Formulated aquafeed holds promise as a safer alternative to fresh feed, offering numerous advantages in large-scale farming of crustacean and fin fish . However, the current limitations of PCR-based detection methods hinder their broad application for testing a diverse range of aquafeed and feed ingredients. In response to this challenge, our objective is to develop a robust and validated PCR-based diagnostic assay for detecting WSSV in aquafeed . Th is assay will play a pivotal role in facilitating disease-free certifications of formulated aquafeeds , thereby ensuring the health and well-being of farmed aquatic animals.
Experimental shrimp feeds were produced by incorporating WSSV-infected tissue containing a viral load of 3.07 x 107 copies/mg of tissue into a commercial-type shrimp feed formulation prior to extrusion . In order to find an optimal method of DNA isolation from aquafeed, total genomic DNA was isolated using f our different methods. T he viral polymerase gene was selected as a target gene to develop a PCR protocol for WSSV detections since the polymerase gene plays an important role in WSSV replication . Four sets of primers/probes (corresponding to three conventional PCR protocols and one real-time PCR protocol) targeting the polymerase gene of WSSV were developed. The specificity and limit of detection of the newly developed PCR protocols were determined. Diagnosis sensitivity (Dse) and Diagnosis specificity (Dsp ) of the PCR methods were determined using known WSSV-spiked feed .
The newly developed primers and probes detected only WSSV in the specificity test. To determine the limit of detection (LOD), three independent assays were conducted for each primer pair. The LOD was 100 copies/reaction and 10 copies/reaction for conventional PCR and real-time PCR assays, respectively.
The PCR assay using WSSV p olymerase as a target gene for PCR amplification has a high sensitivity and specificity in detecting WSSV in formulated aquafeed. Since polymerase is a critical gene in viral replication, a lack of amplification of polymerase would indicate that aquafeed does not contain infectious WSSV. The WSSV detection method described here can provide an effective means of determining the infectivity of aquafeed products. By providing a 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.