Cytomegalovirus DNA quantification using an automated platform for nucleic acid extraction and real-time PCR assay setup

Abstract

Analytical performance characteristics of the QIAsymphony RGQ system with artus cytomegalovirus (CMV) reagents were determined. Measurable range spanned 2.0 to ≥ 7.0 log(10) copies/ml. The detection limit was 23 copies/ml. Intrarun and interrun coefficients of variation were ≤ 2.1% at 3.0 and 5.0 log(10) copies/ml. In clinical specimens, RGQ values were ~0.2 log(10) copies/ml higher than those in an assay using a BioRobot M48 extraction/manual reaction setup/7500 Real-Time PCR instrument. No cross-contamination was observed.

Fig. 1.

Measurable range of CMV DNA quantification using two different CMV preparations. The dotted line indicates the theoretical trend line of complete agreement between expected and observed measurements.

Fig. 2.

Limit of detection. Probability of detection corresponds to percentage of replicates detected (n = 20 tested at each concentration). Open circles, LOD data (percentage of replicates detected). Arched solid line, probit regression line. Arched dashed lines, 95% confidence intervals of the regression line. Solid vertical line, predicted concentration of 95% detection limit. Dashed vertical lines, 95% confidence intervals for 95% detection limit.

Fig. 3.

(A) Comparison of quantification by QIAsymphony RGQ and M48/manual setup/ABI7500 platforms throughout the measuring range, using clinical plasma specimens (n = 157). Solid line, Deming regression; dotted/dashed line, identity. (B) Difference in quantification between QIAsymphony RGQ and M48/manual setup/ABI7500 platforms. Mean, average of QIAsymphony RGQ and M48/manual setup/ABI7500 platforms; difference, M48/manual setup/ABI7500 platforms minus QIAsymphony RGQ; SD, standard deviation (0.21).