Detection of equine arteritis virus by real-time TaqMan reverse transcription-PCR assay.
A one-tube real-time TaqMan reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of equine arteritis virus (EAV). The test was validated using the seminal plasma and nasal secretions of infected horses that were proven to contain EAV by traditional virus isolation in rabbit kidney thirteen (RK-13) cells, as well as a variety of cell culture-propagated European and North American strains of EAV. The primers and a fluorogenic TaqMan probe were designed to amplify and detect a highly conserved region of open reading frame 7 (ORF7) of EAV. The real-time TaqMan PCR assay detected EAV RNA in all samples that were confirmed to contain infectious EAV by virus isolation. The assay had an analytical sensitivity of 10 molecules of EAV RNA allowing the detection of EAV in clinical samples or tissue culture fluid (TCF) containing at least 200 viral RNA copies per ml. Thus, the one-tube real-time TaqMan RT-PCR assay provides a rapid, accurate, quantitative, convenient and high sample throughput system for diagnosis of EAV infection, in a closed-tube format that minimizes the risk of cross-contamination.
Balasuriya
Udeni B R
UB
Bernard and Gloria Salick Equine Viral Disease Laboratory, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, One Shields Avenue, University of California, Davis, CA 95616, USA. ubbalasuriya@ucdavis.edu
Leutenegger
Christian M
CM
Topol
J B
JB
McCollum
William H
WH
Timoney
Peter J
PJ
MacLachlan
N James
NJ
D000818:Animals; D018174:Arterivirus Infections / Q000175:diagnosis / Q000662:veterinary*; D002460:Cell Line; D017124:Conserved Sequence; D007240:Equartevirus / Q000235:genetics / Q000302:isolation & purification*; D005060:Europe; D006734:Horse Diseases / Q000821:virology*; D006736:Horses; D008297:Male; D009297:Nasal Mucosa / Q000378:metabolism / Q000821:virology; D009656:North America; D016366:Open Reading Frames; D012367:RNA, Viral / Q000032:analysis*; D011817:Rabbits; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D012661:Semen / Q000821:virology; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000737:chemistry*; D013997:Time Factors
D012367:RNA, Viral; D019914:Taq Polymerase