Abstract:To achieve a simple, rapid and efficient detection of two pathogens in shrimp products, a duplex fluorescent recombinase polymerase amplification(RPA) detection method was developed for infectious hypodermal and hematopoietic necrosis virus(IHHNV) and Enterocytozoon hepatopenaei (EHP). Primers and probes were designed based on the conserved sequences of IHHNV and EHP, and the duplex fluorescent RPA method was established. The specificity, sensitivity and repeatability of the method were validated. The results showed that the established method exhibited distinct amplification only for IHHNV and EHP. Detection using diluted plasmids of IHHNV and EHP demonstrated that the sensitivity of the duplex fluorescent RPA method for detecting IHHNV and EHP plasmids could reach 1×102 copies/μL. The method showed good repeatability with ten repeated plasmid samples. The method was employed to examine imported frozen shrimp samples, resulting in the discovery of one positive EHP and one positive IHHNV. Comparison of the detection results with fluorescent PCR showed consistency between the two methods. The method established in this research has important clinical significance for the distinguishing and detection of the two diseases, and can be applied to rapid screening methods for disease diagnosis in aquaculture farms, shrimp product production enterprises, and port laboratories.
