Vet. Res. 34 (2003) 165-176
DOI: 10.1051/vetres:2002063

Use of an internal standard in a closed one-tube RT-PCR for the detection of equine arteritis virus RNA with fluorescent probes

David G. Westcott^a, Donald P. King^a, Trevor W. Drew^a, Norbert Nowotny^{b, c}, Johanna Kindermann^b, Duncan Hannant^d, Sándor Beláke^a and David J. Paton<sup>a

a</sup>  Department of Virology, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey, KT15 3NB, UK
^b  Clinical Virology Group, Institute of Virology, University of Veterinary Medicine, Vienna, Veterinärplatz, 1210, Vienna, Austria
^c  Department of Medical Microbiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, UAE
^d  Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU, UK

(Received 12 July 2002; accepted 7 October 2002)

Abstract
Routine detection of equine arteritis virus (EAV) can be achieved by virus isolation (VI) in cell culture, or by the amplification of viral genome by molecular methods. To simplify molecular diagnosis, a number of different Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and RT-nested PCR (RT-nPCR) assays were compared, and a one-tube method was developed and optimised utilizing a fluorogenic probe (TaqMan®). An artificial RNA template (Mimic) and associated probe were also constructed to provide in-tube validation of the RT-PCR system. To assess the utility of the RT-PCR TaqMan® assay, 28 different isolates of EAV representing different genetic groups of American and European strains were tested. Furthermore, the ability of VI and RT-PCR TaqMan® assay to detect EAV in different biological matrices such as semen, nasal and faecal swabs and blood was compared. All 28 EAV strains were detected by the RT-PCR TaqMan® assay. The results of TaqMan® and VI testing were in agreement for 30 of the 33 semen samples and all of the 50 other clinical specimens examined: the RT-PCR TaqMan® assay detected 18 positive semen samples, three more than VI. In conclusion, the one-tube RT-PCR TaqMan® assay is a rapid, reliable method for the detection of EAV.

Key words: equine arteritis virus / RT-PCR / mimic / fluorescent probe / TaqMan®

Correspondence and reprints: David G. Westcott Tel.: 44 (0) 1932 357440; fax: 44 (0) 1932 357239;
    e-mail: d.g.westcott@vla.defra.gsi.gov.uk

© INRA, EDP Sciences 2003