Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp

Abstract

Quantitative real time PCR, recently developed in molecular biology, is applied in this paper to quantify the white spot syndrome virus (WSSV) in infected shrimp tissue. The WSSV content in moribund shrimp of all species tested (Penaeus stylirostris, P. monodon, P. vannamei) ranged from 2.0 × 104 to 9.0 × 1010 WSSV copies μg–1 of total DNA (n=26). In whole moribund post‐larvae, 4.3 × 109 WSSV copies μg–1 of DNA were detected which is equivalent to 5.7 × 1010 WSSV copies g–1 of post‐larvae. The comparison of WSSV content between different tissues showed that muscle and hepatopancreas tissues contained 10 times less virus than gills, pleopods and haemolymph. With inocula of known virus content, bioassays by immersion challenge showed that a minimum of five logs of WSSV copies was necessary to establish disease in the challenged shrimp. In contrast, five logs of WSSV copies injected into shrimp muscle produced a LT‐50 of 52 h. This real time polymerase chain reaction (PCR) technique is sensitive (four copies), specific (negative with DNA from shrimp baculoviruses and parvoviruses), dynamic (seven logs) and easy to perform (96 tests in <4 h).