Double-nested polymerase chain reaction for detection of caprine arthritis-encephalitis virus proviral DNA in blood, milk, and tissues of infected goats

Jeff Barlough, Nancy East, Joan D Rowe, Karen Van oosear, Elfriede Derock, Luisa Bigornia, Espen Rimstad

Research output: Contribution to journalArticle

53 Scopus citations

Abstract

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 (κ = 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.

Original languageEnglish (US)
Pages (from-to)101-113
Number of pages13
JournalJournal of Virological Methods
Volume50
Issue number1-3
DOIs
StatePublished - 1994

Keywords

  • CAEV
  • Caprine arthritis encephalitis virus
  • DNA sequencing
  • ELISA
  • Goat diseases
  • Milk transmission
  • Polymerase chain reaction
  • Seroconversion
  • Southern hybridization
  • Viral diagnosis

ASJC Scopus subject areas

  • Virology

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