Quantitative detection of a marine fish iridovirus isolated from large yellow croaker, Pseudosciaena crocea, using a molecular beacon

Abstract

A real-time polymerase chain reaction (PCR) assay utilizing a molecular beacon for the quantitative detection of a marine fish iridovirus isolated from large yellow croaker, Pseudosciaena crocea (LYCIV), was developed, which involved the amplification of a 122 bp DNA fragment from a conserved region of LYCIV ATPase gene. The specific probe consisting of two short arm and a central loop sequences complementary to the target amplicon was characterized with respect to its efficiency of quenching (E-ff), and signal to background ratio by spectrofluorometric analysis of its hybridization with the complementary oligonucleotide target. The positive control plasmid pFHT-ATPase 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 (C-T) value and logarithmic positive plasmid concentration were close to one (r(2) = 0.998) and the detection limit of the assay was 70 copies of positive plasmid/assay. The specificity of this real-time PCR was also demonstrated by using the genomic DNA templates from the healthy fish, white spot syndrome baculovirus (WSSV), and epizootic heamatopietic necrosis virus (EHNV), respectively. The coefficient of variation (CV) of the assay ranged from 1.16 to 4.42%, depending on the concentration of the positive plasmid. The quantitative detection of different tissues from LYCIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (6.86 x 10(6) and 4.62 x 10(6) viral genome copies/mg tissue, respectively) while no viral DNA was detected in the muscular tissue. These results suggested that the real-time PCR assay reported here could be used for rapid, sensitive, and quantitative detection of LYCIV infection. (c) 2005 Elsevier B.V. All rights reserved.