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Development of a Real-Time Enzymatic Recombinase Amplification Assay (RT-ERA) and an ERA Combined with a Lateral Flow Dipstick (LFD) Assay (ERA-LFD) for Enteric Microsporidian (Enterospora epinepheli) in Grouper Fishes

Minqi Chen, Yongcan Zhou, Shifeng Wang, Jian Luo, Weiliang Guo, Hengwei Deng et al. · 2025 · 3 citationsRead the paper

Enterospora epinepheli 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, R2 = 0.9915), enabling accurate quantification. Both methods displayed a detection limit of 2 × 100 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 E. epinepheli, with broad applicability to pathogen detection in aquaculture systems.

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Working prototype

RT-ERA and ERA-LFD assays achieved a detection limit of 2 copies/μL. — 2 copies/μL (Using recombinant plasmid standards)
RT-ERA demonstrated a strong linear relationship between plasmid copy numbers and cycle threshold (Ct) values. — y = −2.1226x + 19.562, R2 = 0.9915 (Recombinant plasmid standards)
RT-ERA and ERA-LFD showed no cross-reactivity with other common aquaculture pathogens. — 100% specificity (Tested against 9 other pathogens including V. alginolyticus and SGIV)
RT-ERA and ERA-LFD showed 100% concordance with basic ERA in clinical samples. — 100% concordance (42 tissue samples and 26 water samples from grouper fishes)
RT-ERA and ERA-LFD provided faster results than conventional PCR. — RT-ERA: 14-20 min; ERA-LFD: 10 min (Isothermal conditions (39-40 °C))

Setup: Recombinant plasmid standards (2x10^0 to 2x10^8 copies/μL), 42 grouper tissue samples, and 26 pond water samples.
How: Developed two isothermal assays (RT-ERA and ERA-LFD) targeting the 18S rDNA gene of E. epinepheli using HPLC-purified primers and specific probes. RT-ERA uses a real-time fluorescence quantifier, while ERA-LFD utilizes a lateral flow dipstick for visual detection.
Limitations: Requires partitioned isolation operation to avoid false positives or template contamination.

Summarized by Gemma 4 from the open-access full text · 8 Oct 2026. Read it