The use of formulated aquafeed shows potential as a safer option than fresh feed for large-scale farming of crustaceans and fin fish, offering numerous advantages. However, the current limitations of PCR-based detection methods for EHP and other crustacean pathogens hinder their widespread application for testing a diverse range of aquafeed and feed ingredients. In response to this challenge, we are developing a robust and validated PCR-based diagnostic assay for detecting EHP in aquafeed. This assay, once fully developed, will play a crucial role in enabling disease-free certifications of formulated aquafeeds, thereby ensuring the health and well-being of farmed shrimp.
Experimental shrimp diets were produced by incorporating EHP-infected tissue at 0.2% and 2% (w/w) in commercial-type feed formulation before extrusion. To find an optimal method of DNA isolation from aquafeed, total genomic DNA was isolated using four different methods. DNA polymerase gene was selected as a target gene to develop a PCR protocol for EHP detections since the polymerase gene plays a vital role in EHP replication. Four sets of primers/probes (corresponding to three conventional PCR protocols and one real-time PCR protocol) were developed. Three independent assays were conducted for each primer pair to determine the limit of detection (LOD). The LOD was 100 copies/reaction and 10 copies/reaction for conventional PCR and real-time PCR assays, respectively. The newly developed primers and probes detected only EHP, and no other pathogens known to infect crustaceans, in the specificity test.
The PCR assay using EHP polymerase as a target gene for PCR amplification demonstrates an unparalleled sensitivity and specificity in detecting EHP in formulated aquafeed. Since polymerase is a critical gene in EHP replication, a lack of amplification of polymerase would indicate that aquafeed does not contain infectious EHP. The EHP detection method described here provides an effective means of assessing biosecurity risk of formulated aquafeed and feed ingredients. By providing a rapid and robust means to evaluate the infectivity of aquafeed products, we empower industry stakeholders to make more informed choices, reduce waste, and optimize the utilization of resources, ultimately benefiting both the environment and economic sustainability of formulated feed used in shrimp farming.