Journal of Medical Virology 80:1426–1433 (2008)
A Simple One-Step Real-Time RT-PCR for Diagnosis
of Dengue Virus Infection
Harryson Wings Godoy dos Santos,1 Telma Regina Ramos Silva Poloni,1 Kelly Paula Souza,1
Vanessa Danielle Menjon Muller,1 Flávia Tremeschin,1 Lı́via Christensen Nali,2
Leandro Ricardo Fantinatti,2 Alberto Anastacio Amarilla,2 Helda Liz Alfonso Castro,1
Marcio Roberto Nunes,3 Samir Mansour Casseb,3 Pedro Fernando Vasconcelos,3
Soraya Jabur Badra,2 Luiz Tadeu Moraes Figueiredo,2 and Victor Hugo Aquino1*
1
Departamento de Análises Clı´nicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas,
USP, Ribeirão Preto, SP, Brazil
2
Centro de Pesquisa em Virologia, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil
3
Seção de Arbovirologia e Febres Hemorrágicas, Instituto Evandro Chagas, Secretaria de Vigilância em Saúde,
Ministe´rio da Saúde, PA, Bele´m, Brazil
Dengue is the most important arbovirus disease
in tropical and sub-tropical countries, and can
be caused by infection with any of the fourdengue virus (DENV) serotypes. Infection with
DENV can lead to a broad clinical spectrum,
ranging from sub-clinical infection or an influenza-like disease known as dengue fever (DF) to a
severe, sometimes fatal, disease characterized by
hemorrhage and plasma leakage that can lead to
shock, known as dengue hemorrhagic fever/
dengue shock syndrome (DHF/DSS). The diagnosis of dengue is routinely accomplished by
serologic assays, such as IgM and IgG ELISAs, as
well as HI tests, analyzing serum samples
obtained from patients with at least 7 days of
symptoms onset. These tests cannot be used for
diagnosis during the early symptomatic phase. In
addition, antibodies against dengue are broad
reactive with other flaviviruses. Therefore, a
specific diagnostic method for acute DENV
infection is of great interest. In that sense, the
real-time RT-PCR has become an important tool
that can be used for early and specific detection of
dengue virus genome in human serum samples.
This study describes a simple, specific, and
sensitive real-time RT-PCR for early diagnosis of
dengue virus infection. J. Med. Virol. 80:1426–
1433, 2008. ß 2008 Wiley-Liss, Inc.
KEY WORDS: dengue; diagnosis; real-time
RT-PCR
INTRODUCTION
Dengue is the most important arthropod-borne viral
disease in tropical and sub-tropical countries [Monath,
1994; Gubler, 2002; Guzman and Kouri, 2002]. Dengue
is caused by any of the four antigenically related, but
ß 2008 WILEY-LISS, INC.
genetically distinct viruses named dengue virus 1, 2, 3,
and 4 (DENV-1, -2, -3, and -4). Like other members of the
genus Flavivirus, family Flaviviridae, DENV has a
positive-sense single-stranded RNA genome of approximately 10,700 nucleotides, surrounded by a nucleocapsid and covered by a lipid envelope that contains
the viral glycoproteins [Henchal and Putnak, 1990;
Monath and Heinz, 1996]. The RNA genome contains an
m7GppN-cap at the 50 -end but lack a poly(A) tail at the
30 -end. Within the genome there is a single open reading
frame, flaked by 50 - and 30 -untranslated regions (50 - and
30 -UTR), that encodes a polyprotein that is co- and posttranslationally cleaved into three structural proteins
(C-prM/M-E) and seven non-structural proteins (50 NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-30 ). DENV is
transmitted to humans mainly by the bite of the Aedes
aegypti mosquito specie. Infection with any of the four
DENV serotypes can lead to a broad clinical spectrum,
ranging from sub-clinical infection or an influenzalike disease known as dengue fever (DF) to a severe,
sometimes fatal, disease characterized by hemorrhage
and plasma leakage that can lead to shock, known as
dengue hemorrhagic fever/dengue shock syndrome
(DHF/DSS) [Halstead, 1988; Gubler, 2002; Guzman
and Kouri, 2002]. The World Health Organization
estimates that there may be 50–100 million cases of
DENV infections worldwide every year, which result in
250,000–500,000 cases of DHF/DSS and 24,000 deaths
each year [WHO, 1997; Gibbons and Vaughn, 2002].
