Original Article

Yonago Acta medica 2016;59:217–222

Construction and Evaluation of Cytomegalovirus DNA Quantification System with
Real-Time Detection Polymerase Chain Reaction
Yuki Hatayama,* Yuki Hashimoto,* Ayako Hara* and Toru Motokura*†*Division of Clinical Laboratory, Tottori University Hospital, Yonago 683-8504, Japan and †Division of Clinical Laboratory Medicine,
Department of Pathophysiological and Therapeutic Science, School of Medicine, Tottori University Faculty of Medicine, Yonago 6838503, Japan

ABSTRACT

after hematopoietic stem cell transplantation and solidorgan transplantation.1, 2
CMV establishes latency in cells of the myeloid
lineage following primary infection. The CMV antigenemia test is the most widely used quantitative assay forthe diagnosis and follow-up of CMV infection, whichbecomes positive prior to the onset of CMV disease.3–5
The CMV antigenemia test made it possible to start
the administration of antiviral agents as the preemptive
therapy when CMV antigen-positive cells were detected
in a certain amount. However, the CMV antigenemia
test has disadvantages such as long inspection time andcomplicated procedures. Various reports have been
made about the usefulness of real-time PCR to quantify
CMV DNA in recent years. One study comparing theusefulness of quantitative PCR system and CMV antigenemia tests for monitoring CMV reactivation showedthat quantitative PCR have sensitivity equal to or morethan that of CMV antigenemia.6–9 A quantitative PCRsystem for CMV DNA is commercially available and so
expensive that it is not covered by health insurance inJapan. The aim of this study is to construct an original
quantitative PCR system for CMV DNA, and to evaluate
the analytical performance.

Background

For patients with reactivation of humancytomegalovirus (CMV), a highly sensitive and accurate
CMV quantification system is essential to monitor viralload.
Methods We constructed a real-time detection PCR(RTD-PCR) system for CMV DNA and evaluated its
linearity, lower detection limit, dynamic range and accuracy using two CMV standards. We used 219 clinicalsamples derived from 101 patients to compare the system with the pp65 antigen test.
Results The 95% detection limit was determined to be
556 IU/mL (95% CI, 440–797 IU/mL), and the quantification range was between 102 and 106 copies or IU/mL(r = 0.996, 0.999, respectively). The coefficients of variation of inter-assay reproducibility assessed in each threedifferent runs were 2.5% at 1,000 IU/mL and 1.6% at
10,000 IU/mL. The coefficients of variation of intra-assay variability by testing the same samples three times
in a single run were 1.8–3.6% and 0.4–1.9%, respectively. The concordance between antigenemia and plasma
or serum CMV DNA levels was a good correlation (r =0.695, P < 0.01).
Conclusion We constructed the RTD-PCR system
which enables accurate evaluation of CMV reactivation
by monitoring of viral load in immunosuppressed or immunocompromised patients.

MATERIALS AND METHODSStandard and clinical samples

Plasmid DNA, incorporating CMV glycoprotein B (gB)region (UL55), was made as a standard, the copy number of which was calculated based on mass and baselength. The CMV DNA reference panels, which arederived from AD169 strain World Health Organization(WHO) International HCMV standard, were purchasedfrom the National Institute for Biological Standards
and Control (NIBSC, Ridge, Hertfordshire, UK). This
material has been assigned a concentration of 5 × 106
International Units (IU) when reconstituted in 1 mL ofnuclease-free water.10 Archived 219 serum or plasma
samples remaining after CMV antigenemia test wereavailable at Tottori University Hospital, and stored at–20 ℃ until the study. Viral DNA was extracted from200 μL of serum or plasma using commercial DNA isolation reagents (Qiagen, Tokyo, Japan). This study was

Key words CMV antigenemia, cytomegalovirus,
quantification, RTD-PCR

Cytomegalovirus (CMV) is a DNA virus that belongs to
the herpes virus family, and often causes severe disease
in immunosuppressed or immunocompromised patients.
It is one of the most frequently occurring complications
Corresponding author: Toru Motokura, MD, PhD
motokura@med.tottori-u.ac.jp
Received 2016 March 28
Accepted 2016 June 2
Abbreviations: CBT, cord blood transplantation; CMV, cytomegalovirus; Ct, cycle of threshold; DNA, deoxyribonucleic acid; gB,
glycoprotein B; IU, international unit; pp65 antigen, polypeptide 65
antigen; RTD-PCR, real-time detection polymerase chain reaction;
WHO, World Health Organization217

Y. Hatayama et al.

approved by the Ethics Committee at Tottori University
Faculty of Medicine (approval number 2326).

results were reported as log10 pp65 positive cells/30,000
examined and CMV DNA titers were also reported aslog10 IU/mL. The differences between titers obtained byboth quantitative tests were considered statistically significant for P-value < 0.05, according to Mann-Whitney
U test (SPSS statics 21; IBM, Armonk, NY).

