title: Development and application of reverse transcriptase nested polymerase chain reaction test for the detection of exogenous avian leukosis virus.
abstract: A polymerase chain reaction (PCR) assay that utilizes nested primers to amplify a fragment of the long terminal repeat of exogenous avian leukosis virus (ALV) was developed and evaluated for detection of ALV subgroup J directly from clinical samples. Compilation of sequence data from different endogenous and exogenous ALVs allowed the selection of a conserved set of nested primers specific for the amplification of exogenous ALV subgroups A, B, C, D, and J and excluded amplification of endogenous viruses or endogenous viral sequences within the chicken genome. The nested primers were successfully used in both PCR and reverse transcriptase (RT)-PCR assays to detect genetically diverse ALV-J field isolates. Detection limits of ALV-J isolate ADOL-Hc1 DNA by nested PCR and RNA by RT-nested PCR were superior to detection of group-specific antigen by enzyme-linked immunosorbent assay (ELISA) in cell culture. Detection of ALV-J in cloacal swabs by RT-nested PCR was compared with direct detection by antigen-capture (ac)-ELISA; RT-nested PCR detected fewer positive samples than ac-ELISA, suggesting that RT-nested PCR excluded detection of endogenous virus in clinical samples. Detection of ALV-J in plasma samples by RT-nested PCR was compared with virus isolation in C/E chicken embryo fibroblasts; the level of agreement between both assays as applied to plasma samples ranged from low to moderate. The main disagreement between both assays was observed for a group of plasma samples found positive by RT-nested PCR and negative by virus isolation, suggesting that RT-nested PCR detected ALV-J genome in plasma samples of transiently or intermittently infected birds. ALV-J transient and intermittent infection profiles are characterized by inconsistent virus isolation responses throughout the life of a naturally infected flock.
authors.affiliation: Department of Avian Medicine, Poultry Diagnostic and Research Center, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-4875, USA.
authors.forename: Maricarmen
authors.initials: M
authors.lastname: García
authors.forename: John
authors.initials: J
authors.lastname: El-Attrache
authors.forename: Sylva M
authors.initials: SM
authors.lastname: Riblet
authors.forename: Vagner R
authors.initials: VR
authors.lastname: Lunge
authors.forename: André S K
authors.initials: AS
authors.lastname: Fonseca
authors.forename: Pedro
authors.initials: P
authors.lastname: Villegas
authors.forename: Nilo
authors.initials: N
authors.lastname: Ikuta
mesh_terms: D000818:Animals; D001353:Avian Leukosis / Q000097:blood / Q000175:diagnosis* / Q000821:virology*; D001354:Avian Leukosis Virus / Q000235:genetics* / Q000302:isolation & purification*; D002645:Chickens / Q000097:blood / Q000821:virology*; D002988:Cloaca / Q000821:virology; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005814:Genes, Viral; D016679:Genome, Viral; D011201:Poultry Diseases / Q000097:blood / Q000175:diagnosis / Q000821:virology; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D020079:Terminal Repeat Sequences
chemical_list: D017931:DNA Primers