*Correspondence to: Victor Hugo Aquino, Av. Bandeirantes,
Ribeirão Preto, SP, Brazil. E-mail: vhugo@fcfrp.usp.br
Accepted 10 March 2008
DOI 10.1002/jmv.21203
Published online in Wiley InterScience
(www.interscience.wiley.com)
Real-Time RT-PCR for Diagnosis of DVF
Early diagnosis of dengue virus infection is important
for patient management and control of dengue outbreaks. The confirmatory diagnosis of dengue is routinely performed using serologic assays, such as
immunoglobulin M (IgM) and IgG enzyme-linked
immunosorbent assays (ELISAs) or hemmaglutination
inhibition (HI) tests, analyzing serum samples of
patients with at least 7 days of symptoms onset.
Therefore, serologic assays cannot be used for early
diagnosis of dengue virus infection. Virus isolation by
cell culture remains the gold standard for dengue
diagnosis; however it is low sensitive and takes more
than 7 days to complete the test.
Several reverse transcriptase polymerase chain reaction (RT-PCR) methods have been developed in the past
decade for detecting DENV infection during the acute
phase [Deubel et al., 1990, 1997; Lanciotti et al., 1992;
Figueiredo et al., 1997a, 1997b]. However, the conventional RT-PCR is a laborious technique, which has a
high risk of cross-contamination. The real-time RTPCR, a fully automatic assay, has emerged as a more
suitable method for routine diagnosis of dengue virus
infection. Real-time RT-PCR has many advantages
over conventional RT-PCR including rapidity, a
higher sensitivity, a lower cross-contamination rate,
easy standardization, and the possibility of quantitative
measurements. Several authors have reported real-time
RT-PCR assays for the detection of DENV in serum
samples [Callahan et al., 2001; Houng et al., 2001;
Drosten et al., 2002; Chutinimitkul et al., 2005].
However, those studies included the use of expensive
probes, more than a pair of primers, and/or more than
one-step. The present study describes the use of a
simple and highly specific real-time RT-PCR for DENV
detection using a single pair of generic primers.
MATERIALS AND METHODS
Clinical Samples and Virus Isolation
This study included serum samples sent to the
Virology Research Center of the School of Medicine
of Sao Paulo University, Sao Paulo, SP, and to the
Department of Arbovirus and Hemorrhagic Fevers at
the Evandro Chagas Institute/MS/SVS, Belem, PA,
Brazil, for dengue diagnosis. Serum samples (n ¼ 126)
were collected within the first 5 days of fever onset. This
study was approved by the Ethical Committee of the
Clinical Hospital of the School of Medicine of Ribeirao
Preto, Sao Paulo University (HCRP no. 4921/2007).
Aedes albopictus (C6/36) cells contained in a 12-well
plates were inoculated with 20 ml of serum samples for
virus isolation. Cells were incubated for up to 10 days
and then the isolated viruses were identified by
indirect immunofluorescent test as described previously
[Figueiredo et al., 1992].
Definitions
Patients were classified clinically according to the
WHO criteria [WHO, 1986] as DF and DHF/DSS based
1427
on their medical records. The date of onset of fever
was defined as day 1. Dengue-infected patients with
viremia and without antibodies response or having a
ratio IgM/IgG 1.2 (detected by capture IgM and IgG
ELISA) were considered as having primary infection
and those having a ratio IgM/IgG <1.2 were considered
as having secondary infection [Shu et al., 2003a].
Virus
The four strains of DENV that were included in this
study were DENV-1 (Riberiao Preto), DENV-2 (New
Guinea C), DENV-3 (H87) and DENV-4 (Boa Vista).
