Rapid detection of Enterocytospora artemiae in Chinese grass shrimp (Palaemonetes sinensis) through isothermal recombinase polymerase amplification

Enterospora artemiae , an obligate intracellular parasitic microsporidium, severely affects the development of Chinese grass shrimp ( Palaemonetes sinensis ) aquaculture. Currently, no effective drugs or vaccines are available for treatment. To improve the diagnosis and prevention of microsporidia infection in P. sinensis , two recombinase polymerase amplification (RPA) detection methods (visualized by electrophoresis [RPA-AGE] and a colloidal gold lateral flow dip-strip [RPA-LFD], respectively) were established based on the E. artemiae S8 serine protease gene. RPA-AGE showed optimal amplification at 37°C for 30 min, and amplification by RPA-LFD was completed in 10 min at 37°C and produced detection results within 5 min. Regarding specificity, both methods showed specific amplification of E. artemiae but not of other pathogens. Regarding sensitivity, the minimum detection limit for both RPA-AGE and RPA-LFD was 4.7 copies/μL. Using 30 clinical samples, the 70%-positive rate was lower than that of fluorescence quantitation, but accuracy was improved compared with conventional polymerase chain reaction-based amplification (56.7%). Our RPA-AGE and RPA-LFD methods showed high specificity and sensitivity, with short detection time. In particular, the RPA-LFD method can be used for simple on-site detection of E. artemiae in P. sinensis farms without the requirement of experimental equipment, which can facilitate the prevention and control of this microsporidial disease.