title: Development and evaluation of a reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay for detection of equine arteritis virus in equine semen and tissue samples using the POCKIT™ system.
abstract: Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of horses. Most importantly, EAV induces abortion in pregnant mares and can establish persistent infection in up to 10-70% of the infected stallions, which will continue to shed the virus in their semen. The objective of this study was to develop and evaluate a reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) for the detection of EAV in semen and tissue samples. The newly developed assay had a limit of detection of 10 RNA copies and a 10-fold higher sensitivity than a previously described real-time RT-PCR (RT-qPCR). Evaluation of 125 semen samples revealed a sensitivity and specificity of 98.46% and 100.00%, respectively for the RT-qPCR assay, and 100.00% and 98.33%, respectively for the RT-iiPCR assay. Both assays had the same accuracy (99.2%, k=0.98) compared to virus isolation. Corresponding values derived from testing various tissue samples (n=122) collected from aborted fetuses, foals, and EAV carrier stallions are as follows: relative sensitivity, specificity, and accuracy of 88.14%, 96.83%, and 92.62% (k=0.85), respectively for the RT-qPCR assay, and 98.31%, 92.06%, and 95.08% (k=0.90), respectively for the RT-iiPCR assay. These results indicate that RT-iiPCR is a sensitive, specific, and a robust test enabling detection of EAV in semen and tissue samples with very considerable accuracy. Even though the RT-qPCR assay showed a sensitivity and specificity equal to virus isolation for semen samples, its diagnostic performance was somewhat limited for tissue samples. Thus, this new RT-iiPCR could be considered as an alternative tool in the implementation of EAV control and prevention strategies.
authors.affiliation: Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA.
authors.forename: Mariano
authors.initials: M
authors.lastname: Carossino
authors.affiliation: GeneReach USA, Lexington, MA, USA.
authors.forename: Pei-Yu A
authors.initials: PY
authors.lastname: Lee
authors.affiliation: Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA.
authors.forename: Bora
authors.initials: B
authors.lastname: Nam
authors.affiliation: Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA.
authors.forename: Ashley
authors.initials: A
authors.lastname: Skillman
authors.affiliation: Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA.
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Shuck
authors.affiliation: Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Timoney
authors.affiliation: GeneReach USA, Lexington, MA, USA.
authors.forename: Yun-Long
authors.initials: YL
authors.lastname: Tsai
authors.affiliation: GeneReach USA, Lexington, MA, USA.
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Ma
authors.affiliation: GeneReach USA, Lexington, MA, USA.
authors.forename: Hsiao-Fen G
authors.initials: HF
authors.lastname: Chang
authors.affiliation: GeneReach USA, Lexington, MA, USA.
authors.forename: Hwa-Tang T
authors.initials: HT
authors.lastname: Wang
authors.affiliation: Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA. Electronic address: ubalasuriya@uky.edu.
authors.forename: Udeni B R
authors.initials: UB
authors.lastname: Balasuriya
mesh_terms: D000818:Animals; D018174:Arterivirus Infections / Q000175:diagnosis / Q000517:prevention & control / Q000662:veterinary* / Q000821:virology; D007240:Equartevirus / Q000302:isolation & purification*; D005260:Female; D006734:Horse Diseases / Q000175:diagnosis* / Q000821:virology; D006736:Horses; D008297:Male; D016366:Open Reading Frames; D011247:Pregnancy; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012661:Semen / Q000821:virology*; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D012367:RNA, Viral
keywords: Abortion; Equine arteritis virus; Equine semen; Equine viral arteritis; Insulated isothermal RT-PCR; Real-time RT-PCR