This study included also other flaviviruses: Yellow
fever virus (17D), Bussuquara virus (BeAn 4073),
Cacipacore virus (Be An 327600), Iguape virus (SpAn
71686), Ilheus virus (BeH 7445), Rocio virus (SpH
34675) and Saint Louis Encephalitis virus (SpAn
11916). All the viruses were maintained in C6/36 cells
and the infection was confirmed by indirect immunofluorescence [Gubler et al., 1984]. Several aliquots of all
viruses were stocked at 708C until use.
Virus Titration
An aliquot of the four DENV strain was used for viral
title determination using the plaque assay method.
Briefly, the virus stocks were serially diluted 10-fold and
added, in triplicate, in a 24-well plate containing a
monolayer of Vero cells. After 1 hr of infection, each well
was added with 1 ml of L15 medium containing 0.5%
carboximetilcellulose and 2% fetal bovine serum. After
5 days of incubation, the overlay medium was removed
and the cells were stained with 0.1% crystal violet.
The plaque number, at an appropriate dilution, was
counted and the viral titer was expressed as a plaque
forming units per ml (PFU/ml). The virus titers were
as follow: DENV-1, 1.5 10E6 PFU/ml; DENV-2, 6.9 10E6 PFU/ml; DENV-3, 1 10E6 PFU/ml; DENV-4,
1.5 10E6 PFU/ml.
RNA Extraction
Viral RNA was extracted from 140 ml of serum samples
or fluids of infected C6/36 cells using the QIAamp Viral
RNA mini kit (QIAGEN, Hamburg, Germany), following
the manufacturer’s recommendation. The RNA was
eluted with 80 ml of the corresponding elution buffer.
RT-NESTED-PCR for Flaviviruses
RNA obtained from serum samples and fluid of
infected cells was used for genome amplification and
serotyping using the method previously described by
Bronzoni et al. [2005].
Conventional RT-PCR
This test was carried out as described previously using
the 50 -UTR-S (50 -AGT TGT TAG TCT ACG TGG ACC
GA-30 , positions 1–23) and 50 -UTR-C (50 -CGC GTT TCA
GCA TAT TGA AAG-30 , positions 129–149 based on the
J. Med. Virol. DOI 10.1002/jmv
1428
dos Santos et al.
DENV-3 strain H87, GenBank accession no. M93130)
primers [Aquino et al., 2006].
Real-Time RT-PCR
The reaction was carried out with the SuperScript
III Platinum SYBR Green One-Step qRT-PCR kit
(Invitrogen, Carlsbad, CA) in the thermocycler SmartCycler (Cepheid, Sunnyvale, CA). A total of 25 ml
reaction mixture contained 0.5 ml of SuperScript III RT
Platinum Taq Mix, 0.2 mM of each primer, 12.5 ml of 2X
SYBR Green and 5 ml of purified RNA. The amplification
cycle was as follows: 508C for 20 min; 958C for 5 min;
45 cycles: 958C for 15 sec, 548C for 40 sec and 728C for
30 sec. Finally, the melting curve was constructed
incubating the amplification products from 60 to 908C
with an increase of 0.28C/sec. The melting temperature
(Tm) values of the specific amplicons were in the range of
80.57–81.738C, whereas the Tm primer–dimer values
were found to be below, ranging from 75.14 to 76.318C.
Standard Curve
The concentration of each DENV serotype was
adjusted to 1 10E6 PFU/ml, and then the RNA was
purified as described above. The RNA samples were
diluted serially 10-fold and each dilution was used in the
real-time RT-PCR for the construction of a standard
curve for all DENV serotypes.
Capture IgM and IgG ELISA
with those obtained between the fourth and fifth. The
difference was analyzed using the Student’s t-test and
considered significant when P < 0.05.
