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

Abstract

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.

Keywords: ERA with a lateral flow dipstick; Enterospora epinepheli; enzymatic recombinase amplification (ERA); grouper (Epinephelus spp.); real-time ERA.

Figure 1

Hybrid groupers (E. fuscoguttatus♀ × E. lanceolatus♂) infected by E. epinepheli. (A): Relatively healthy grouper (top) and severe diseased grouper (bottom). (B): Intestinal content discharge from a diseased grouper. (C): Intestinal conditions of diseased grouper (a, b, c, and d), showing thinning and transparency of intestinal walls, residual mass content, and white feces discharge, and relatively healthy groupers (e and f). Red arrow indicates abdominal concavity, and blue arrow indicates flattened intestine.

Figure 2

Optimized primer screening results for E. epinepheli. M: Maker D2000, 1~24: primer pairs from group 1 to 24 in Table S1.

Figure 3

Optimization of ERA reaction condition for E. epinepheli detection. (A): RT-ERA temperature optimization test. (B): ERA-LFD temperature optimization test. (C): ERA-LFD time optimization test. Three replicates were performed for each RT-ERA temperature. In ERA-LFD, a blue control line indicates a negative result, while both a blue control and red test line indicate a positive result. Reaction intensity is determined by the test line’s red color intensity. NTC, Negative control.

Figure 4

(A): Sensitivity testing results for E. epinepheli PCR sensitivity test. (B): Basic ERA sensitivity test. (C): RT-ERA sensitivity test. (D): RT-ERA standard curve. (E): ERA-LFD sensitivity test. M: Marker D2000. NTC: negative control.

Figure 5

(A): Specificity testing results for E. epinepheli RT-ERA specific experiment. (B): ERA-LFD specific experiment. PDD, Photobacterium damselae subsp. damselae; VNN: viral nervous necrosis; SGIV: Singapore grouper iridovirus; NTC: negative control.