A single-step silver enhancement method permitting rapid diagnosis of cytomegalovirus infection in formalin-fixed, paraffin-embedded tissue sections by in situ hybridization and immunoperoxidase detection.

Abstract

A single-step silver enhancement method was developed which intensifies the polymerized nickel-complexed diaminobenzidine (Ni-DAB) reaction product of peroxidase. With such enhancement, an in situ hybridization procedure can be performed in less than 8 hr by using a 2-hr hybridization incubation and direct detection. Cytomegalovirus (CMV)-infected lung sections were hybridized in situ for 2 hr with a biotinylated CMV genomic-length probe. The probe was detected directly with avidin-biotinylated peroxidase using Ni-DAB as the substrate, and the reaction product was enhanced with silver. Silver was deposited only on the Ni-DAB and not on normally argyrophilic substances. Indirect detection of the probe using a sandwich technique before silver enhancement proved more sensitive, but the length of the procedure was increased without substantially changing the result (infection vs. no infection). Sensitivity was also improved by omitting the dehydration step before applying the probe, and by increasing the temperature and duration of denaturation. In a blinded study of 21 open-lung biopsies, 13 of 13 culture-negative specimens were negative by hybridization, and 7 of 8 culture-positive specimens were positive by hybridization. Modified short hybridization with a biotinylated probe and silver-enhanced direct detection therefore provides a rapid but sensitive method for diagnosis of viral infection.