Comparison of radioactive (32P and 35S) and biotinylated probes for detection of cytomegalovirus DNA

Abstract

Cytomegalovirus (CMV) DNA was detected in a dot-blot assay by hybridization to a DNA probe labeled with radioisotopes (32P or 35S) or biotin. Limits of detection were established for both the radioisotopically labeled DNA probes as well as the biotin-labeled probe. Hybridization of the radioisotopically labeled probes was detected by autoradiography and liquid scintillation while the biotin-labeled probe was detected after coupling to one of three enzymes (e.g., horseradish peroxidase, alkaline phosphatase, or acid phosphatase). In addition, several different substrates were evaluated with the nonisotopic detection enzymes. Detection limits (and times for detection) were 1 pg (4 h) for 32P, approximately 1 pg (96 h) for 35S, 5 pg (1-3 h) for the phosphatases, and 25-50 pg for peroxidase. Thus, 32P-labeled probes appear to provide the best sensitivity whereas the avidin-linked phosphatases provide the best sensitivity among the nonisotopic detection systems.