Double-nested polymerase chain reaction for detection of caprine arthritis-encephalitis virus proviral DNA in blood, milk, and tissues of infected goats.
A nested polymerase chain reaction (PCR) for detecting proviral DNA of caprine arthritis-encephalitis virus (CAEV) in biological samples was developed. Primers for both gag and pol sequences of the CAEV genome were included in a single tube for simultaneous amplification ('double' PCR), and the resulting bands were resolved visually in ethidium bromide-stained agarose gels. Internal gag and pol probes were used to verify the identity of the amplified products by non-radioactive Southern hybridization. Final confirmation of the identity of representative PCR bands was provided by DNA sequence analysis. A comparison between the PCR and an antibody ELISA (with recombinant CAEV p28 as target) using 141 caprine blood samples indicated very strong agreement between the two assays (kappa = 0.912). Four of 7 goats with indeterminate ELISA results were PCR-positive as were 5 of 40 (12.5%) seronegative goats, most probably indicating delayed seroconversion. Eleven of 27 goats (41%) PCR-positive on blood had detectable CAEV proviral DNA in milk. Proviral DNA was also detected in lung, mesenteric lymph node, bone marrow, synovial membrane, and mammary gland of a seropositive, clinically affected goat, but not in equivalent tissues of a healthy seronegative goat.
Barlough
J
J
Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis 95616.
East
N
N
Rowe
J D
JD
Van Hoosear
K
K
DeRock
E
E
Bigornia
L
L
Rimstad
E
E
D000818:Animals; D016088:Arthritis-Encephalitis Virus, Caprine / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D001826:Body Fluids / Q000821:virology; D018076:DNA, Complementary; D004279:DNA, Viral / Q000032:analysis*; D015511:Goat Diseases / Q000821:virology*; D006041:Goats; D016180:Lentivirus Infections / Q000662:veterinary*; D008892:Milk / Q000821:virology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D014766:Viremia / Q000662:veterinary
D018076:DNA, Complementary; D004279:DNA, Viral