title: Development and application of a real-time PCR assay for the detection and quantitation of lymphocystis disease virus.
abstract: Lymphocystis disease virus (LCDV) is responsible for a chronic self-limiting disease that affects more than 125 teleosts. Viral isolation of LCDV is difficult, time-consuming and often ineffective; the development of a rapid and specific tool to detect and quantify LCDV is desirable for both diagnosis and pathogenic studies. In this study, a quantitative real-time PCR (qPCR) assay was developed using a Sybr-Green-based assay targeting a highly conserved region of the MCP gene. Primers were designed on a multiple alignment that included all known LCDV genotypes. The viral DNA segment was cloned within a plasmid to generate a standard curve. The limit of detection was as low as 2.6DNA copies/μl of plasmid and the qPCR was able to detect viral DNA from cell culture lysates and tissues at levels ten-times lower than conventional PCR. Both gilthead seabream and olive flounder LCDV has been amplified, and an in silico assay showed that LCDV of all genotypes can be amplified. LCDV was detected in target and non-target tissues of both diseased and asymptomatic fish. The LCDV qPCR assay developed in this study is highly sensitive, specific, reproducible and versatile for the detection and quantitation of Lymphocystivirus, and may also be used for asymptomatic carrier detection or pathogenesis studies of different LCDV strains.
authors.affiliation: Department of Veterinary Medical Sciences, University of Bologna, Viale Vespucci, 2, 47042 Cesenatico, FC, Italy. Electronic address: sara.ciulli@unibo.it.
authors.forename: Sara
authors.initials: S
authors.lastname: Ciulli
authors.affiliation: Department of Veterinary Medical Sciences, University of Bologna, Viale Vespucci, 2, 47042 Cesenatico, FC, Italy.
authors.forename: Ana Cristina de Aguiar Saldana
authors.initials: AC
authors.lastname: Pinheiro
authors.affiliation: Department of Veterinary Medical Sciences, University of Bologna, Viale Vespucci, 2, 47042 Cesenatico, FC, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Volpe
authors.affiliation: Panittica Pugliese, Str. Comunale del Procaccio, 72010 Torrecanne, BR, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Moscato
authors.affiliation: Aquatic Biotechnology Center of WCU Project Lab. of Aquatic Animal Diseases College of Veterinary Medicine, Gyeongsang National University, 900 Gazwadong, Jinju, Gyeongnam 660-701, South Korea.
authors.forename: Tae Sung
authors.initials: TS
authors.lastname: Jung
authors.affiliation: Department of Food Sciences, University of Udine, Via Sondrio, 2, 33100 Udine, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Galeotti
authors.affiliation: Cesena Campus, University of Bologna, Viale Vespucci, 2, 47042 Cesenatico, FC, Italy.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Stellino
authors.affiliation: Cesena Campus, University of Bologna, Viale Vespucci, 2, 47042 Cesenatico, FC, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Farneti
authors.affiliation: Department of Veterinary Medical Sciences, University of Bologna, Viale Vespucci, 2, 47042 Cesenatico, FC, Italy.
authors.forename: Santino
authors.initials: S
authors.lastname: Prosperi
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004266:DNA Virus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology*; D005399:Fishes; D005838:Genotype; D007497:Iridoviridae / Q000302:isolation & purification*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014730:Veterinary Medicine / Q000379:methods*
chemical_list: D017931:DNA Primers; D004279:DNA, Viral
keywords: Carrier detection; Genotyping; Lymphocystis disease virus; MPC gene; Real-time PCR; Sparus aurata