RESULTS
Specificity of the One-Step Real-Time
RT-PCR Assay
A generic pair of primers for DENV was tested for
specificity against various Brazilian flaviviruses such as
yellow fever virus, Bussuquara virus, Cacipacore virus,
Iguape virus, Ilheus virus, Rocio virus and Saint
Louis Encephalitis virus. These viruses co-circulate in
Brazil producing very similar symptoms to DENV. The
real-time RT-PCR using the generic pair of primers was
found to be highly specific for DENV (Fig. 1). Laboratory
strains corresponding to each of the four DENV
serotypes showed a Ct of 17–21 and a Tm of 81, which
indicate a specific amplification. Although the other
flaviviruses were detected at a Ct of 36–39, their
amplicons showed a Tm of 75 that corresponds to
primer dimers (Fig. 1B).
Evaluation of the Real-Time RT-PCR Assay
Analyzing Serum Samples
The applicability of the real-time RT-PCR assay was
evaluated using 126 serum samples collected from
suspected dengue patients. These samples were also
A modified capture IgM and IgG ELISA was used to
measure antibodies against DENV in serum samples
as described previously by Shu et al. [2003a]. The
modification included the use of a pool of mice immune
ascitic fluids (MIAF) prepared against the four DENV
serotypes. Briefly, each microtiter 96-well plate was
coated overnight at 48C with affinity-purified goat antihuman IgM (m chain specific) or IgG (g chain specific)
antibodies (KPL, USA) at 5 mg/ml (100 ml/well) in 0.1 M
carbonate buffer (Na2CO3/NaHCO3, pH 9.5). After
washing and blocking, the wells were incubated with
100 ml of 1:40-diluted serum in PBST–1% BSA–5%
normal goat serum for 1 h at 378C. After washing, the
wells were incubated with 100 ml of a cocktail containing
1:20-diluted pooled virus antigens from the brain of
DENV-1-, DENV-2-, DENV-3-, and DEN-4-infected
mice for 1 h at 378C. After washing, the wells were
incubated with 1:100-diluted pooled of DENV-1-,
DENV-2-, DENV-3-, and DENV-4 MIAF and incubated
for 1 h at 378C. After washing, the wells were incubated
with alkaline phosphatase-conjugated goat anti-mouse
IgG (g chain specific) (Sigma, USA). The enzyme activity
was developed, and the OD was measured 1 h later.
Appropriate control sera were included in each plate.
IgM was not investigated in one sample and IgG in
11 samples because insufficient volumes were available.
Statistical Analysis
Viral load of serum samples collected between the 1st
and 3rd days after the symptoms onset was compared
J. Med. Virol. DOI 10.1002/jmv
Fig. 1. A: Optical graph showing the Ct of DENV-1 to -4 and the other
flaviviruses: yellow fever virus, Bussuquara virus, Cacipacore virus,
Iguape virus, Ilheus virus, Rocio virus and Saint Louis Encephalitis
virus. B: Tm of de amplicon corresponding to each virus.
Real-Time RT-PCR for Diagnosis of DVF
tested by a conventional RT-PCR, a capture ELISA for
IgM and IgG detection, and a cell culture inoculation for
viral isolation attempt (Table I). DENV genome was
detected in 104 (82.5%) samples by real-time RT-PCR
and in 59 (46.8%) samples by conventional RT-PCR.
Anti-dengue IgM was found in 17 (13.6%) of 125 serum
samples and anti-dengue IgG in 9 (7.8%) of 115 serum
samples. DENV was isolated from 34 (45.9%) of 74 serum
samples inoculated in cell culture.
The laboratory diagnosis of dengue virus infection
was established by virus isolation, virus genome
amplification, and/or specific IgM detection. According
to these criteria, 112 (88.9%) of 126 patients were
infected with DENV. However, 6 of the 14 negative
samples were not inoculated in cell culture due to
insufficient sample volumes; therefore, it was not
possible to establish whether they were truly negative
or not. The real-time RT-PCR was not able to detect
virus genome in two samples with positive results by the
conventional RT-PCR, three with virus isolation, and
three with positive IgM (Table I). Thus, real-time RTPCR was positive in 92.8% (104/112) of the infected
patients. The real-time RT-PCR was found to be
more sensitive than virus isolation, IgM detection, and
conventional RT-PCR (Table II).
