Development of real-time NASBA assays with molecular beacon detection to quantify mRNA coding for HHV-8 lytic and latent genes

Background: Human herpesvirus-8 (HHV-8) is linked to the pathogenesis of Kaposi's sarcoma (KS), and the HHV-8 DNA load in peripheral blood mononuclear cells ( PBMC) is associated with the clinical stage of KS. To examine the expression of HHV-8 in PBMC, four HHV-8 mRNA specific NASBA assays were developed Methods: We have developed four quantitative nucleic acid sequence-based amplification assays (NASBA-QT) specifically to detect mRNA coding for ORF 73 (latency-associated nuclear antigen, LANA), vGCR (a membrane receptor), vBcl-2 ( a viral inhibitor of apoptosis) and vIL-6 (a viral growth factor). The NASBA technique amplifies nucleic acids without thermocycling and mRNA can be amplified in a dsDNA background. A molecular beacon is used during amplification to enable real-time detection of the product. The assays were tested on PBMC samples of two AIDS-KS patients from the Amsterdam Cohort. Results: For all four assays, the limit of detection (LOD) of 50 molecules and the limit of quantification ( LOQ) of 100 molecules were determined using in vitro transcribed RNA. The linear dynamic range was 50 to 10 7 molecules of HHV-8 mRNA. We found HHV-8 mRNA expression in 9 out of the 10 tested samples. Conclusion: These real-time NASBA assays with beacon detection provide tools for further study of HHV-8 expression in patient material