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Development of real-time NASBA assays with molecular beacon detection to quantify mRNA coding for HHV-8 lytic and
Abeltje M Polstra1, J Goudsmit, M Cornelissen
1Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. a.m.polstra@amc.uva.nl
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.