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 2.6 DNA copies/l 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 is highly sensitive, specific, reproducible and versatile for the detection and quantification of Lymphocystivirus, and may also be used for asymptomatic carrier detection or pathogenesis studies of different LCDV strains.

Ciulli, S., Pinheiro, A.C.d.A.S., Volpe, E., Moscato, M., Jung, T.S., Galeotti, M., et al. (2015). Development and application of a real-time PCR assay for the detection and quantitation of lymphocystis disease virus. JOURNAL OF VIROLOGICAL METHODS, 213, 164-173 [10.1016/j.jviromet.2014.11.011].

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

CIULLI, SARA;VOLPE, ENRICO;FARNETI, RICCARDO;PROSPERI, SANTINO;DE AGUIAR SALDANHA PINHEIRO, ANA CRISTINA
2015

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 2.6 DNA copies/l 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 is highly sensitive, specific, reproducible and versatile for the detection and quantification of Lymphocystivirus, and may also be used for asymptomatic carrier detection or pathogenesis studies of different LCDV strains.
2015
Ciulli, S., Pinheiro, A.C.d.A.S., Volpe, E., Moscato, M., Jung, T.S., Galeotti, M., et al. (2015). Development and application of a real-time PCR assay for the detection and quantitation of lymphocystis disease virus. JOURNAL OF VIROLOGICAL METHODS, 213, 164-173 [10.1016/j.jviromet.2014.11.011].
Ciulli, Sara; Pinheiro, Ana Cristina de Aguiar S.aldana; Volpe, Enrico; Moscato, Michele; Jung, Tae S.ung; Galeotti, Marco; Stellino, Sabrina; Farneti...espandi
File in questo prodotto:
Eventuali allegati, non sono esposti

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/522637
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? 6
  • Scopus 26
  • ???jsp.display-item.citation.isi??? 21
social impact