Primer sets

Primers for the CMV gB region sequence were designed(Takara Bio, Kusatsu, Japan) as follows : forward primer
5’-CCCTTGAGGTAGGGCGGTAG-3’, reverse primer
5’-TGCGCGAATTCAACTCGTACA -3’, and TaqMan
probe (FAM) 5’-ACCTTGTCCTCCACGTACTTTACCCGCT-3’ (TAMRA).

RESULTS
Standards calibration and linearity

For the calibration of standards, two standard panels(pCMV gB, NIBSC) were cross-validated in the same
reaction. Standard curves for CMV quantification were
established for each panel by serially diluting the standards from 106 to 102 copies/mL (IU/mL for NIBSC
standard) and by amplification in triplicates. We generated a log-linear regression plot by plotting thecycle ofet al., 18
Y. Hatayama
threshold (Ct) value. The correlation coefficients of thetwo different standards were 0.996 and 0.999, respec-

ProtocolThe RTD-PCR reaction was carried out in a 50 μL reaction mixture containing 900 nM forward and reverse
primer, 250 nM TaqMan Probe, 2 X TaqMan Universal
PCR Master Mix (Applied Biosystems, Waltham, MA)
and 5 μL template. The mixture was incubated at 50 ℃
for 2 minutes for initial activation of uracil-N-glycosylase, followed by inactivation of uracil-N-glycosylase atFig. 1.
95 ℃ for 5 minutes. Subsequent PCR amplification consisted of 60 cycles of denaturation at 95 ℃ at 15 seconds
and annealing and extension at 60 ℃ for 1 minute in an
ABI7300 Real-time PCR System (Applied Biosystems).

45
40

y = –3.1011x + 47.308
R² = 0.99636

35

Evaluation of the RTD-PCR assay

30
Ct Value

Conversion of CMV DNA international units (IU) to
copies/mL was evaluated by serial dilution of NIBSC
standards from 106 to 102 IU/mL, with triplicates at every dilution. Linearity of quantification by RTD-PCR
system was evaluated as the log recovery for each member of quality control panel. The lower detection limit
was determined by 10-fold serial dilutions from 103 to
101 IU/mL in 16 replicates.

25
y = –3.5835x + 49.441
R² = 0.9993

20
15
WHO

10

pCMV_gB5

CMV antigenemia test

0

The CMV antigenemia test is used for detecting viral
matrix phosphoprotein (pp65) in peripheral blood leukocytes (PBLs) by indirect immunostain assay. Brieﬂy,
EDTA-treated whole blood samples were fractionatedby dextran sedimentation and lysis of erythrocytes. The
granulocytes were then centrifuged to prepare a cytospin
slide (15,000 granulocytes per slide). The cells were thenfixed with formaldehyde, sequentially immunostainedby monoclonal antibodies C10/C11 (LSI CMV; LSI
Medience, Tokyo, Japan), and reacted with goat alkaline
phosphatase-labeled anti-mouse immunoglobulin as thesecond reaction. The examination was performed in duplicate. Measurement of the CMV antigenemia test was
performed by LSI Medience.

0.0

1.0

2.0

3.0 4.0 5.0 6.0
Log10 copies or IU/mL

7.0

8.0

9.0

Fig. 1. Standard curves generated by linear regression analysis of
the Ct value measured for each amplification versus the log10 virus
copy number for each standard dilution. , WHO standard panel
ranging from 106 IU/mL ; , plasmid pCMV_gB with original 106
copies/mL. Ct, cycle of threshold; CMV, cytomegalovirus; gB,
glycoprotein B; IU, international unit; WHO, World Health Organization; y, Ct value; R2, coefficient of determination.

tively (Fig. 1). The two standards exactly corresponded
to each other; the conversion factor was 1.02–1.07 copies/IU for each concentration (3–7 log copies or IU/mL).
Low detection limit