Primary and secondary dengue infections were
defined based on the detection of viral genome and/or
viral isolation, and on IgM/IgG ratio. Eleven dengueinfected patients without IgG determination were
excluded from this analysis. In 101 patients with dengue
infection, 93 (92%) had primary and 8 (7.9%) secondary
dengue infection.
DENV serotypes were determined by RT-NESTEDPCR directly from serum samples or from fluids
of infected C6/36 cells. DENV-3 was detected in
115 (91.3%), DENV-2 in 6 (4.8%), and DENV-1 in 5
(3.9%) serum samples.
Viral Load
Real-time RT-PCR standard curves were constructed
for all DENV serotypes using 10-fold serial dilutions
of RNA purified from seed viruses, which were titrated
by a plaque forming assay. Figure 2 shows an example
of the standard curve for DENV-3, which was used
to determine the viral load in the serum samples. The
limit of detection was 1 10E1 PFU/ml for all four
serotypes. The same generic pair of primers was
also used in a conventional RT-PCR and found to
have a detection limit of 1 10E4 PFU/ml; it was
found to be 1000 times less sensitive than the real-time
RT-PCR.
Viral load detected in the serum samples ranged from
2.55 10E0 to 7.5 10E6 PFU/ml (Table I). The average
of viral load in serum samples collected within the
first 3 days after the symptoms onset was 1.8 10E3
PFU/ml, while those collected between the 4th and
5th days after the symptoms onset showed an average of
viral load of 5.0 10E1 PFU/ml. These viral loads were
significantly different (P ¼ 0.0001).
1429
It was not possible to analyze the correlation between
viral load and disease severity because only three serum
samples of DHF/DSS patients were obtained. Those
patients had a viral load of 2.7 10E1, 6.39 10E2
and 9.0 10E2 PFU/ml, respectively. Likewise, the
difference in viral load in primary and secondary DENV
infection could not be examined because of the lack of an
appropriate number of serum samples collected on the
same day after the onset of symptoms.
DISCUSSION
It is well known that DF and DHF/DSS can be
produced by infection with any of the four DENV
serotypes [Halstead, 1988; Gubler, 2002; Guzman and
Kouri, 2002]. The major pathophysiological abnormality
seen in DHF/DSS is an acute increase in vascular
permeability leading to a loss of plasma from vascular
compartment. Plasma leakage can lead to shock, which,
if uncorrected, leads to tissue anoxia, metabolic acidosis,
and death. Early and effective replacement of plasma
losses with plasma expander or fluid and electrolyte
solution results in a favorable outcome in most cases
[WHO, 1997]. Therefore, early diagnosis, independently
of serotype determination, would ensure a more efficient
management of patients infected with dengue.
This study describes a simple SYBR Green-based onestep real-time RT-PCR for early diagnosis of dengue
infection. Real-time RT-PCR for the diagnosis of acute
dengue virus infection has many advantages over
conventional methods, such as virus isolation, antiDENV IgM detection, and conventional RT-PCR. Virus
isolation by cell culture, followed by identification of the
isolate using fluorescent antibody, is considered the
‘‘gold standard’’ for diagnosis of viral infection [Vorndam
and Kuno 1997]. However, it has the disadvantage of
low sensitivity and that longer than 7 days is usually
required to complete the test. Hence, serological diagnosis based on capture IgM and IgG ELISA has been
used for the diagnosis of dengue infection in many
laboratories. However, IgM can only be detected
approximately 5 days after the onset of illness, and it
is sometimes not detected in secondary infections. In
addition, strong antibody cross-reactivity occurs among
members of the Flaviviridae family, which may complicate the interpretation of serological results [Laue et al.,
1999]. Compared to conventional RT-PCR, real-time
RT-PCR has several advantages. Besides its rapidity,
low risk of false positive results, high sensitivity, and
specificity, real-time PCR allows quantitative measurements. The assay described in this study was shown to
be highly specific, discriminating DENV from other
flaviviruses. The real-time RT-PCR was able to detect
laboratory-adapted DENV of all serotypes in cell culture
fluids and DENV-1, DENV-2, and DENV-3 in serum
samples. The high specificity is likely due to the use of a
generic pair of primers that was selected from a highly
conserved region located at the 50 -end of dengue virus
genome. It is well known that the 50 -end of flavivirus
genome has secondary structures [Monath and Heinz,
J. Med. Virol. DOI 10.1002/jmv
1430
dos Santos et al.
