Development of a sensitive and quantitative assay for spring viremia of carp virus based on real-time RT-PCR

Abstract

A one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) using a TaqMan probe to quantitatively detect spring viremia of carp virus (SVCV) is described. In this assay, a pair of primers amplifying an 81-bp DNA fragment and a TaqMan probe was designed targeting the conserved region at the SVCV glycoprotein ( G) gene. To avoid the disadvantages arising from plasmids, an extension adding a T7 phage polymerase promoter to the 5′ end of the antisense primer was carried out to obtain viral cRNA. Standardized cycle threshold (Ct) values for 10-fold serial dilutions of SVCV cRNA were achieved by real-time RT-PCR and used to create standard curves. A regression line between the mean Ct values and viral template concentrations over a 1:10 7 dilution range with an r 2 value (0.9916) and a slope (−3.36) and the coefficient of variation (intra- or inter-assay is <2% and <4%, respectively) indicated that the assay was highly reproducible. The assay was specific to SVCV and there was no cross-reactivity with other fish viruses (viral hemorrhagic septicemia virus, VHSV; infectious pancreatic necrosis virus, IPNV; grass carp reovirus, GCRV; epizootic haematopoietic necrosis virus, EHNV). The standard curve allows precise absolute quantitation and shows that the detection limit of the assay is 40 copies of the viral RNA. This one-step RT-PCR assay was evaluated using 60 clinical common carp samples collected during the year 2006, indicating such technology offers considerable advantages over conventional RT-PCR methods in current routine use for SVCV surveillance. This is the first report of the development of a one-step TaqMan ® RT-PCR for SVCV detection.