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




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BMC Infectious Diseases

, 2:18

First Online: 04 September 2002Received: 24 May 2002Accepted: 04 September 2002

Abstract

BackgroundHuman 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

MethodsWe 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.

ResultsFor 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 molecules of HHV-8 mRNA. We found HHV-8 mRNA expression in 9 out of the 10 tested samples.

ConclusionThese real-time NASBA assays with beacon detection provide tools for further study of HHV-8 expression in patient material.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2334-2-18 contains supplementary material, which is available to authorized users.

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Autor: Abeltje M Polstra - J Goudsmit - M Cornelissen

Fuente: https://link.springer.com/



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