TABLE I. Details of the 126 Patients
RT-PCR
Patient
1652
2090
1498
1568
1570
1573
1604
1690
2040
2160
2127
2404
2408
2535
1677
2065
2089
2121
2131
2179
2183
2333
1567
1661
1667
2039
2068
2078
2128
2174
2177
2198
2340
2401
2417
2514
1610
1652
1666
2138
2154
2167
2170
2331
2410
2425
2580
2591
2593
510
544
549
550
554
597
599
2419
2586
2598
2581
2595
2603
2604
Standard Real-time
P
P
N
P
N
P
P
P
P
N
P
P
N
N
N
P
P
P
P
P
P
N
N
P
N
N
P
P
P
P
P
P
N
N
P
N
N
P
N
P
P
P
P
N
P
P
N
P
N
N
P
P
P
P
P
N
P
N
N
N
N
N
N
P
P
P
P
P
P
P
P
P
P
P
P
P
P
N
P
N
P
P
N
P
N
P
P
P
N
P
P
P
P
P
P
P
N
P
P
P
P
P
P
P
P
P
P
P
P
N
P
P
P
P
P
P
P
P
P
P
P
P
N
P
P
P
Tm
Serology
Viral load Viral
(PFU/ml) isolation
81.02 27,285
81.22 12,759
81.45
6
81.37 538,518
81.4
8
81.46 1,378,605
81.06 148,511
81.39 520,092
81.29
1,059
81.56
23
81.49
2,774
81.52 140,139
81.58
8
81.47
28
81.4
107,934
81.31
81.48
9,007
55,060
81.31
3,861
81.61
7
81.46 1,410,977
81.53
39
81.73
81.63
81.39
81.59
81.53
80.98
81.62
1,022
71
3,750
3,998
60
4,622
8
81.49
81.6
81.77
81.93
81.46
81.42
81.28
81.47
81.34
81.49
81.31
81.53
2,225
20
18
79,360
32
93,903
287,766
10,908
892,154
9
484
28
81.36
81.5
81.22
81.3
81.48
81.24
81.43
81.49
81.39
81.44
81.51
81.31
2,186
4
104
93
7,882
49
3,043
473,980
5
2,161
18
37
81.2
81.4
81.48
13
13
299,695
ND
ND
N
N
N
P
P
P
N
N
ND
P
N
ND
N
P
N
ND
P
N
N
P
N
N
N
N
N
N
N
N
N
ND
P
N
ND
N
P
ND
N
ND
ND
P
P
P
P
N
N
ND
N
N
ND
P
N
P
P
N
P
N
N
P
N
N
P
IgM
IgG
Infection
0.36
0.37
0.4
0.40
0.40
0.36
0.36
0.43
0.43
0.44
0.38
0.38
0.43
0.49
0.45
0.49
0.36
0.43
0.37
0.45
0.40
0.40
0.47
0.45
0.41
0.47
0.49
0.38
0.40
0.43
0.45
0.45
0.41
0.38
0.34
0.47
0.40
0.36
0.44
0.42
0.43
0.43
0.39
0.39
0.45
0.52
0.55
0.48
0.4
0.37
0.39
0.39
0.4
0.43
0.48
0.44
0.44
0.51
0.53
0.47
0.39
0.42
0.41
0.35
0.36
0.38
0.38
0.46
0.34
0.32
0.34
0.43
0.39
0.40
0.34
0.41
0.49
0.49
0.32
0.33
0.42
0.36
0.35
0.39
0.37
0.32
0.33
0.39
0.40
0.45
0.43
0.43
0.41
0.49
0.41
0.47
0.47
0.49
0.44
0.41
0.35
0.46
0.32
0.36
0.33
0.42
0.40
0.36
0.38
0.71
0.41
0.42
0.37
0.39
0.39
0.47
0.45
0.42
0.44
0.42
0.43
0.38
0.35
0.34
0.43
0.47
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
SEC
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
Fever
day
DENV
serotype
2
2
5
2
3
3
1
3
3
4
4
1
4
5
2
1
2
3
3
