Biomedical Engineering Reference
In-Depth Information
Mutirangura et al. showed that EBV DNA could be detected in the serum of 14
of 42 NPC patients but none of the 82 control subjects
(
5
)
. This observation
suggests that the composition of the serum and the cellular compartments are
significantly different. Later, our group developed a quantitative real-time poly-
merase chain reaction (PCR) assay for the detection of plasma EBV DNA, and
we were able to detect EBV DNA in the plasma of 96% of NPC patients
(
6
)
.
On the contrary, only 7% of healthy individuals showed positive results, and
their plasma EBV DNA levels were much lower than the NPC patients
(
6
)
.
Furthermore, the levels of plasma EBV DNA showed a strong correlation with
the clinical stages of the patients
(
6
)
. The median concentration of the plasma
EBV DNA of late-stage patients (stages III and IV) was almost eight times
higher than that of the early-stage (stages I and II) patients
(
6
)
. These observa-
tions suggested that EBV DNA is tumor-derived and its concentration reflects
the tumor burden. To date, quantitative analysis of circulating EBV DNA has
been shown to be a very important tool for the detection
(
6
)
, monitoring
(
7
)
,
and prognostication
(
8
)
of NPC and other EBV-associated malignancies
(
9
,
10
)
.
Despite a growing number of clinical applications of circulating EBV DNA
analysis, the molecular nature of these DNA species was unclear. Regarding
this issue, we used different approaches to study the nature of these EBV DNA
molecules
(
11
)
. To discriminate whether the circulating EBV DNA molecules
exist as DNA fragments or as a component of intact virions, we subjected the
plasma of NPC patients to DNase digestion and ultracentrifugation
(
11
)
. First,
we showed that viral particles were resistant to DNase digestion and are
pelletable by ultracentrifugation
(
11
)
. In contrast, spiked extracted EBV DNA
could no longer be detected in the plasma of NPC patients after DNase diges-
tion and, after ultracentrifugation, most of the extracted EBV DNA remained
in the supernatant
(
11
)
. The observation that circulating EBV DNA in NPC
patients is susceptible to DNase digestion but not pelletable by ultracentrifuga-
tion suggests that plasma EBV DNA exists as DNA fragments instead of a part
of an intact virion.
Furthermore, we developed a method for the quantitative analysis of the frag-
ment size of circulating EBV DNA fragments. We have shown that 87% of
circulating EBV DNA in NPC and lymphoma patients are below 181 bp in size
(
11
)
. This method has further been modified for the analysis of the size of
circulating DNA in pregnant women by targeting a gene in the human genome
(e.g., the
Leptin
gene)
(
12
)
. We showed that the circulating DNA in pregnant
women is longer than that of the nonpregnant counterparts, and the fetal-
derived DNA in maternal circulation is shorter than the maternally derived
DNA
(
12
)
. The principle and protocols of the quantitative analysis of the size
of circulating EBV DNA are discussed later.
For the measurement of the length of plasma EBV DNA molecules, a panel
of 10 quantitative real-time PCR assays targeting a region encoding the EBV-
