JOURNAL OF CLINICAL MICROBIOLOGY, July 2000, p. 2734–2737 0095-1137/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Vol. 38, No. 7
NOTES Detection of Human Cytomegalovirus DNA by Real-Time Quantitative PCR ANDREAS NITSCHE,1 NINA STEUER,1 CHRISTIAN ANDREAS SCHMIDT,1 OLFERT LANDT,2 HEINZ ELLERBROK,3 GEORG PAULI,3 AND WOLFGANG SIEGERT1* Klinik fu ¨r Innere Medizin m.S. Ha ¨matologie und Onkologie, Charite´-Campus Virchow Klinikum, Humboldt Universita ¨t zu Berlin,1 TIB Molbiol,2 and Department of Virology, Robert Koch-Institut,3 Berlin, Germany Received 4 October 1999/Returned for modification 18 January 2000/Accepted 17 April 2000
A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients. al. (7). Recently, we compared this optimized TaqMan chemistry-based assay with the HybProbe format for real-time PCR detection (9). In this study, we present the development of a quantitative TaqMan-based PCR assay which can detect CMV DNA with high precision and reproducibility. In a first attempt to prove the applicability of the assay, we analyzed plasma samples of bone marrow transplant (BMT) patients. A total of 194 samples of heparinized plasma were obtained
Human cytomegalovirus (CMV) infections can cause severe complications in immunocompromised individuals (2, 16). In these patients, there is a need for early, rapid, and sensitive diagnosis because new antiviral drugs can reduce the frequency and severity of CMV disease (3, 4, 6, 14). We therefore established a real-time PCR assay for the detection and quantification of CMV DNA using TaqMan chemistry. The methodological basis for the assay system was first described by Holland et
FIG. 1. Schematic representation of primer-exonuclease probe combinations used in the TaqMan-based assay. The black bar represents a 500-bp fragment of the CMV major immediate-early gene locus (accession no. M21295). Oligonucleotide locations are as follows: MIE5, 3170 to 3147; MIE4, 2736 to 2760; 1F, 3168 to 3151; 1B, 2742 to 2760; 2B, 2942 to 2960; 3F, 3120 to 3100; 3B, 3025 to 3048; 4F, 2971 to 2950; 4B, 2799 to 2818; TM1, 2873 to 2894; TM2, 3086 to 3061; and TM3, 2835 to 2860. Bold numbers indicate the amplicon size.
* Corresponding author. Mailing address: Klinik fu ¨r Innere Medizin m.S. Ha¨matologie und Onkologie, Charite´-Campus Virchow Klinikum, Humboldt Universita¨t zu Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Phone: 49-30-45053673. Fax: 49-30-45053925. E-mail:
[email protected]. 2734
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FIG. 2. Calibration curve. Using primer-exonuclease set E, we measured 107 to 101 copies of plasmid pMIE per assay. Using the threshold cycle (CT) values for the corresponding plasmid copy numbers, we constructed a calibration curve. Input plasmid pMIE copy number per assay is plotted versus threshold cycle. The inset shows the PCR products after agarose gel electrophoresis and ethidium bromide staining.
from 10 BMT patients prior to and at weekly intervals after transplantation and stored frozen at ⫺20°C until use. DNA for PCR was prepared using an InViSorb Spin DNA Micro Kit III (InVitek, Berlin, Germany). The elution volume was chosen to
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obtain 1 l of DNA solution per l of prepared plasma. For the pp65 antigen detection assay, buffy coat leukocytes were prepared from the same samples using previously reported methods (12, 13). As a positive control template, a plasmid containing the target sequence from the major immediate-early region of CMV (pMIE) was constructed as described elsewhere (9). Serial dilutions of 107 to 101 plasmids per assay were prepared by using plasma from anti-CMV immunoglobulin G-negative blood donors and human placental DNA as a carrier (Sigma, Deisenhofen, Germany). PCR was performed with a Perkin-Elmer model 7700 sequence detection system. Optimum conditions were obtained with 16 mM (NH4)2SO4, 50 mM Tris-HCl (pH 8.8), 0.01% Tween 20, 2.0 mM Mg2⫹, 0.8 M each primer (1F and 1B), 80 M each deoxynucleoside triphosphate (n ⫽ 4), 1 M 6-carboxy-X-rhodamine, 50 nM exonuclease probe TM2, and 1 U of InViTaq DNA polymerase (InVitek). Five microliters of template was added, to result in a 50-l reaction mixture. Amplifications were performed with 3 min of template denaturation at 94°C, followed by 45 cycles at 94°C for 20 s, 64°C for 20 s, and 72°C for 1 min. Variables were compared using the Mann-Whitney test. P values of ⱕ0.005 were considered significant. For assay optimization, 10 primer-exonuclease probe combinations were tested to detect serial dilutions of plasmid pMIE (Fig. 1). We could not confirm some of the guidelines given by Perkin-Elmer for the design of primer-exonuclease probe sets: neither reducing the amplicon size nor increas-
FIG. 3. Time courses for pp65-positive cells in the antigenemia assay (bars) and CMV DNA load in plasma determined by quantitative PCR (circles) using the TaqMan-based assay. Data for four typical BMT patients are shown before and after transplantation (TX). PBL, peripheral blood leukocytes.
