Detection and quantification of infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp by real-time PCR

A real-time PCR method using a fluorogenic 5' nuclease assay and a PE Appli ed Biosystems GeneAmp 5700 sequence detector was developed to detect infect ious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp. A pair of PCR primers to amplify an 81 bp DNA fragment and a fluorogenic p robe (TaqMan probe) were selected from ORF1 (open reading frame 1) of the I HHNV genome. The primers and TaqMan probe used in this assay were shown to be specific for IHHNV and did not react with either hepatopancreatic parvov irus (HPV), white-spot syndrome virus (WSSV), or shrimp DNA. A plasmid, pIH HNV-P4, containing the target IHHNV sequence was constructed and used as a positive control. The concentration of pIHHNV-P4 was determined through spe ctrophotometric analysis and the plasmid was used for quantitative studies. This real-time PCR assay had a detection limit of 10 copies and a log-line ar range up to 5 x 10(7) copies of IHHNV DNA. The assay was then used to qu antify IHHNV in infected shrimp collected from 5 locations: Hawaii, Panama, Mexico, Guam, and the Philippines. The quantitative analysis showed that w ild-caught, large juvenile Penaeus stylirostris collected from the Gulf of California (Mexico) in 1996 were naturally infected with IHHNV and containe d up to 10(9) copies of IHHNV mug(-1) of DNA. Similar quantities of IHHNV w ere detected in hatchery-raised, small juvenile P. stylirostris collected f rom Guam in 1995 and in farm-raised, post-larval P. monodon from the Philip pines in 1996. Laboratory-infected P. stylirostris contained approximately 10(8) copies of IHHNV 31 d after being fed with IHHNV-infected shrimp tissu e. In contrast, individuals of Super Shrimp(R), a line of P. stylirostris s elected for IHHNV resistance, showed no signs of infection 32 d after inges ting IHHNV-infected shrimp tissue. Laboratory-infected P, vannamei also con tained approximately 10(8) copies of IHHNV 30 d after being fed infected sh rimp tissue. A time-course study of IHHNV replication in juvenile P. vannam ei showed that the doubling time in the exponential growth phase was approx imately 22 h.