NUCLEIC-ACID SEQUENCE-BASED AMPLIFICATION, A NEW METHOD FOR ANALYSIS OF SPLICED AND UNSPLICED EPSTEIN-BARR-VIRUS LATENT TRANSCRIPTS, AND ITS COMPARISON WITH REVERSE-TRANSCRIPTASE PCR

Nucleic acid sequence-based amplification (NASBA) assays were develope d for direct detection of Epstein-Barr virus (EBV) transcripts encodin g EBV nuclear antigen 1 (EBNA1), latent membrane proteins (LMP) 1 and 2, and BamHIA rightward frame 1 (BARF1) and for the noncoding EBV earl y RNA 1 (EBER1), The sensitivities of all NASBAs were at least 100 cop ies of specific in vitro-generated RNA. Furthermore, 1 EBV-positive JY cell in a background of 50,000 EBV-negative Ramos cells (the relative sensitivity) was detected by using the EBNA1, LMP1, and LMP2 NASBA as says. The relative sensitivity of the EBER1 NASBA was 100 EBV-positive cells, which was probably related to the loss of small RNA molecules during the isolation. The BARF1 and LMP2 NASBAs were evaluated on clin ical material. BARF1 expression was found in 6 of 7 nasopharyngeal car cinomas (NPC) but in 0 of 22 Hodgkin's disease (HD) cases, whereas LMP 2 expression was found in 7 of 7 NPCs and in 17 of 22 HD cases. For de tection of EBNA1 transcripts in HLs (n = 12) and T- and B-cell non-Hod gkin's lymphomas (n = 3 and n = 2, respectively), NASBA was compared w ith reverse transcriptase (RT) PCR. Two samples were positive only,vit h NASBA, and two other samples were positive only with RT-PCR; for all other samples, the RT-PCR and NASBA results were in agreement. We con clude that NASBA is suitable for sensitive and specific detection of t he above-mentioned EBV transcripts, regardless of their splicing patte rns and the presence of EBV DNA. The EBNA1, LMP2, and BARF1 NASBAs dev eloped in this study proved to be reliable assays for detection of the corresponding transcripts in EBV-positive clinical material.