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FIG. 4. CMV DNA load in plasma of healthy blood donors, pp65-negative (neg) BMT patients, and pp65-positive (pos) BMT patients. The median DNA concentration for blood donors and pp65-negative BMT patients is 0 ge/ml of plasma, whereas the median DNA level (horizontal bar) for pp65-positive BMT patients is 8,838 ge/ml of plasma.
ing the Mg2⫹ concentration led to an improvement in the assay. Primer-exonuclease probe combinations E and F detected as few as 10 plasmids per assay, whereas the remaining combinations showed higher detection limits, for example, ⬎1,000 plasmids per assay for combination J. We found that reducing the amplicon size did not result in an improvement in the detection limit (combinations G, H, J, and K). Mg2⫹ concentrations were varied from 1.0 to 10.0 mM and determined to be optimal at 2.0 mM for combination E and 8.0 mM for combination F. However, the detection limit could not be improved to plasmid amounts below 10 plasmids per assay. Increasing the exonuclease probe concentration had no beneficial effect on PCR performance. Finally, the ratio of sense primer to antisense primer was optimal when the primers were used in equimolar amounts. To determine the assay precision with these assay conditions and serial dilutions of plasmid pMIE, a calibration curve was constructed (9), with a correlation (r2) of 0.99 for 101 to 107 plasmids per assay (Fig. 2). This range is sufficient for the detection of CMV DNA in clinical samples (5). No crossreactivity with DNAs from herpes simplex virus types 1 and 2, varicella-zoster virus, Epstein-Barr virus, and human herpesviruses 6, 7, and 8 could be demonstrated (data not shown). To determine the intra-assay precision of the CMV DNA TaqMan-based assay, DNA was isolated from three plasma samples, containing 1,151, 437, and 12.5 genome equivalents (ge)/ml, as previously measured with the TaqMan-based assay. Each plasma sample was assayed four times. The resulting standard deviations were 58 ge/ml (5%), 49 ge/ml (11%), and 4 ge/ml (32%) for the respective DNA concentrations. The interassay precision was determined by measuring identical DNA preparations on six consecutive days in duplicate. The resulting standard deviations were 172 ge/ml (15%), 66 ge/ml (15%), and 2 ge/ml (16%), respectively. Thus, the variability of this assay is low compared to those of previously published assays (1, 10, 11).
To explore the clinical applicability of the CMV DNA TaqMan-based assay, we studied 194 consecutive plasma samples from 10 patients after BMT. Three patients were known to be negative in the pp65 antigenemia assay throughout the posttransplantation course, and seven patients were positive in the pp65 antigenemia assay. The pp65-positive BMT patients (Fig. 3, patients 1, 2, and 3) displayed higher levels of CMV DNA in plasma than did the pp65-negative BMT patients (Fig. 3, patient 4) (15). While the correlation of the pp65 peak and the DNA peak was good in most patients (patients 2 and 3), in patient 1 the peaks were shifted. The discordance between the detection of antigen and the detection of DNA may be due to the fact that two distinct viral components in different materials (leukocytes and plasma) were being tested. Preliminary data suggest a higher correlation when DNA from peripheral blood leukocytes is used for the TaqMan-based assay. Of the 194 plasma samples tested, 27 plasma samples were from a control group of healthy blood donors including 5 seropositive and 22 seronegative individuals; also included were 122 pp65-negative and 45 pp65-positive samples. As expected, we demonstrated that pp65-positive samples contained significantly higher concentrations of CMV DNA than did pp65-negative samples (P ⬍ 0.0001) and samples from healthy blood donors (P ⬍ 0.0001) (Fig. 4). Although 5 of 27 healthy blood donors (19%) were DNA positive, the level of CMV DNA was significantly lower than that in samples from pp65negative BMT patients (P ⫽ 0.0046). Interestingly, the five DNA-positive blood donors were seronegative, and none of the five seropositive blood donors was DNA positive. Larsson et al. demonstrated recently that CMV DNA can be detected in peripheral blood leukocytes of all seropositive and most seronegative blood donors investigated over time (8). Hence, it is not surprising that we detected CMV DNA in the plasma of healthy, seronegative individuals. In summary, we have presented a precise and rapid PCR assay for the quantification of CMV DNA which may prove useful for routine clinical testing.
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