Validation of a qPCR assay for the detection of Ictalurid herpesvirus-2 (IcHV-2) in fish tissues and cell culture supernatants.
Ictalurid herpesvirus-2 (IcHV-2) is a pathogen of cultured black bullhead, Ameiurus melas (Rafinesque), and has been shown to produce high mortality in experimental exposures of channel catfish, Ictalurus punctatus (Rafinesque). During acute infections, the virus grows readily in cell cultures but produces a cytopathic effect (CPE) similar to that of Ictalurid herpesvirus-1 (IcHV-1) and the channel catfish reovirus. We have developed a quantitative PCR assay that can be used to detect IcHV-2 in fish tissues and cell culture supernatants. The assay does not amplify other fish herpesviruses tested or host DNA. It is quantitative over a range of eight logs, and the limit of detection is <10 copies per reaction. In replicate assays carried out on different days, the coefficient of variability was 10%. The best organs for the detection of acute IcHV-2 infections by our assay are the spleen and kidney. This assay should be useful for the diagnosis of IcHV-2 disease, the identification of syncytial CPEs in cell cultures, and for the detection of latent infections in carrier fish.
Goodwin
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University of Arkansas at Pine Bluff Aquaculture/Fisheries Center, Pine Bluff, AR 71601, USA. agoodwin@uaex.edu
Marecaux
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D000818:Animals; D001483:Base Sequence; D002397:Catfishes / Q000821:virology; D002478:Cells, Cultured; D003470:Culture Media / Q000032:analysis; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D052656:Ictalurivirus / Q000235:genetics / Q000302:isolation & purification*; D007668:Kidney / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013154:Spleen / Q000821:virology
D003470:Culture Media