Supplemental file 1
Quantification of plasma EBV DNA by quantitative polymerase chain reaction (Q-PCR)
with a World Health Organization (WHO) standard 09/260
Q-PCR for EBV EBNA1 gene
Plasma DNA was extracted from the peripheral blood with a commercial kit (QIAamp
DNA mini kit, Qiagen GmbH, Hilden, Germany). Q-PCR for EBV was performed on plasma
DNA as previously reported.1 Briefly, plasma DNA was amplified in triplicates with a pair of
primers (EBNA1F: 5’-TCA TCA TCA TCC GGG TCT CC-3’; EBNA1R: 5’- CCT ACA
GGG TGG AAA AAT GGC-3’) and a TaqMan probe (6FAM-CGC AGG CCC CCT CCA
GGT AGA A) targeting the EBNA-1 gene. The reaction was set up in a volume of 50 L with
the TaqMan Universal PCR Master Mix (PE Biosystems, Foster City, CA, USA), containing
5 L purified DNA, 300 nM of primers EBNA1F/EBNA1R, and 200 nM of the TaqMan
probe. Thermal cycling was initiated with a 2-minute incubation at 50°C, followed by a first
denaturation step of 10 minutes at 95°C, and then 40 cycles of 95°C (denaturation) for 15
seconds and 60°C (re-annealing and extension) for 1 minute. Real-time PCR data were
collected continuously and analyzed with the ABI Prism 7700 Sequence Detector (PE
Biosystems). Cycle thresholds (CT), at which a significant increase in fluorescence signal was
first detected, were set at a minimum of 10 standard deviations above the mean baseline
fluorescence, calculated from cycles 1 to 15. As the larger the starting quantity of target
sequence there was, the earlier a significant increase in fluorescence would be observed, the
CT was inversely proportional to the starting target gene copy number. Strict adherence to
quality assurance and controls was as previously described.1
1
EBV Q-PCR with the WHO standard 09/260
The EBV Q-PCR was calibrated against a WHO standard 09/260 (Epstein-Barr Virus
for Nucleic Acid Amplification Techniques, NIBSC; Hertfordshire, United Kingdom)
according to the recommended procedures.2,3 The standard 09/260 was reconstituted with 1
mL of de-ionized, nuclease-free molecular-grade water to a final stock concentration of 5 x
106 IU/mL. A serial dilution of 09/260 was then made, covering a 4-log range from 5 x 106
IU to 5 x 102 IU/mL over 9 dilutions (5 x 106, 2.5 x 106, 5 x 105, 2.5 x 105, 5 x 104, 2.5 x 104,
5 x 103, 2.5 x 103, 5 x 102 IU/mL). From each of these diluted standards, 5 L (corresponding
to an absolute amount of 2.5 x 104, 1.25 x 104, 2.5 x 103, 1.25 x 103, 2.5 x 102, 1.25 x 102, 2.5
x 10, 1.25 x 10, 2.5 IU) was amplified by Q-PCR in triplicates. A standard curve was then
constructed, by plotting the CTs against the logarithm of the starting quantities of 09/260
(Figure 1).
EBNA1- Ct vs log IU
40.00
y = -3.268x + 36.939
2
R = 0.998
Avg. Ct
30.00
20.00
10.00
0.00
0.000
2.000
log IU
4.000
WHO International Standard (NIBSC)
Figure 1. Standard curve constructed with WHO standard 09/260.
2
Patient samples were assayed in triplicates. The CTs of the patient samples were
determined, and the initial starting amount was calculated from the standard curve.
Calibration of the pB-EBNA1 plasmid against WHO standard 09/260
Some of the patient samples were initially quantified by using an in-house plasmid
standard pB-EBNA1.1 To calibrate pB-EBNA1 against the WHO standard 09/260, validation
experiments were conducted, by plotting the standard curve of pB-EBNA1 against that of
WHO standard 09/260 over a similar 9-point range, so as to demonstrate that the efficiencies
of amplification of the two standards were comparable.
EBNA1- Ct vs log IU
40.00
30.00
y = -3.264x + 41.059
Avg. Ct
y = -3.268x + 36.939
2
2
R = 0.999
R = 0.998
20.00
10.00
0.00
0.00
2.00
4.00
log IU
6.00
8.00
10.00
p1 EBNA1
WHO International Standard
(NIBSC)
Figure 2. Amplification efficiencies of pB-EBNA1 and WHO 09/260.
As shown in Figure 2, the standard plots of pB-EBNA1 and the WHO standard
09/260 were parallel. As stipulated by the manufacturer,2,3 the absolute value of the slope of
the standard plot (logarithm of input versus CT) should be <0.1. In Figure 2, the calculated
3
slope was 0.0639, thus validating the calibration experiment. Hence, the plasmid pB-EBNA1
was also valid for determining the IU of samples previously assayed in comparison with the
WHO standard 09/260.1
References
1. Au WY, Pang A, Choy C, Chim CS, Kwong YL. Quantification of circulating EpsteinBarr virus (EBV) DNA in the diagnosis and monitoring of natural killer cell and EBVpositive lymphomas in immunocompetent patients. Blood. 2004;104(1):243-9.
2. Fryer JF, Heath AB, Wilkinson DE, Minor PD, and the Collaborative Study Group.
Collaborative study to evaluate the proposed 1st WHO international standard for EpsteinBarr Virus (EBV) for nucleic acid amplification technology (NAT)-based assays.
http://www.who.int/biologicals/expert_committee/BS2011.2172Epstein_Barr_Virus.pdf. Last
accessed on January 2, 2013.
3. National Institute for Biological Standards and Control. WHO International Standard. 1st
WHO international standard for Epstein-Barr Virus for nucleic acid amplification techniques
NIBSC
code:
09/260.
Instructions
for
use
(Version
2.0,
Dated
12/01/2012).
http://www.nibsc.ac.uk/documents/ifu/09-260.pdf. Last accessed January 2, 2013.
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