Detection of Metschnikowia bicuspidata in Chinese Mitten Crabs (Eriocheir sinensis) Using Recombinase Polymerase Amplification.

The fungal pathogen Metschnikowia bicuspidata causes "milky disease" in the Chinese mitten crab (Eriocheir sinensis), which poses substantial challenges to sustainable aquaculture development considering the current lack of effective treatment interventions. To address this issue, in laboratory validation, we developed two rapid recombinase polymerase amplification (RPA) detection methods for M. bicuspidata in E. sinensis targeting the histone acetyltransferase B-type subunit 2 gene (HAT-B2): an electrophoretic assay (RPA-AGE) and a colloidal gold lateral flow dipstick assay (RPA-LFD). We optimized RPA-AGE and RPA-LFD protocols for specific pathogen detection. Target detection was achieved within 35 min using RPA-AGE (30 min amplification at 37 °C followed by 5 min agarose gel electrophoresis), whereas RPA-LFD provided results in 15 min with high specificity (10 min amplification at 37 °C plus 5 min strip reading). Both methods exhibited exclusive specificity to M. bicuspidata, with no cross-reactivity with six pathogens, including Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, Candida albicans, Saccharomyces cerevisiae, and Microsporidia sp. The detection sensitivity of both platforms reached 4.8 copies/μL in laboratory validation. For field testing, the detection results from 30 field samples showed that although the 70% detection rate was lower than the 83.3% achieved by quantitative PCR, these approaches surpassed the detection rate of conventional PCR (53.3%). Notably, the RPA-LFD platform is applicable under field conditions as no specialized equipment is required. These rapid, sensitive, and specific detection systems provide practical tools for the early diagnosis and containment of M. bicuspidata infections in aquaculture settings.