Normalized quantification of human cytomegalovirus DNA by competitive real-time PCR on the LightCycler instrument

Abstract

The development of a novel normalized quantitative competitive real-time PCR on the LightCycler instrument (NQC-LC-PCR) and its application to the quantification of cytomegalovirus (CMV) DNA in clinical samples are described. A heterologous competitor DNA was spiked into test samples and served as an internal amplification control. The internal control (IC) DNA in the test samples was coamplified with the CMV DNA and was tested against a calibrator sample that contained equal amounts of IC DNA and CMV reference standard DNA. An algorithm was developed to normalize possible varying amplification efficiencies between the standard and the samples. After normalization, CMV DNA copy numbers were determined in absolute terms. In a routine clinical setting, normalized quantification by NQC-LC-PCR using a single IC concentration led to results ranging from 500 to 50,000 CMV DNA copies/ml. The results obtained with conventional real-time quantification on the LightCycler instrument were almost identical to those obtained with the NQC-LC-PCR-based quantification. This was the case only for samples in which the PCR was not inhibited. With partially inhibited samples, NQC-LC-PCR was still able to correctly quantify CMV DNA copy numbers even when the PCR was inhibited by about 70%. By analyzing 80 undefined clinical samples, we found that NQC-LC-PCR was suitable for the routine assessment of CMV DNA in clinical plasma samples. Since the ICs were already added to the samples during the DNA purification, almost the entire assay was controlled for sample adequacy. Thus, false negative results were precluded. The NQC-LC-PCR approach developed should be adaptable for additional microbiological applications.

FIG. 1.

Schematic sketch of the CMV-specific IC (A) and CMV-specific PCR product (B). The IC DNA fragment consists of the CMV-specific primer sequences flanking the 5′ and 3′ termini of the neo gene sequence, which was used as heterologous DNA. IC DNA and CMV-specific DNA were amplified with the same set of primers. The sizes of the IC-specific and CMV-specific PCR products were 325 and 278 bp, respectively. The 3′ FRET hybridization probe specific for neo was labeled with LC-Red 640 and detected on channel F2 of the LightCycler instrument, whereas the 3′ FRET hybridization probe specific for CMV was labeled with LC-Red 705 and detected on channel F3.

FIG. 2.

Real-time fluorescence plot of the CMV-specific amplification (A) and the IC-specific amplification (B). One hundred copies of the IC and the indicated number of copies of CMV DNA were applied to each capillary. Note in panel B that with increasing amounts of CMV DNA, the IC amplification was competitively inhibited.

FIG. 3.

Normalized quantification of CMV reference standard DNA. Ten, 20, 100, 200, and 1,000 copies of CMV reference standard DNA per capillary (n = 8) were assessed by NQC-LC-PCR and conventional LightCycler PCR. (A) CMV copies measured by NQC-LC-PCR were plotted against applied CMV copies (r = 0.972; P < 0.001; 95% CI, 0.946 to 0.985). (B) CMV copies measured by NQC-LC-PCR were plotted against CMV copies measured by conventional LightCycler PCR (r = 0.971; P < 0.001; 95% CI, 0.945 to 0.984; linear regression analysis, y = 1.02x − 0.05). Note that none of the samples was inhibited.

FIG. 4.

Normalized quantification of CMV DNA in PCR-inhibited samples by NQC-LC-PCR. Approximately 100 copies of CMV DNA and 100 copies of IC DNA were amplified in the presence of hemoglobin added in graded amounts. Quantification was performed in normalized fashion (open squares) and by the conventional real-time LightCycler method (closed squares). Quantified CMV copies in samples with hemoglobin were compared with quantified CMV copies in samples without hemoglobin by Student's t test. Symbols marked with an asterisk indicate significantly different CMV copy numbers (P < 0.005) between samples with and without hemoglobin; symbols without an asterisk indicate that results were not significantly different (P > 0.05).

FIG. 5.

Correlation between quantitative CMV DNA values from clinical samples analyzed by NQC-LC-PCR assay and by conventional LightCycler assay. The scatter diagram and the regression line show the relation of mean numbers of CMV DNA copies (squares) for 28 CMV-positive clinical samples assessed by both methods (r = 0.973; P < 0.001; 95% CI, 0.943 to 0.987; linear regression analysis, y = 1.07x − 0.18). The NQC-LC-PCR assay used a single calibrator sample for normalized quantification. For the conventional LightCycler assay, a serial dilution of CMV reference standard DNA was used as the external standard. In the NQC-LC-PCR, the results for four samples exceeded the range of quantification. These samples were diluted 1:10 and retested (triangles). Note that none of the samples appeared to be inhibited.