title: Rapid and sensitive detection of Feline immunodeficiency virus using an insulated isothermal PCR-based assay with a point-of-need PCR detection platform.
abstract: Feline immunodeficiency virus (FIV) is an important infectious agent of cats. Clinical syndromes resulting from FIV infection include immunodeficiency, opportunistic infections, and neoplasia. In our study, a 5' long terminal repeat/gag region-based reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) was developed to amplify all known FIV strains to facilitate point-of-need FIV diagnosis. The RT-iiPCR method was applied in a point-of-need PCR detection platform--a field-deployable device capable of generating automatically interpreted RT-iiPCR results from nucleic acids within 1 hr. Limit of detection 95% of FIV RT-iiPCR was calculated to be 95 copies standard in vitro transcription RNA per reaction. Endpoint dilution studies with serial dilutions of an ATCC FIV type strain showed that the sensitivity of lyophilized FIV RT-iiPCR reagent was comparable to that of a reference nested PCR. The established reaction did not amplify any nontargeted feline pathogens, including Felid herpesvirus 1, feline coronavirus, Feline calicivirus, Feline leukemia virus, Mycoplasma haemofelis, and Chlamydophila felis. Based on analysis of 76 clinical samples (including blood and bone marrow) with the FIV RT-iiPCR, test sensitivity was 97.78% (44/45), specificity was 100.00% (31/31), and agreement was 98.65% (75/76), determined against a reference nested-PCR assay. A kappa value of 0.97 indicated excellent correlation between these 2 methods. The lyophilized FIV RT-iiPCR reagent, deployed on a user-friendly portable device, has potential utility for rapid and easy point-of-need detection of FIV in cats.
authors.affiliation: Department of Biomedical and Diagnostic Sciences, University of Tennessee, Knoxville, TN (Wilkes, Kania)GeneReach USA, Lexington, MA (Tsai, Lee, Chang, Ma, Chang, Wang) beckpen@utk.edu.
authors.forename: Rebecca Penrose
authors.initials: RP
authors.lastname: Wilkes
authors.affiliation: Department of Biomedical and Diagnostic Sciences, University of Tennessee, Knoxville, TN (Wilkes, Kania)GeneReach USA, Lexington, MA (Tsai, Lee, Chang, Ma, Chang, Wang).
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Kania
authors.affiliation: Department of Biomedical and Diagnostic Sciences, University of Tennessee, Knoxville, TN (Wilkes, Kania)GeneReach USA, Lexington, MA (Tsai, Lee, Chang, Ma, Chang, Wang).
authors.forename: Yun-Long
authors.initials: YL
authors.lastname: Tsai
authors.affiliation: Department of Biomedical and Diagnostic Sciences, University of Tennessee, Knoxville, TN (Wilkes, Kania)GeneReach USA, Lexington, MA (Tsai, Lee, Chang, Ma, Chang, Wang).
authors.forename: Pei-Yu Alison
authors.initials: PY
authors.lastname: Lee
authors.affiliation: Department of Biomedical and Diagnostic Sciences, University of Tennessee, Knoxville, TN (Wilkes, Kania)GeneReach USA, Lexington, MA (Tsai, Lee, Chang, Ma, Chang, Wang).
authors.forename: Hsiu-Hui
authors.initials: HH
authors.lastname: Chang
authors.affiliation: Department of Biomedical and Diagnostic Sciences, University of Tennessee, Knoxville, TN (Wilkes, Kania)GeneReach USA, Lexington, MA (Tsai, Lee, Chang, Ma, Chang, Wang).
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Ma
authors.affiliation: Department of Biomedical and Diagnostic Sciences, University of Tennessee, Knoxville, TN (Wilkes, Kania)GeneReach USA, Lexington, MA (Tsai, Lee, Chang, Ma, Chang, Wang).
authors.forename: Hsiao-Fen Grace
authors.initials: HF
authors.lastname: Chang
authors.affiliation: Department of Biomedical and Diagnostic Sciences, University of Tennessee, Knoxville, TN (Wilkes, Kania)GeneReach USA, Lexington, MA (Tsai, Lee, Chang, Ma, Chang, Wang).
authors.forename: Hwa-Tang Thomas
authors.initials: HT
authors.lastname: Wang
mesh_terms: D000818:Animals; D002415:Cats; D016181:Feline Acquired Immunodeficiency Syndrome / Q000175:diagnosis* / Q000821:virology; D016089:Immunodeficiency Virus, Feline / Q000235:genetics / Q000302:isolation & purification*; D019095:Point-of-Care Systems; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012367:RNA, Viral / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D012367:RNA, Viral
keywords: Detection; Feline immunodeficiency virus; insulated isothermal polymerase chain reaction; point of need