Abstract
Branched DNA (bDNA) is a signal amplification technology used in clinical and research laboratories to quantitatively detect nucleic acids. An overnight incubation is a significant drawback of highly sensitive bDNA assays. The VERSANT® HIV-1 RNA 3.0 Assay (bDNA) (“Versant Assay”) currently used in clinical laboratories was modified to allow shorter target incubation, enabling the viral load assay to be run in a single day. To dramatically reduce the target incubation from 16–18 h to 2.5 h, composition of only the “Lysis Diluent” solution was modified. Nucleic acid probes in the assay were unchanged. Performance of the modified assay (assay in development; not commercially available) was evaluated and compared to the Versant Assay. Dilution series replicates (>950 results) were used to demonstrate that analytical sensitivity, linearity, accuracy, and precision for the shorter modified assay are comparable to the Versant Assay. HIV RNA-positive clinical specimens (n = 135) showed no significant difference in quantification between the modified assay and the Versant Assay. Equivalent relative quantification of samples of eight genotypes was demonstrated for the two assays. Elevated levels of several potentially interfering endogenous substances had no effect on quantification or specificity of the modified assay. The modified assay with drastically improved turnaround time demonstrates the viability of signal-amplifying technology, such as bDNA, as an alternative to the PCR-based assays dominating viral load monitoring in clinical laboratories. Highly sensitive bDNA assays with a single day turnaround may be ideal for laboratories with especially stringent cost, contamination, or reliability requirements.
Highlights
Branched DNA technology, first developed over twenty years ago [1], is today widely used in clinical [2] and research [3] laboratories to quantitatively detect specific nucleic acid sequences. bDNA quantitative hybridization technology has a wide dynamic range and is sensitive enough for applications intended to reliably detect very few target molecules [4]
Unlike PCR, where a region of the intended target is exponentially amplified in order to generate detectable signal, in bDNA assays only signal is amplified. bDNA assays are not susceptible to contamination risks associated with PCR-based assays [5]
We have modified the Versant HIV-1 RNA 3.0 Assay (‘‘Versant Assay’’) currently in use in clinical laboratories to allow the viral load assay to be run in a single day
Summary
Branched DNA (bDNA) technology, first developed over twenty years ago [1], is today widely used in clinical [2] and research [3] laboratories to quantitatively detect specific nucleic acid sequences. bDNA quantitative hybridization technology has a wide dynamic range and is sensitive enough for applications intended to reliably detect very few target molecules [4]. Branched DNA (bDNA) technology, first developed over twenty years ago [1], is today widely used in clinical [2] and research [3] laboratories to quantitatively detect specific nucleic acid sequences. BDNA quantitative hybridization technology has a wide dynamic range and is sensitive enough for applications intended to reliably detect very few target molecules [4]. A reduction in the processing time allowing the single day turnaround standard in clinical PCR assays, would improve the productivity of laboratories utilizing bDNA. We have modified the Versant HIV-1 RNA 3.0 Assay (bDNA) (‘‘Versant Assay’’) currently in use in clinical laboratories to allow the viral load assay to be run in a single day (assay in development; not commercially available). An evaluation of the modified assay consisting of analytical performance testing, a method comparison with clinical specimens, a genotype equivalence study, and testing of potentially interfering endogenous substances is presented
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