Abstract:Enterocytozoon hepatopenaei (EHP) has caused serious losses in shrimp aquaculture in China in recent years. For cultured shrimp, disease prevention cannot be carried out by vaccination because of the lack of specific immune mechanisms. The most effective measure to prevent disease outbreaks is to conduct on-site rapid pathogen detection as far as possible for relatives or seedlings or to carry out on-site detection early in the onset of shrimp, timely detection, and identification of pathogens. There is, therefore, an urgent demand for rapid detection techniques and kits for controlling and preventing EHP. A novel, highly sensitive kit, developed in our laboratory, can achieve rapid detection of EHP in the field by optimizing the three steps of tissue nucleic acid extraction, nucleic acid amplification, and nucleic acid detection, and provide a practical solution for early rapid screening and detection of shrimp EHP disease. To validate the newly developed highly sensitive kit for rapid EHP detection in the field, a systematic evaluation of the six performance parameters of the kit was carried out in this study. The analytical specificity results showed that the kit did not cross-react with the DNA/RNA extracted from healthy shrimp or shrimp infected with five other pathogens, including WSSV, CMNV, SHIV, VpAHPND, and IHHNV. The analytical sensitivity analysis showed that the detection limit was 101 copies/μL with shrimp DNA preparation. The diagnostic sensitivity and diagnostic specificity were determined to be 91.7% and 99.2%, respectively, in tests of 298 clinical samples, in comparison with the TaqMan qPCR protocol of EHP. The repeatability was 100% for strongly positive and negative samples and 95.8% for weakly positive samples. The period of validity of the kit was tested and found to be over 7 months at –20℃ storage and over 12 months at –40℃. The study results demonstrated that the kit is a simple, sensitive, specific, and accurate tool for the rapid detection of EHP in practical applications.
