Rapid and sensitive detection of Enterocytozoon hepatopenaei in real-time recombinase polymerase amplification assay

Abstract

Enterocytozoon hepatopenaei (EHP) is a very serious pathogen in cultured shrimp, which brings huge economic losses to the shrimp industry every year. In this study, we established a real-time recombinase polymerase amplification (real-time RPA) assay based on the spore wall protein (SWP) gene sequence for the quantitative detection of the shrimp microsporidian EHP. The real-time RPA assay results indicated a high sensitivity and no cross-reaction with other aquatic pathogens. The limit of detection (LOD) was as low as 174 copies/μL in 95% of cases, as determined by probit analysis of eight independent experiments. The established standard curves showed correlation coefficient values (R2 = 0.9418) in the range of 1.0 × 100 to 1.0 × 108 copies/uL. Fifty samples of Litopenaeus vannamei were collected in shrimp farms and subjected to detection with real-time RPA and qPCR. Eight negative and 42 positive samples were detected by real-time RPA. Compared with those of qPCR (43/50), the diagnostic sensitivity and specificity of real-time RPA were 97.7% and 100%, respectively. Consequently, this rapid real-time RPA method has great application potential for field use or point-of-care diagnostics.