Development and validation of a two-step real-time RT-PCR for the detection of eel virus European X in European eel, Anguilla anguilla.
Eel virus European X (EVEX) is one of the most common pathogenic viruses in farmed and wild European eel (Anguilla anguilla) in the Netherlands. The virus causes a hemorrhagic disease resulting in increased mortality rates. Cell culture and antibody-based detection of EVEX are laborious and time consuming. Therefore, a two-step real-time reverse transcriptase (RT-)PCR assay was developed for rapid detection of EVEX. Primers and probe for the assay were designed based on a sequence of the RNA polymerase or L gene of EVEX. The real-time RT-PCR assay was validated both for use with SYBR Green chemistry and for use with a TaqMan probe. The assay is sensitive, specific, repeatable, efficient and has a high r²-value. The real-time RT-PCR assay was further evaluated by testing field samples of European eels from the Netherlands, which were positive or negative for EVEX by virus isolation followed by an indirect fluorescent antibody test. The real-time RT-PCR assay allows rapid, sensitive and specific laboratory detection of EVEX in RNA extracts from 10% eel organ suspensions and cell cultures with cytopathic effects, and is a valuable contribution to the diagnosis of viral diseases of eel.
Laboratory for Fish and Shellfish Diseases, Central Veterinary Institute, part of Wageningen UR, P.O. Box 65, 8200 AB Lelystad, The Netherlands.
Steven J
SJ
van Beurden
Michal A
MA
Voorbergen-Laarman
Ineke
I
Roozenburg
Annette S
AS
Boerlage
Olga L M
OL
Haenen
Marc Y
MY
Engelsma
D000811:Anguilla / Q000821:virology*; D000818:Animals; D052160:Benzothiazoles; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D009426:Netherlands; D015345:Oligonucleotide Probes / Q000235:genetics; D009930:Organic Chemicals; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012209:Rhabdoviridae / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D015345:Oligonucleotide Probes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I