title: Translation of a laboratory-validated equine herpesvirus-1 specific real-time PCR assay into an insulated isothermal polymerase chain reaction (iiPCR) assay for point-of-need diagnosis using POCKIT™ nucleic acid analyzer.
abstract: Equine herpesvirus myeloencephalopathy (EHM), a major problem for the equine industry in the United States, is caused by equine herpesvirus-1 (EHV-1). In addition, EHV-1 is associated with upper respiratory disease, abortion, and chorioretinal lesions in horses. Here we describe the development and evaluation of an inexpensive, user-friendly insulated isothermal PCR (iiPCR) method targeting open reading 30 (ORF30) to detect both neuropathogenic and non-neuropathogenic strains on the field-deployable POCKIT™ device for point-of-need detection of EHV-1. The analytical sensitivity of the EHV-1 iiPCR assay was 13 genome equivalents per reaction. The assay did not cross react with ten non-target equine viral pathogens. Performance of the EHV-1 iiPCR assay was compared to two previously described real-time PCR (qPCR) assays in two laboratories by using 104 archived clinical samples. All 53 qPCR-positive and 46 of the 51 qPCR-negative samples tested positive and negative, respectively, by the iiPCR. The agreement between the two assays was 95.19% (confidence interval 90.48-99.90%) with a kappa value of 0.90. In conclusion, the newly developed EHV-1 iiPCR assay is robust to provide specificity and sensitivity comparable to qPCR assays for the detection of EHV-1 nucleic acid in clinical specimens.
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
authors.affiliation: GeneReach USA, Lexington, MA, USA. Electronic address: peiyu329@genereachbiotech.com.
authors.forename: Pei-Yu Alison
authors.initials: PA
authors.lastname: Lee
authors.affiliation: GeneReach USA, Lexington, MA, USA. Electronic address: fiwind.tsai@genereachbiotech.com.
authors.forename: Yun-Long
authors.initials: YL
authors.lastname: Tsai
authors.affiliation: GeneReach USA, Lexington, MA, USA. Electronic address: marktsai@genereachbiotech.com.
authors.forename: Chuan-Fu
authors.initials: CF
authors.lastname: Tsai
authors.affiliation: GeneReach USA, Lexington, MA, USA. Electronic address: yuki7510zz@genereachbiotech.com.
authors.forename: Yu-Han
authors.initials: YH
authors.lastname: Shen
authors.affiliation: GeneReach USA, Lexington, MA, USA. Electronic address: gracechang@genereachbiotech.com.
authors.forename: Hsiao-Fen Grace
authors.initials: HG
authors.lastname: Chang
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: ashley.skillman@uky.edu.
authors.forename: Ashley
authors.initials: A
authors.lastname: Skillman
authors.affiliation: GeneReach USA, Lexington, MA, USA. Electronic address: thomaswang@genereachbiotech.com.
authors.forename: Hwa-Tang Thomas
authors.initials: HT
authors.lastname: Wang
authors.affiliation: LABÉO(-)Frank Duncombe, U2RM, EA4655 Normandie Université, UNICAEN, 1 Route de Rosel, 14053 Caen, France. Electronic address: Stephane.PRONOST@laboratoire-labeo.fr.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Pronost
authors.affiliation: Animal Disease Diagnostic Laboratory, Ohio Department of Agriculture, Reynoldsburg, OH, USA. Electronic address: YZhang@agri.ohio.gov.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004679:Encephalomyelitis / Q000175:diagnosis / Q000662:veterinary / Q000821:virology; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D004861:Herpesvirus 1, Equid / Q000235:genetics / Q000302:isolation & purification*; D006734:Horse Diseases / Q000175:diagnosis* / Q000821:virology; D006736:Horses; D016366:Open Reading Frames / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D004279:DNA, Viral
keywords: EHM; Equine herpesvirus myeloencephalopathy; Equine herpesvirus-1; iiPCR