title: Production of mono- and polyclonal antibodies to Citrus leprosis virus C2 and their application in triple antibody sandwich ELISA and immunocapture RT-PCR diagnostic assays.
abstract: The newly discovered Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) is one of the causal virus of citrus leprosis disease complex; which leads to substantial loss of citrus production in the states of Meta and Casanare of Colombia. Specific and sensitive detection methods are needed to monitor the dissemination of CiLV-C2 in Colombia, and to prevent introduction of CiLV-C2 to other citrus growing countries. Toward this end, putative coat protein gene (CPG) of CiLV-C2 was amplified from CiLV-C2 infected citrus tissues. The CPG was cloned, expressed and purified a recombinant coat protein of ∼31kDa which used to generate monoclonal antibodies and polyclonal antisera. Four monoclonal antibodies and two polyclonal antisera were selected as being specific following Western blotting. The monoclonal antibody MAb E5 and polyclonal antiserum PAb UF715 were selected testing with an extract of CiLV-C2 infected leaves using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). In addition, an immunocapture RT-PCR was standardized using MAb E5 for specific and sensitive detection of CiLV-C2. The standardized TAS-ELISA and IC-RT-PCR were able to detect CiLV-C2 in the extracts of symptomatic citrus leprosis tissues up to the dilutions of 1:160 and 1:2580, respectively. Result demonstrated that CiLV-C2 is present in citrus orchards in Meta and Casanare citrus growing areas of Colombia. TAS-ELISA could be used for routine detection of CiLV-C2, epidemiological studies, and for border inspections for quarantine purposes. IC-RT-PCR could be valuable for CiLV-C2 validation and viral genome analysis.
authors.affiliation: University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, Lake Alfred, FL 33850, USA. Electronic address: nandlalc@gmail.com.
authors.forename: Nandlal
authors.initials: N
authors.lastname: Choudhary
authors.affiliation: USDA-ARS, Foreign Disease-Weed Science Research, Fort Detrick, MD, USA.
authors.forename: Avijit
authors.initials: A
authors.lastname: Roy
authors.affiliation: Centro de Investigacion La Libertad, CORPOICA, Villavicencio, Colombia.
authors.forename: M G
authors.initials: MG
authors.lastname: Leon
authors.affiliation: USDA-APHIS-PPQ, Center for Plant Health Science and Technology, Beltsville Laboratory, Beltsville, MD 20705, USA.
authors.forename: G
authors.initials: G
authors.lastname: Wei
authors.affiliation: USDA-APHIS-PPQ, Center for Plant Health Science and Technology, Beltsville Laboratory, Beltsville, MD 20705, USA.
authors.forename: M K
authors.initials: MK
authors.lastname: Nakhla
authors.affiliation: USDA-APHIS-PPQ, Center for Plant Health Science and Technology, Director's Office, Riverdale, MD 20737, USA.
authors.forename: L
authors.initials: L
authors.lastname: Levy
authors.affiliation: University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, Lake Alfred, FL 33850, USA. Electronic address: rhby@ufl.edu.
authors.forename: R H
authors.initials: RH
authors.lastname: Brlansky
mesh_terms: D002957:Citrus / Q000821:virology*; D003105:Colombia; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D010935:Plant Diseases / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012209:Rhabdoviridae / Q000276:immunology / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
keywords: Antibodies; CiLV-C2; Citrus leprosis disease; Citrus virus; TAS-ELISA