Development and validation of TaqMan quantitative PCR for detection of frog virus 3-like virus in eastern box turtles (Terrapene carolina carolina)

• Published in: Journal of virological methods Vol. 188; no. 1-2; pp. 121 - 125
• Main Authors: Allender, Matthew C., Bunick, David, Mitchell, Mark A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2013
• Subjects: Animals; blood; coat proteins; disease detection; disease outbreaks; disease surveillance; Epidemiology; fish; frogs; Molecular; mortality; plasmids; population; quantitative analysis; quantitative polymerase chain reaction; Ranavirus; Ranavirus - isolation & purification; Real-Time Polymerase Chain Reaction - methods; Reptile; Sensitivity and Specificity; turtles; Turtles - virology; Virology - methods; viruses; Reptile; Ranavirus; Molecular; Epidemiology
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2012.12.012

► qPCR is 1000 more sensitive than conventional PCR. ► Assay has similar efficiency in plasmid, cell lysates, and dilutions of whole turtle blood. ► Assay targets a conservative portion of the major capsid protein gene. Ranavirus has caused disease epidemics and mass mortality events globally in free-ranging fish, amphibian, and reptile populations. Viral isolation and conventional PCR are the most common methods for diagnosis. In this study, a quantitative real-time PCR (qPCR) assay was developed using a TaqMan probe-based assay targeting a highly conserved region of the major capsid protein of frog virus 3-like virus (FV3-like) (Family Iridoviridae, genera Ranavirus). Standard curves were generated from a viral DNA segment cloned within a plasmid. The assay detected viral DNA 1000 times lower than conventional PCR. Thirty-one clinical samples (whole blood and oral swabs) from box turtles were tested using these assays and the prevalence of the virus determined. Quantitative PCR allows for a superior, rapid, sensitive, and quantitative method for detecting FV3-like virus in box turtles, and this assay will be useful for early detection and disease monitoring.