A non-destructive method based on the polymerase chain reaction for detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp

Current methods to detect hepatopancreatic parvovirus (HPV) infection of pe naeid shrimp depend on invasive techniques that require dissecting the orga ns infected by this virus. However, sacrificing valuable stocks in order to determine their HPV status can be a drawback in the case of breeding progr ams. A method was developed for HPV detection by applying a polymerase chai n reaction (PCR) assay to fecal samples collected from live HPV-infected sh rimp Penaeus chinensis. A pair of PCR primers, 1120F/1120R, which amplify a 592 base pair (bp) region from the virus genome, was designed from previou sly known HPV sequence information (HPV clone HPV8). PCR amplification with these primers generated a product of the expected size directly from the c rude feces of HPV-infected shrimp but not from the feces of specific pathog en-free (SPF) shrimp. The HPV origin of the amplified product was validated by means of an in situ hybridization assay where the product of the amplif ication, labeled with digoxigenin (DIG)-11-dUTP, showed an intense reaction within hepatopancreatic cells displaying characteristic HPV lesions on HPV -infected shrimp. No reaction to this probe was observed when reacted in si tu with sections of the hepatopancreas of SPF specimens or to sections of s hrimp infected by the infectious hypodermal and hematopoietic necrosis viru s (IHHNV), another parvovirus of penaeid shrimp. These primers were tested for specificity against homologous and nonhomologous viruses and no product was amplified. A fragment of the expected size was obtained only when puri fied HPV or purified HPV8 plasmid was used as template DNA. Under optimized conditions, these primers detected as little as 1 fg of purified HPV8 plas mid DNA, equivalent to approximately 300 HPV particles. Analysis of fecal s amples by PCR may prove useful for non-lethal screening of valuable shrimp of unknown HPV status. This same strategy also might be used for detection of other enteric viruses that infect penaeid shrimp.