title: Comparison of electron microscopy, ELISA, real time RT-PCR and insulated isothermal RT-PCR for the detection of Rotavirus group A (RVA) in feces of different animal species.
abstract: There is no gold standard for detection of Rotavirus Group A (RVA), one of the main causes of diarrhea in neonatal animals. Sensitive and specific real-time RT-PCR (rtRT-PCR) assays are available for RVA but require submission of the clinical samples to diagnostic laboratories. Patient-side immunoassays for RVA protein detection have shown variable results, particularly with samples from unintended species. A sensitive and specific test for detection of RVA on the farm would facilitate rapid management decisions. The insulated isothermal RT-PCR (RT-iiPCR) assay works in a portable machine to allow sensitive and specific on-site testing. The aim of this investigation was to evaluate a commercially available RT-iiPCR assay for RVA detection in feces from different animal species. This assay was compared to an in-house rtRT-PCR assay and a commercially available rtRT-PCR kit, as well as an ELISA and EM for RVA detection. All three PCR assays targeted the well-conserved NSP5 gene. Clinical fecal samples from 108 diarrheic animals (mainly cattle and horses) were tested. The percentage of positive samples by ELISA, EM, in-house rtRT-PCR, commercial rtRT-PCR, and RT-iiPCR was 29.4%, 31%, 36.7%, 51.4%, 56.9%, respectively. The agreement between different assays was high (81.3-100%) in samples containing high viral loads. The sensitivity of the RT-iiPCR assay appeared to be higher than the commercially available rtRT-PCR assay, with a limit of detection (95% confidence index) of 3-4 copies of in vitro transcribed dsRNA. In conclusion, the user-friendly, field-deployable RT-iiPCR system holds substantial promise for on-site detection of RVA.
authors.affiliation: Department of Veterinary Medicine, Infectious Diseases Division, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt; Clinical Virology Laboratory, Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, 2407 River Drive, Room A239, Knoxville, TN 37996, USA.
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Soltan
authors.affiliation: GeneReach USA, 27 Revere Street, Lexington, MA 02420, USA.
authors.forename: Yun-Long
authors.initials: YL
authors.lastname: Tsai
authors.affiliation: GeneReach USA, 27 Revere Street, Lexington, MA 02420, USA.
authors.forename: Pei-Yu A
authors.initials: PA
authors.lastname: Lee
authors.affiliation: GeneReach USA, 27 Revere Street, Lexington, MA 02420, USA.
authors.forename: Chuan-Fu
authors.initials: CF
authors.lastname: Tsai
authors.affiliation: GeneReach USA, 27 Revere Street, Lexington, MA 02420, USA.
authors.forename: Hsiao-Fen G
authors.initials: HG
authors.lastname: Chang
authors.affiliation: GeneReach USA, 27 Revere Street, Lexington, MA 02420, USA.
authors.forename: Hwa-Tang T
authors.initials: HT
authors.lastname: Wang
authors.affiliation: Clinical Virology Laboratory, Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, 2407 River Drive, Room A239, Knoxville, TN 37996, USA; Veterinary Diagnostic and Investigational Laboratory, College of Veterinary Medicine, University of Georgia, 43 Brighton Road, Tifton, GA, USA. Electronic address: beckpen@uga.edu.
authors.forename: Rebecca P
authors.initials: RP
authors.lastname: Wilkes
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000821:virology; D003967:Diarrhea / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay; D005243:Feces / Q000821:virology*; D006734:Horse Diseases / Q000175:diagnosis / Q000821:virology; D006736:Horses; D008854:Microscopy, Electron; D019095:Point-of-Care Systems; D012330:RNA, Double-Stranded; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012680:Sensitivity and Specificity; D013696:Temperature; D019562:Viral Load; D017361:Viral Nonstructural Proteins / Q000235:genetics
chemical_list: C434828:NSP5 protein, rotavirus group A; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D017361:Viral Nonstructural Proteins
keywords: Diagnosis; Insulated isothermal PCR; Point of need; Real-time RT-PCR; Rotavirus