Development of a Melting Curve-Based Triple Eva Green Real-Time PCR Assay for Simultaneous Detection of Three Shrimp Pathogens

Simple Summary Shrimp aquaculture is an important industry due to its advantages of short production cycles and good economic benefits, but it faces challenges from diseases caused by tiny organisms that can lead to slow growth or large-scale shrimp deaths. Three such harmful pathogens are Enterocytozoon hepatopenaei (EHP), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Decapod iridescent virus 1 (DIV1). It is essential to quickly identify these organisms. In this study, we created a new test that can detect all three pathogens at once by using a melting curve-based Eva Green real-time PCR assay. We used this test on 190 shrimp samples from different regions in China and found that a significant number of shrimp were affected by these organisms. Our test is as accurate as the current standard test but faster because it looks for all three organisms simultaneously. This new method can help shrimp farmers identify diseases early, which is crucial for treating sick shrimp and preventing the spread of disease. This work is valuable because it offers a better way to keep shrimp healthy, which benefits the shrimp industry and supports aquaculture biosecurity.Abstract Infections with Enterocytozoon hepatopenaei (EHP), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Decapod iridescent virus 1 (DIV1) pose significant challenges to the shrimp industry. Here, a melting curve-based triple real-time PCR assay based on the fluorescent dye Eva Green was established for the simultaneous detection of EHP, IHHNV, and DIV1. The assay showed high specificity, sensitivity, and reproducibility. A total of 190 clinical samples from Shandong, Jiangsu, Sichuan, Guangdong, and Hainan provinces in China were evaluated by the triple Eva Green real-time PCR assay. The positive rates of EHP, IHHNV, and DIV1 were 10.5%, 18.9%, and 44.2%, respectively. The samples were also evaluated by TaqMan qPCR assays for EHP, DIV1, and IHHNV, and the concordance rate was 100%. This illustrated that the newly developed triple Eva Green real-time PCR assay can provide an accurate method for the simultaneous detection of three shrimp pathogens.