Real-time PCR assay for sensitive organ detection and epidemic investigation of Turbot reddish body iridovirus

Abstract

A rapid and sensitive Real-time PCR assay coupled with SYBR Green Ⅰchemistry was developed for the quantitative detection of Turbot reddish body iridovirus (TRBIV) isolated from farmed turbot (Scophthalmus maximus). A 152 bp DNA fragment from TRBIV major capsid protein (MCP) gene was involved in the Real-time PCR assay using the Roter Gene 3000 sequence detection system. The PCR mixture contained a fluorescence dye, SYBR Green Ⅰ, which binding to dsDNA exhibited fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. The positive control plasmid pUCm-T/TRBIV MCP containing the target sequence was quantified to make the standard curve for sample detection after serial 10-fold dilution. Linear coefficient correlations between cycle threshold (CT) value and logarithmic positive plasmid concentration were close to one (r2=0.9952) and the detection limit of the assay was 102 copies of positive plasmids. The quantitative detection of different tissues from TRBIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (5.23×106 and 2.18×106 viral genome copies/mg tissue, respectively) while no viral DNA was detected in the muscular tissue. The molecular epidemic investigation of TRBIV showed that many cultured turbots were infected and TRBIV has been spread widely in turbot farms locating at Shandong peninsula. The virus number was varied from 1.27×102 to 2.33×106 of viral genome copies/mg tissue in spleen of infected turbot. These results suggest that the Real-time PCR assay reported here can be used as a rapid, sensitive and quantitative method for TRBIV.