Chen Minqi, Zhou Yongcan, Wang Shifeng, Luo Jian, Guo Weiliang, Deng Hengwei, Zheng Pei, Zhong Zhihong, Su Baofeng, Zhang Dongdong, Ye Zhi
School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), Hainan University, Sanya 572025, China.
Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, School of Marine Biology and Fisheries, Collaborative Innovation Center of Marine Science and Technology, Hainan University, Haikou 570228, China.
Biology (Basel). 2025 Mar 25;14(4):330. doi: 10.3390/biology14040330.
poses a severe threat to grouper aquaculture due to the absence of effective prevention and treatment strategies. To address this challenge, we developed and validated two isothermal diagnostic tools, the real-time enzymatic recombinase amplification (RT-ERA) assay and the enzymatic recombinase amplification combined with a lateral flow dipstick (ERA-LFD) assay, targeting the 18S rDNA gene of the parasite. These assays operate under isothermal conditions at ≤40 °C and offer rapid detection, with RT-ERA yielding results in 14~20 min and ERA-LFD in approximately 10 min. The RT-ERA assay demonstrated a strong linear relationship between plasmid copy numbers and cycle threshold (Ct) values (y = -2.1226x + 19.562, R = 0.9915), enabling accurate quantification. Both methods displayed a detection limit of 2 × 10 copies/μL and no cross-reactivity with other aquaculture pathogens. Validation using grouper tissue and water samples from Hainan, China, demonstrated 100% concordance rates with basic ERA and outperformed compared to conventional PCR. These assays provide sensitive, specific, and rapid detection tools for effective monitoring and pathogen load assessment of , with broad applicability to pathogen detection in aquaculture systems.
由于缺乏有效的预防和治疗策略，对石斑鱼养殖构成了严重威胁。为应对这一挑战，我们开发并验证了两种等温诊断工具，即实时酶促重组酶扩增（RT-ERA）检测法和酶促重组酶扩增结合侧向流动试纸条（ERA-LFD）检测法，靶向该寄生虫的18S rDNA基因。这些检测法在≤40°C的等温条件下运行，提供快速检测，RT-ERA在14~20分钟内得出结果，ERA-LFD约在10分钟内得出结果。RT-ERA检测法在质粒拷贝数与循环阈值（Ct）值之间显示出很强的线性关系（y = -2.1226x + 19.562，R = 0.9915），能够进行准确定量。两种方法的检测限均为2×10拷贝/μL，且与其他水产养殖病原体无交叉反应。使用来自中国海南的石斑鱼组织和水样进行验证，结果表明与基本ERA的符合率为100%，且优于传统PCR。这些检测法为石斑鱼的有效监测和病原体载量评估提供了灵敏、特异且快速的检测工具，在水产养殖系统中的病原体检测具有广泛的适用性。
