Competitive DNA hybridization in microtitre plates for chicken anaemia virus

Abstract

Unlabelled chicken anaemia virus (CAV) DNA probe, produced by PCR, was immobilized onto nitrocellulose discs that then were fitted into microtitre plate wells in order to develop a competitive, non-radioactive hybridization test for detection of CAV. The discs were hybridized with either DNA extracts of buffy coats or dilutions of CAV DNA (for standard curves), followed by hybridization with biotin-labelled CAV DNA probe in excess of the immobilized, capture probe. Thus, CAV from sample DNA extracts and standard DNA preparations competed with the biotin-labelled CAV DNA probe for the immobilized, capture probe, decreasing subsequent colour development by an avidin-biotin-alkaline phosphatase detection system. Standard curves were log linear from 5–100 ng viral DNA with r2≥0·91. Tests were considered positive at 2 SD less than mean absorbence of samples from uninfected chickens, and ranged from 52 to 108 μm viral DNA or 2 to 4·2×1010virions μg−1buffy 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 microtitre plates, dot-blots, enzyme-linked immunosorbent assay (ELISA), and in situ hybridization on blood smears. None of the tests was positive for uninfected chickens. Viral isolation from buffy coats, though expensive and lengthy, was the most sensitive method. It detected virus in buffy coat from each infected chicken, while competitive hybridization detected 72% of infected chickens, in situ hybridization 69%, dot-blots 67%, and ELISA 36%. Sensitivity 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.