Competitive DNA hybridization in microtitre plates for chicken anaemia virus

Unlabelled chicken anaemia virus (CAV) DNA probe, produced by PCR, was immo bilized onto nitrocellulose discs that then were fitted into microtitre pla te wells in order to develop a competitive, non-radioactive hybridization t est for detection of CAV. The discs were hybridized with either DNA extract s of buffy coats or dilutions of CAV DNA (for standard curves), followed by hybridization with biotin-labelled CAV DNA probe in excess of the immobili zed, capture probe. Thus, CAV from sample DNA extracts and standard DNA pre parations competed with the biotin-labelled CAV DNA probe for the immobiliz ed, capture probe, decreasing subsequent colour development by an avidin-bi otin-alkaline phosphatase detection system. Standard curves were log linear from 5-100ng viral DNA with r(2)greater than or equal to0.91. Tests were c onsidered positive at 2 SD less than mean absorbence of samples from uninfe cted chickens, and ranged from 52 to 108 muM viral DNA or 2 to 42 x10(10) v irions mug(-1) buffy coat DNA. Blood samples from chickens infected and not infected with CAV at one day of age were tested for evidence of infection until 28 days of age by viral isolation, competitive hybridization in micro titre plates, dot-blots, enzyme-linked immunosorbent assay (ELISA), and in situ hybridization on blood smears. None of the tests was positive for unin fected chickens. Viral isolation from buffy coats, though expensive and len gthy, was the most sensitive method. It detected virus in huffy coat from e ach infected chicken, while competitive hybridization detected 72% of infec ted chickens, in situ hybridization 69%, dot-blots 67%, and ELISA 36%. Sens itivity of competitive hybridization was 0.78, and its specificity was 1.00 . Three chickens must be sampled from an infected flock for a 90% chance of detecting a positive chicken at the 0.025 one-tailed level of significance , assuming 100% prevalence. (C) 2001 Academic Press.