Development and application of a real-time PCR assay for the detection and quantitation of lymphocystis disease virus

• Published in: Journal of virological methods Vol. 213; pp. 164 - 173
• Main Authors: Ciulli, Sara, Pinheiro, Ana Cristina de Aguiar Saldana, Volpe, Enrico, Moscato, Michele, Jung, Tae Sung, Galeotti, Marco, Stellino, Sabrina, Farneti, Riccardo, Prosperi, Santino
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2015
• Subjects: Animals; Carrier detection; carrier state; cell culture; detection limit; DNA Primers - genetics; DNA Virus Infections - diagnosis; DNA Virus Infections - veterinary; DNA Virus Infections - virology; DNA, Viral - chemistry; DNA, Viral - genetics; Fish Diseases - diagnosis; Fish Diseases - virology; Fishes; flounder; genes; Genotype; Genotyping; Iridoviridae - isolation & purification; Lymphocystis disease virus; Lymphocystivirus; Molecular Diagnostic Techniques - methods; Molecular Sequence Data; MPC gene; Paralichthys olivaceus; pathogenesis; plasmids; Pleuronectiformes; quantitative polymerase chain reaction; Real-time PCR; Real-Time Polymerase Chain Reaction - methods; Reproducibility of Results; Sensitivity and Specificity; Sequence Analysis, DNA; Sparus aurata; Teleostei; tissues; Veterinary Medicine - methods; viruses; MPC gene; Lymphocystis disease virus; Carrier detection; Real-time PCR; Genotyping; Sparus aurata
• ISSN: 0166-0934; 1879-0984; 1879-0984
• DOI: 10.1016/j.jviromet.2014.11.011

•A qPCR assay was developed to detect and quantify lymphocystis disease virus (LCDV)•Primers were designed on a multiple alignment that included all known LCDV genotypes•The first S. aurata Italian LCDV strain was identify as genotype VII•The qPCR assay showed high sensitivity to quantify LCDV in S. aurata and P. olivaceus•Naturally infected S. aurata subjects showed a systemic and persistent infection 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.