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Comparative Study
. 2009 Jan;11(1):54-59.
doi: 10.2353/jmoldx.2009.080073. Epub 2008 Dec 12.

Detection of cytomegalovirus in whole blood using three different real-time PCR chemistries

Erica Vincent  1 , Zhengming Gu  1 , Markus Morgenstern  1 , Candace Gibson  1 , Jianmin Pan  2 , Randall T Hayden  3
Affiliations
Comparative Study

Detection of cytomegalovirus in whole blood using three different real-time PCR chemistries

Erica Vincent et al. J Mol Diagn. 2009 Jan.

Abstract

Several different primer-probe chemistries have been produced commercially for real-time PCR detection and quantification of cytomegalovirus, but there are few studies evaluating their relative performance. We assessed three such commercial reagents with respect to analytical and clinical operating characteristics. The samples included 149 clinical whole blood specimens that were de-identified and assayed in parallel with all primer-probe systems. Individual methods used TaqMan, dual fluorescence resonance energy transfer hybridization probes, and labeled primer chemistries. Method comparability was determined both qualitatively, based on pair-wise assessment of concordance, and quantitatively, based on pair-wise linear regression analysis. Analytical sensitivity and the lower end of the linear dynamic range reached 10 target copies per reaction for the TaqMan and labeled primer systems and 100 target copies per reaction for the dual fluorescence resonance energy transfer probe system. Quantitative linearity reached an upper limit of 10(5) copies per reaction for all methods. No assay cross-reactivity was seen with other common viral pathogens (100% analytical specificity). Pair-wise analysis of qualitative results from clinical samples showed no significant differences in sensitivity between the three sets of reagents, and linear regression analysis indicated that the quantitative values achieved were comparable in all positive specimens. The findings demonstrate that similar analytical and clinical performance characteristics can be demonstrated for quantitative detection of cytomegalovirus in clinical whole blood extracts using a wide variety of real-time PCR chemistries.

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Figures

Figure 1
Figure 1
Ten-fold serial dilutions of CMV DNA from 10 to 1 × 105 copies/reaction demonstrating comparative linearity of optimized TaqMan, Labeled Primer, and Dual Probe methods.
Figure 2
Figure 2
A: Comparison of quantitative results between Labeled Primer and TaqMan methods. B: Comparison of quantitative results between Labeled Primer and Dual Probe methods. C: Comparison of quantitative results between TaqMan and Dual Probe methods.
See this image and copyright information in PMC

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