Rapid Detection of Enterocytozoon hepatopenaei Infection in Shrimp With a Real-Time Isothermal Recombinase Polymerase Amplification Assay

Hepatopancreas microsporidian was first discovered in Penaeus monodon in Thailand in 2002, also known as monodon slow growth syndrome. Years later, this fungal microsporidian parasite was classified within the family Enterocytozoonidae called Enterocytozoon hepatopenaei (EHP). It is becoming a major danger to the shrimp aquaculture sector due to the lack of unambiguous pathognomonic signs other than slow growth rate and wide size variation upon visual inspection. Although EHP does not cause significant mortality in shrimp as infectious diseases, it results in economic losses due to growth retardation and lower productivity. Many studies have lately been undertaken on how to detect EHP infection in larval shrimp, feed, and water sources which are light microscopy, histological examination, in situ hybridization and PCR, but improvements on detection speed and convenience are still in demand. In this article, the authors develop a technique using isothermal recombinase polymerase amplification (RPA) assay derived from the recombination-dependent DNA replication (RDR) mechanism of bacteriophage T4. This study combined fluorescence analysis with the RPA assay and offered a real-time RPA assay for the detection of EHP. Impressively, the detection procedure can be accomplished in 3–7 min at 39°C and showed good specificity, which is comparable to the current PCR- and LAMP-based methods.