapid and convenient detection of LYCIV is an urgent demand for diagnosis. In this study, rapid and simple recombinase polymerase amplification (RPA), real-time RPA and RPA combined with lateral flow dipstick (RPA-LFD) methods were developed for the detection of LYCIV based on the conserved sequence of the LYCIV major capsid protein (MCP) gene. With these optimized RPA analyses, LYCIV detection could be completed within 20 min at 40°C. Both RPA and real-time RPA could detect viral DNA as low as 10 copies/μL, while the detection limit of RPA-LFD was 10 copies/μL, and there was no cross-reaction with other aquatic pathogens (KHV, CyHV-2, GCRV-JX01, SVCV, LCDV and LMBV). In practical evaluation of RPA, real-time RPA and RPA-LFD methods, the results showed consistency with the general PCR detection. In short, the developed RPA, real-time RPA and RPA-LFD analyses could be simple, rapid, sensitive and reliable methods for field diagnosis of LYCIV infection and have significant potential in the protection of LYCIV infection.
大黄鱼（Larimichthys crocea）是中国重要的海水养殖品种。大黄鱼虹彩病毒（LYCIV）可导致大黄鱼死亡率升高。因此，快速便捷地检测 LYCIV 是诊断的迫切需求。本研究基于 LYCIV 主要衣壳蛋白（MCP）基因的保守序列，建立了快速简便的重组酶聚合酶扩增（RPA）、实时 RPA 和 RPA 与侧向流动试纸条（RPA-LFD）联合检测方法。优化后的 RPA 分析可在 40°C 下 20min 内完成 LYCIV 检测。RPA 和实时 RPA 均可检测到低至 10 拷贝/μL 的病毒 DNA，而 RPA-LFD 的检测限为 10 拷贝/μL，与其他水产病原体（KHV、CyHV-2、GCRV-JX01、SVCV、LCDV 和 LMBV）无交叉反应。在 RPA、实时 RPA 和 RPA-LFD 方法的实际评估中，结果与通用 PCR 检测一致。总之，本研究建立的 RPA、实时 RPA 和 RPA-LFD 分析方法可用于现场诊断 LYCIV 感染，具有简单、快速、灵敏和可靠的特点，对 LYCIV 感染的防控具有重要意义。
