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Comparative Study
. 2003 Feb;5(1):15-20.
doi: 10.1016/S1525-1578(10)60446-1.

Development of real-time PCR assays for the quantitative detection of Epstein-Barr virus and cytomegalovirus, comparison of TaqMan probes, and molecular beacons

Jiska Jebbink  1 , Xin Bai, Beverly Barton Rogers, D Brian Dawson, Richard H Scheuermann, Rana Domiati-Saad
Affiliations
Comparative Study

Development of real-time PCR assays for the quantitative detection of Epstein-Barr virus and cytomegalovirus, comparison of TaqMan probes, and molecular beacons

Jiska Jebbink et al. J Mol Diagn. 2003 Feb.

Abstract

Human Epstein-Barr virus (EBV) and cytomegalovirus (CMV) can cause serious complications in immunocompromised patients. Rapid diagnosis of EBV and CMV infection is critical in the management of the disease so that anti-viral therapy can be started early. Here we describe the development of real-time PCR assays using TaqMan probes and molecular beacons and compare the performance of both assays with a well-established, validated, gel-based PCR method for the quantification of EBV and CMV in patients' samples. The TaqMan and molecular beacon assays were linear between 10 to 10(7) viral genomes/reaction. Both assays generated calibration curves with strong correlation and low intra-assay and interassay variation. Results of EBV and CMV viral load determination inpatient samples obtained by the gel-based and real-time PCR were very similar. The real-time PCR assays showed increases in viral load before clinical measures of viral disease and decreases in viral load during anti-viral therapy in two of six pediatric patients. The data indicate that these TaqMan and molecular beacon approaches are accurate, rapid, and reliable assays for the diagnosis and monitoring of EBV and CMV infections in patients.

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Figures

Figure 1.
Figure 1.
Real-time PCR assays for the detection of EBV and CMV using molecular beacon or TaqMan probes. Amplification plots (A, B, C, D) and standard curves (E, F, G, H) of EBV and CMV real-time PCR. Tenfold serial dilutions of quantitative EBV and CMV DNA ranging from 107 to 101 copies/reaction were amplified in duplicate. Amplification plots show the detection of the serially diluted DNA. PCR cycles are plotted against the fluorescence intensity. The cycle at which the fluorescence reaches a threshold value is called the threshold cycle (Ct). Standard curves were obtained by plotting the Ct values against the copy number. The correlation coefficients and linear regression equations are shown.
Figure 2.
Figure 2.
Correlation between the gel-based quantitative PCR and molecular beacon real-time assay or TaqMan real-time assay for EBV (A, B) and CMV (C, D). DNA from 23 patients’ samples was subjected to the conventional quantitative PCR and to real-time PCR and their EBV or CMV viral load quantitated. The copy numbers of EBV DNA or CMV DNA measured by the conventional quantitative PCR and the real-time PCR were plotted. A regression analysis was used for the comparison. The linear regression plots and corresponding values are shown.
Figure 3.
Figure 3.
EBV viral levels in two pediatric patients. A: EBV levels in sequential samples from pediatric transplant recipient were determined using the Taqman real-time PCR assay. B: EBV levels in sequential samples from a pediatric patient with acute EBV infection were determined using the molecular beacon real-time PCR assay.
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