A Sensitive Branched DNA HIV-1 Signal Amplification Viral Load Assay with Single Day Turnaround

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

Multicenter Evaluation of the VERSANT Hepatitis B Virus DNA 3.0 Assay

The VERSANT hepatitis B virus (HBV) 3.0 Assay (branched DNA [bDNA]) (referred to herein as VERSANT 3.0) was evaluated at four external sites for analytical sensitivity, specificity, reproducibility, linearity of quantification, and limits of detection. In addition, each of the test evaluation sites provided HBV DNA-positive clinical samples that were previously analyzed by one of three commercially available HBV DNA quantitative tests: Digene Hybrid Capture II HBV DNA Test (Digene); VERSANT HBV DNA 1.0 Assay (bDNA) (VERSANT 1.0); and COBAS AMPLICOR HBV Monitor Test (COBAS AMPLICOR). These samples were reexamined using VERSANT 3.0. The results from these studies showed that VERSANT 3.0 has high specificity (99.3%), excellent reproducibility (between-run coefficient of variation [CV] = 1.6 to 9.4%; within-run CV = 6.5 to 20.7%), and a broad linear range of quantification (2.0 × 10(3) to 1.0 × 10(8) HBV DNA copies/ml) that facilitate the monitoring of HBV DNA levels at diagnosis and throughout the course of treatment. In general, correlation was very good between results obtained from clinical samples analyzed by VERSANT 3.0 and the comparative HBV DNA quantitative assays (VERSANT 1.0, R(2) = 0.900; Digene, R(2) = 0.985; COBAS AMPLICOR, R(2) = 0.771). The greatest differences in comparative quantitation occurred at HBV DNA levels approaching the limits of the dynamic ranges for the comparative assays. The performance characteristics of the new VERSANT 3.0 assay demonstrated that it provides a reliable and robust method for routinely monitoring serum HBV DNA levels in assessing disease activity and determining response to antiviral treatment

Analytical performance summary for the modified HIV-1 bDNA assay.

<p>Analytical performance summary for the modified HIV-1 bDNA assay.</p

Cumulative effect of lower volume and PVSA addition.

<p>Three conditions were compared in this experiment: Versant HIV-1 RNA 3.0 Assay with standard volume target incubation (140 µL), low volume target incubation (80 µL), or low volume target incubation plus PVSA (80 µL, PVSA). Two concentrations of 8E5 (n = 4 for each level of each condition) were analyzed on a single plate with 2 h target incubation. Enhanced signal generation (RLU average) was observed for low volume target incubation, and further improvement was observed upon addition of PVSA to the low volume target incubation. Although signal increased slightly for negative samples (n = 6 for each condition) in the low volume conditions, signal for the low concentration positive samples in the low volume conditions increased more than enough to compensate.</p

Polyanions in target incubation solution.

<p>In this example, addition of 1% (w/V) of polyacrylic acid (PAA), polyantholesulfuric acid (PASA), or polyvinylsulfonic acid (PVSA) to the Lysis Diluent (LD, control) caused different enhancement of signal generation relative to the control condition. An 8E5 sample at 80,000 copies/mL (n = 5 for each condition) was analyzed here on a single plate with 3 h target incubation. Superior signal enhancement by PVSA relative to alternative polyanions was observed throughout our experiments. No polyanion caused discernable increase in signal with negative samples (n = 1 for each condition).</p

Signal generation in standard and low volume assays.

<p>The standard assay was run with 3 and 15 h target incubation (140 µL) and the low volume assay was run with 3 h target incubation (80 µL). Identical lots of all components except Lysis Diluent were used on a single instrument for the three conditions. A dilution series of 8E5 in Seracon II at three concentrations (n = 10 for each condition) generated nearly identical average Relative Light Units (RLUs) for standard volume target incubation at 15 h and low volume target incubation volume at 3 h. Signal in negative samples remained unchanged (n = 6 for each condition).</p

Specificity with individual HIV-negative clinical specimens: determination of 29 copies/mL as the negative cutoff and subsequent verification in the modified assay.

<p>Specificity with individual HIV-negative clinical specimens: determination of 29 copies/mL as the negative cutoff and subsequent verification in the modified assay.</p

Bland-Altman plot of method comparison data between the modified bDNA assay and the VERSANT HIV-1 bDNA 3.0 Assay.

<p>No evidence of proportional bias across the linear range of the assay is apparent (regression slope = −0.026; 95% CI from −0.086 to 0.035).</p