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

Published online by Cambridge University Press:  24 April 2009

Wu Cheng-Long ,
Shi Cheng-Yin ,
Huang Jie  and
Kong Xiao-Yu
Wu Cheng-Long
Affiliation:
Key Laboratory for Sustainable Utilization of Marine Fisheries Resource, Ministry of Agriculture; Yellow Sea Fisheries Research Institute, Qingdao 266071, China Key Laboratory of Mariculture, Ministry of Education; Ocean University of China, Qingdao 266003, China
Shi Cheng-Yin*
Affiliation:
Key Laboratory for Sustainable Utilization of Marine Fisheries Resource, Ministry of Agriculture; Yellow Sea Fisheries Research Institute, Qingdao 266071, China
Huang Jie
Affiliation:
Key Laboratory for Sustainable Utilization of Marine Fisheries Resource, Ministry of Agriculture; Yellow Sea Fisheries Research Institute, Qingdao 266071, China
Kong Xiao-Yu
Affiliation:
Key Laboratory of Mariculture, Ministry of Education; Ocean University of China, Qingdao 266003, China
*
*Corresponding author. E-mail: shicy@ysfri.ac.cn
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Abstract

A rapid and sensitive real-time polymerase chain reaction (PCR) assay coupled with SYBR Green I chemistry was developed for the quantitative detection of Turbot reddish body iridovirus (TRBIV) isolated from farmed turbot (Scophthalmus maximus). A 152 bp DNA fragment from the TRBIV major capsid protein (MCP) gene was involved in the real-time PCR (RT-PCR) assay using the Roter Gene 3000 sequence detection system. The PCR mixture contained a fluorescent dye, SYBR Green I, which exhibited fluorescence enhancement when bound to double-stranded (ds) DNA. 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 a standard curve for sample detection after serial tenfold dilution. Linear coefficient correlations between the 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 virus in 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 become epidemic in turbot farms located along the Shandong peninsula. The virus number varied from 1.27×102 to 2.33×106 viral genome copies/mg tissue in spleens of infected turbot. These results suggest that the RT-PCR assay reported here can be used as a rapid, sensitive and quantitative method for TRBIV.

Keywords

Turbot reddish body iridovirus (TRBIV)real-time PCRSYBR Green Iquantitative detectionsensitive organ
Type
Research Papers
Information
Chinese Journal of Agricultural Biotechnology , Volume 6 , Issue 1 , April 2009 , pp. 61 - 67
Copyright
Copyright © China Agricultural University 2009

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