Development and validation of TaqMan-based real-time PCR assays for translucent post larvae disease (TPD) detection in Penaeus vannamei.

Translucent Post larvae Disease (TPD), caused by Vibrio parahaemolyticus, has emerged as a critical disease impacting shrimp hatcheries across several Asian countries. This disease severely affects the early larval stages and can lead up to 100 % mortality. Thus, controlling TPD is essential to reduce losses in shrimp hatcheries. Although PCR-based assays have recently been reported for the detection of TPD-causing V. parahaemolyticus (VpTPD), these methods have not yet undergone comprehensive validation in accordance with the Aquatic Animal Health Code of the World Organization for Animal Health (WOAH). In this study, we developed TaqMan real-time PCR assays targeting three candidate virulence genes of VpTPD (i.e., vhvp-1, -2, and - 3) following the WOAH guidelines for diagnostic assay development. All three assays demonstrated 100 % diagnostic sensitivity (DSe) and specificity (DSp), with analytical sensitivity (i.e., limit of detection, LOD) of 10 copies per reaction. Experimental challenges were conducted using VpTPD and Specific Pathogen Free (SPF) Penaeus vannamei shrimp. We evaluated the performance of the newly developed assays in comparison to a published protocol. The results confirmed that the newly developed assays reliably detected VpTPD in infected P. vannamei. The availability of a validated real-time PCR assay for detecting VpTPD will provide a valuable diagnostic tool for screening P. vannamei stocks prior to domestic or international movement, thereby supporting biosecurity measures and helping to prevent the spread of disease beyond its current geographic boundary.