Further validation of the lower limit of detection of
RTD-PCR system was obtained by testing multiple replicates of dilutions of the standard. Dilutions (equal to

Statistical analysis

For correlation analysis of obtained data, antigenemia
218

CMV DNA quantification system with RTD-PCR

1000, 500, 250, 125, 63, 31 IU/mL) were tested for 16
replicates at each dilution. Using Probit analysis (IBMSPSS 21), the number of positive results at each dilution
was used to calculate the CMV DNA concentration having a 95% probability of detection (Table 1). The 95%
detection limit was determined to be 556 IU/mL (95%CI, 440–797 IU/mL).

1.8–3.6% and 0.4–1.9% for the low and high concentrations, respectively.
Comparison with CMV antigenemia test

Table 1. RTD-PCR assay data used for probit analysis
CMV
concentration(IU/mL)

NumberNumberpositive/tested expected
positive

Probability

1000
16/16
15.999
1.000
500
14/16
14.583
0.911
250
9/16
8.248
0.516
125
5/16
4.301
0.269
62.5
4/16
2.761
0.173
31.3
2/16
2.143
0.134
0.0
0/16
1.628
0.102
Fig. 2.
CMV, cytomegalovirus; IU, international unit; RTD-PCR, reA
al-time detection polymerase chain reaction.

Specificity

Twelve normal human sera were tested by both assays
and all gave negative results.

All 219 clinical samples derived from 101 patients wereanalyzed both by the pp65 antigenemia test and the
RTD-PCR system. Results of 90 were negative and 75
were positive with both tests. On the contrary, 42 samples (19.1%) that tested negative by pp65 antigenemia
were positive by PCR and showed moderate levels of
CMV DNA (mean viral load 2.9 Log copies/mL) (Table2). Twelve samples were positive by the antigenemia test
and negative by RTD-PCR.
In the case of PCR-positive samples more than orequal to 556 (2.7 log10) IU/mL, the CMV DNA levels
Y. Hatayamasamet al., 19
of 62 pp65-positive samples and 32 pp65-negative
ples were compared. The CMV DNA were significantly
higher in pp65 positive samples than in negative samples(Fig. 2A). In cases of samples with concordant positive
results, the correlation between the number of CMVDNA log copies/mL of plasma or serum and the numberof pp65 positive cells per 30,000 PBLs was statisticallysignificant (r = 0.695, P < 0.01) (Fig. 2B). None of thepatients developed
CMV-related symptoms.
*P < 0.05
Table 2. Comparison between CMV DNA and pp65
antigen results in the examined samples

Reproducibility study

pp65 – pp65 + Total
Inter-assay reproducibility was assessed by repeating 2
CMV DNA –90
12102
samples of different concentrations, 1,000 IU/mL and10,000 IU/mL, in each of three different runs of RTD13
23
CMV DNA + (< 2.7 Log10 IU/mL) 10
PCR. The coefficients of variations were 2.5% at 1,000
6294
CMV DNA + (≥ 2.7 Log10 IU/mL) 32
Y. Hatayama et al., 19
IU/mL and 1.6% at 10,000 IU/mL. Intra-assay variCMV, cytomegalovirus; IU, international unit ; pp65 antigen,ability was evaluated by testing the same samples threepolypeptide
real-time detection polyn = 32 65 antigen ; RTD-PCR,
n = 62
merase chain reaction.
times in a single run. The coefficients of variation were
Fig. 2.

B
pp65 Antigen (Log10/30,000)

A*P < 0.05

2.5
r = 0.695, **P < 0.01

2.0
1.5
1.0
0.5
0.0

2.0

3.0

4.0
CMV DNA (Log10 IU/mL)

n = 32

g10/30,000)

B
2.5
2.0
1.5

r = 0.695, **P < 0.01

n = 625.0

6.0

Fig. 2. Result of clinical samples. (A) CMV DNA loads of RTD-PCR in PCR-positive samples in relation to pp65 antigen results. (B) Correlation between CMVDNA load (log10 IU/mL) in serum or plasma samples and pp65 antigen values (log10
positive cells/30,000 examined) among all samples testing positive by RTD-PCR
assay. CMV, cytomegalovirus; IU, international; unit; pp65 antigen, polypeptide 65
antigen; RTD-PCR, real-time detection polymerase chain reaction.

