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| Real-time PCR assay for sensitive organ detection and epidemic investigation of turbot reddish body iridovirus |
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Abstract A rapid and sensitive real-time PCR assay using the Roter Gene 3000 sequence detection system coupled with SYBR Green І chemistry was developed for the quantitative detection of turbot reddish body iridovirus (TRBIV) isolated from farmed turbot, Scophthalmus maximus. The assay involved the amplification of a 152 bp DNA fragment from a conserved region of TRBIV MCP gene. The PCR mixture contains a fluorescence dye, SYBR Green І, which upon binding to dsDNA exhibits 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=0.99806) 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 (5.23106 and 2.18106 viral genome copies/mg tissue, respectively) while no viral DNA was detected in the muscular tissue. The molecular epidemic investigation of TRBIV showed large number of cultured turbots were infected and TRBIV has been epidemic widely in turbot farms locating at Shandong peninsula. These results suggested that the real-time PCR assay reported here could be used as a rapid, sensitive and quantification method for TRBIV.
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Received: 22 January 2008
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