FLOW CYTOMETRIC DETECTION OF EBV (EBER SNRNA) USING PEPTIDE NUCLEIC-ACID PROBES

The application of peptide nucleic acid (PNA) probes for detection of Epstein-Barr Virus (EBV) snRNA in fixed cells is described. Fluorescei n labelled PNA probes were used to detect EBER1 and EBER2 snRNA in Raj i, Daudi and MS-Sultan cells. The fixation and permeabilization of cel ls were optimized. The optimal fixation was found to be 5% acetic acid plus 4% paraformaldehyde in PBS and the optimal permeabilization 0.5% Tween 20 in PBS whereas no proteolytic digestion was needed. The hybr idization time needed with the PNA probes was only 1 h. When running m ixed samples of Ramos (EBV neg.) Raji, Daudi and MS-Sultan (EBV pos.) cells in flow cytometry a strong fluorescence signal was seen in Raji, Daudi and MS-Sultan cells whereas no fluorescence signal was seen in the Ramos cells. In total 0.5% EBER positive Raji cells could easily b e identified in a mixture of Raji and Ramos cells. The results were ve rified by fluorescence microscopy. It is concluded that PNA probes can be used for in situ hybridization in solution and the analysis can be done using flow cytometry or fluorescence microscopy. PNA probes ther efore may facilitate and enhance the potential use of the in situ hybr idization/flow cytometry combination. (C) 1998 Elsevier Science B.V. A ll rights reserved.