A Multiplex PCR Approach for Accurate Detection of Key Pathogens to Improve Disease Surveillance in Shrimp Aquaculture

Background: White spot syndrome virus (WSSV), Infectious hypodermal and haematopoietic necrosis virus (IHHNV) and Enterocytozoon hepatopenaei (EHP) are widely prevalent diseases affecting cultured and wild shrimp. These diseases lead to significant production and economic losses. There have been reports of co-infection of WSSV with IHHNV, WSSV with EHP and EHP with IHHNV. Since there are no proper management and treatment methods, early and timely detection of diseases is crucial. This study aimed to develop a multiplex PCR assay for timely and accurate diagnosis of WSSV, IHHNV and EHP, which is essential for minimizing production and economic losses in the shrimp aquaculture industry. Methods: This study involves the screening and confirmation of penaeid shrimp samples for diseases viz, WSSV, IHHNV and EHP, designing of PCR primers, optimization of PCR parameters such as annealing temperature, primer concentration and validation of the assay. Result: The multiplex PCR assay detected WSSV, IHHNV and EHP in a single PCR reaction. Analytical sensitivity was found to be 100 pg/µl of WSSV, 100 pg/µl of IHHNV and 10 pg/µl of EHP. Diagnostic sensitivity of the developed assay was 100%. Multiplex PCR assay developed in this study can be used for the screening of broodstock, post larvae, live food and environmental samples for early detection of WSSV, IHHNV and EHP.