219

Fig. 3

Y. Hatayama et al.

Patient A

GCV
VGCV

Patient B

GCV
PSL

pp65 antigen

CBTFig. 3. Patterns of pp65 antigen ( ) and CMV DNA ( ) in two patients. Patient A with diffuse large B-cell lymphoma
developed bone marrow suppression. It became impossible to undergo the pp65 antigenemia test because of WBC ( )decreased in number. Patient B underwent CBT. The period and dose of preemptive and steroid therapies are shown. CBT,
cord blood transplantation; CMV, cytomegalovirus; GCV, ganciclovir; IU, international unit; pp65 antigen, polypeptide 65
antigen; PSL, prednisolone; VGCV, valganciclovir; WBC, white blood cell.

220

CMV DNA quantification system with RTD-PCR

Case study

leukopenia or myelosuppression than antigenemia test
(patient A in Fig. 3). Both antigenemia and CMV DNA
load deceased after the initiation of therapy. In addition,
antigenemia assay and RTD-PCR system showed asimilar trend (patient B in Fig. 3). These results indicatethat using the RTD-PCR system to monitor quantities of
CMV DNA could be useful in ganciclovir treatment.
Twelve samples were pp65 positive and CMV DNA
negative while 42 samples were reverse. The targets ofthe two methods are different; RTD-PCR amplifies free
CMV DNA in plasma and the CMV antigenemia test
detects pp65 antigens in peripheral blood leukocytes.
Which target is appropriate for monitoring CMV infection for clinical use remains to be determined. Their
combination may explain virus status more accurately.
In conclusion, we constructed a RTD-PCR system
which has sufficient sensitivity, specificity, accuracy,
and reproducibility. Quantification can be performed
within three hours without false-positive results. Basedon our results, the RTD-PCR system appears to be as
useful as any commercial CMV quantification system.
Furthermore, it may be useful for patients to consider
comprehensively the results of RTD-PCR and CMV antigenemia test.

We followed two immunosuppressed patients who had
both tests (Fig. 3). Patient A with diffuse large B-cell
lymphoma developed bone marrow suppression. By
anti-viral therapy, pp65 antigen decreased immediately,while CMV DNA also decreased, however, remaining athigh levels. After a while, it became impossible to perform the pp65 antigenemia test because of leukopenia.
Patient B underwent cord blood transplantation (CBT).
After CBT, both tests showed similar transient positive
results.
DISCUSSION
The amount of CMV DNA in blood is a result of the
dynamic state between viral replication and clearance.
Real-time PCR is a promising diagnostic approach for
CMV infection after hematopoietic stem cell transplantation or during chemotherapy since it is more suitable
for standardization and easier to handle than the conventional antigenemia assay. Furthermore, PCR system using plasma or serum can be used during leukocytopenia
while the antigenemia assay does not produce reliabledata without sufficient leukocytes.
Currently, after the introduction of pre-emptive
therapy with ganciclovir by monitoring with the CMV
antigenemia test, CMV disease including CMV-related
pneumonia is nearly preventable.11, 12 We developed anovel CMV DNA quantification system using an RTDPCR system targeting the gB gene. The coefficient ofcorrelation between DNA quantity and Ct value was r >0.99, indicating good linearity. And good reproducibility
and a wide dynamic range (102–106 IU/mL) were shown.
The lower limit of quantification (556 IU/mL) is inferior to that of the commercial PCR kit (150 copies/mL,
Roche Diagnostics, Basel, Switzerland). There are two
methods of RTD-PCR that amplify CMV US17 region(US17-PCR) and immediate early (IE) gene (IE-PCR).
Different sensitivity and threshold to start ganciclovir
were reported for each method.13 And the thresholds ofthe viral load to initiate preemptive therapy were also
different according to clinical situations. For example, inthe study of solid organ transplant recipients evaluating
RTD-PCR of plasma and pp65 antigenemia, an antigenemia threshold value of 20 positive cells/20,000, wasequivalent to 2500 copies/mL (2275 IU/mL) for antiviraltreatment.14 In case of hematopoietic stem cell transplant
recipients from unrelated donors, a much lower thresholdof CMV DNA titer was used for preemptive therapy.15
The present study shows a good correlation between
antigenemia and plasma or serum CMV DNA levels (r= 0.695). The novel assay also showed more usefulness
in monitoring the CMV reactivation of patients with

The authors declare no conflict of interest.

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