APPLICATION OF A LOW-LIGHT IMAGING DEVICE AND CHEMILUMINESCENT SUBSTRATES FOR QUANTITATIVE DETECTION OF VIRAL-DNA IN HYBRIDIZATION REACTIONS

In this quantitative dot-blot hybridization assay for detecting B19 pa rvovirus DNA, we used three different chemiluminescent substrates [ada mantyl-1,2-dioxetane phenyl phosphates (PPD and the new PPD-Plus) and the chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD) plus Emerald enhancer] and a high-performance, low-intensity-light imaging luminograph apparatus. The hybridization test uses digoxigenin-labele d DNA probes, which are immunoenzymatically revealed by anti-digoxigen in Fab fragments conjugated with alkaline phosphatase. All the detecti on systems with the various chemiluminescent substrates gave sensitive and reproducible results for calibrators and positive or negative ref erence clinical samples, with high reproducibility (CV 4-17%). The sig nal was measured after 45 min of incubation. The luminograph apparatus could detect 10 fg of homologous DNA with the PPD-Plus substrate, whe reas the detection limit with the CSPD and PPD substrates was 20 fg an d 20-50 fg, respectively. Analysis of 26 samples with the three substr ates showed good sensitivity and specificity for viral detection.