CHEMILUMINESCENT IN-SITU HYBRIDIZATION FOR THE DETECTION OF CYTOMEGALOVIRUS DNA

A chemiluminescent in situ hybridization assay that could combine the sensitivity of chemiluminescent substrates, the specificity of digoxig enin-labeled probes, and the spatial morphological resolution and loca lization of the signal of the in situ hybridization was developed for the detection of cytomegalovirus (CMV) DNA. CMV DNA in cultured CMV-in fected cells and in different clinical samples (tissue sections and ce llular smears) was detected using digoxigenin-labeled Probes construct ed in our laboratory that were immunoenzymatically visualized employin g antidigoxigenin Fab fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrat e for alkaline phosphatase. The luminescent signal front the hybrid fo rmation was detected analyzed, and measured with a high performance, l ow light level imaging luminograph apparatus connected to an optical m icroscope and to a personal computer for quantitative image analysis. Increasing values of emitted photons per second per infected cell corr esponding to the presence of hybridized CMV DNA, could be found in inf ected cells fixed at various times after infection, following the CMV replication cycle. When the assay was performed on different clinical samples from patients with acute CMV infections, CMV DNA was detected in all positive samples tested, both in cellular samples and in frozen and paraffin-embedded tissue sections, proving specific and sensitive . The chemiluminescent in situ hybridization assay developed in this w ork: can be a useful tool for a sensitive and specific diagnosis of vi ral infection and can be easily adapted to detect and study any specif ic gene sequence inside the cells. The assay may also be promising for an estimation and quantification of nucleic acids present in tissue s amples or cellular smears and for imaging gene expression in cells.