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

Abstract

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 inP. 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 theE. artemiaeS8 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 ofE. artemiaebut 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 ofE. artemiaeinP. sinensisfarms without the requirement of experimental equipment, which can facilitate the prevention and control of this microsporidial disease.