Comparison of a novel in situ polymerase chain reaction (ISPCR) method to other methods for white spot syndrome virus (WSSV) detection in Penaeus vannamei

Penaeus vannamei were experimentally injected with white spot syndrome virus (WSSV) and tested for WSSV at different times post-injection (p.i.) by 1-step polymerase chain reaction (PCR), 2-step PCR, in situ hybridization (ISH) and in situ polymerase chain reaction (ISPCR) in order to compare sensitivity of the methods. With 1-step PCR, 4 of 15 shrimp tested positive for WSSV at 12 h p.i., and all tested positive by 24 h p.i. With 2-step PCR, 13 out of 15 samples tested positive at 2 h p.i. and all were positive by 4 h p.i. Using in situ hybridization, 1 sample tested positive at 18 h p.i. and all were positive by 36 h p.i. With ISPCR, 1 out of 5 samples was positive at 2 h p.i. and all were positive by 8 h p.i. Two-step PCR showed the highest sensitivity, followed by ISPCR, 1-step PCR and ISH. Although ISPCR revealed WSSV in 9 of 10 P. vannamei that tested positive for WSSV using 2-step PCR, none of the shrimp examined showed clinical signs of WSSV infection or detectable WSSV with 1-step PCR. The major infected organs were muscle and the hepatopancreas.