Enterocytozoon hepatopenaei real-time and Shrimp MultiPath™ PCR assay validation for South-East Asian and Latin American strains of Penaeid shrimp

Enterocytozoon hepatopenaei (EHP) infections are a global challenge for the Penaeid shrimp industry with a sharp rise in prevalence over the last 10 yr. EHP is known to cause sub-optimal growth, large size variation and reduced survival of shrimp. Molecular methods development has mainly focussed on 18S rRNA or spore wall protein 1 (SWP1). Due to the specificity and sensitivity issues with previously designed assays for both targets, new molecular assays are needed by the global shrimp industry and regulators to help manage the risks posed by EHP. This paper describes new real-time PCR (qPCR) methods developed for the novel EHP gene targets polar tube protein 2 (PTP2) and spore wall protein 26 (SWP26), whilst also presenting performance metrics of the new Shrimp MultiPath (TM) technology EHP assay. qPCR assays PTP2G and SWP26G show high amplification efficiency, a limit of detection (LOD) of between 1 and 4 copies, low assay variation and high diagnostic sensitivity (DSe) and specificity (DSp) compared to imperfect reference assays. Similar performance is seen with Shrimp MultiPath (TM) EHP showing an LOD of 8 copies, low assay variation and high DSe and DSp. These novel molecular targets for EHP and Shrimp MultiPath (TM) EHP strengthen global efforts to monitor and mitigate risks of EHP infections and outbreaks. Moreover, this study presents novel data on distribution of EHP in shrimp populations from South-East Asia and Latin America, and how sequence variations need to be considered when monitoring EHP in different geographies.