2
3
3
4
2
2
5
3
2
4
1
4
3
4
3
2
1
3
2
4
2
1
3
3
5
2
2
4
3
1
3
1
2
4
2
1
3
2
2
5
2
5
5
1
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Sex Age Disease
F
M
M
M
M
F
F
M
F
F
F
F
M
M
M
M
F
F
M
M
F
M
M
F
F
M
M
F
M
F
F
F
M
M
F
M
M
F
M
M
F
M
M
M
F
F
M
M
F
M
M
F
M
F
M
F
M
F
M
M
F
M
M
25
66
31
40
27
24
63
26
35
31
40
58
30
47
43
22
31
14
40
40
42
15
21
31
32
40
35
11
18
27
40
30
16
35
50
20
43
52
38
30
42
12
27
6
51
45
22
58
14
22
17
52
25
48
41
48
14
48
20
17
53
19
9
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
UN
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
UN
DF
DF
DF
DF
DF
DF
DF
UN
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
(Continued)
J. Med. Virol. DOI 10.1002/jmv
Real-Time RT-PCR for Diagnosis of DVF
1431
TABLE I. (Continued)
RT-PCR
Patient
2046
2253
2273
2275
2292
2320
2346
2353
2418
2436
2497
2513
2520
2533
1877
2254
2259
2263
2291
2294
2295
2297
2355
2409
2424
2430
2443
2546
1893
2044
2251
2279
2284
2308
2318
2325
2381
2389
2409
2451
RN A
RN B
RN C
RN D
RN E
RN F
RN G
RN H
JH
GMM
AAF
MEI
POR 1164
MAR3818
BEL 78824
BEL 78921
BEL 78946
ROR 5853
ROR 5861
ROR 5867
MAO 12032
MAO 12033
VAL
Standard Real-time
P
N
P
P
N
N
N
N
N
N
N
N
N
N
P
P
N
N
P
N
N
N
N
N
N
N
P
N
N
N
N
P
N
N
N
N
N
P
P
N
N
N
N
N
P
N
N
N
N
N
N
N
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
N
P
P
P
P
P
N
N
P
P
N
N
P
P
N
P
P
N
P
N
P
P
P
P
P
P
P
P
P
P
N
N
N
N
P
N
P
N
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
Tm
Serology
Viral load Viral
(PFU/ml) isolation
81.39
81.32
81.29
81.18
81.55
81.46
81.28
81.46
81.41
81.31
187
28
104,848
895
22
14
23
9
22
6
81.29
81.34
81.25
81.41
81.42
28
85
22
106,689
141
2,186
81.55
81.39
62
4
81.5
81.03
27
3
81.62
81.32
9
446
81.49
7
81.44
81.46
81.65
81.81
81.4
81.15
81.51
81.59
81.42
81.5
13
1,645
24
20
25
13
25
377,990
1,016
32
80.58
27
81.73
14
80.9
639
81.68
160
81.54
358
81.47
24
81.55 34,613
81.59 14,750
81.54 365,056
81.41 7,503,778
81.53 1,952,720
80.60 121,216
80.57 65,151
80.80 14,750
80.70 164,862
80.75 30,289
80.57
900
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
N
P
N
N
N
N
N
N
P
P
P
N
P
P
P
P
P
P
P
P
P
P
P
IgM
IgG
Infection
0.39
0.46
0.42
0.41
0.44
0.40
0.44
0.38
0.64
0.46
0.72
0.39
0.53
0.66
0.41
0.49
0.53
0.47
0.43
0.49
0.47
0.48
0.54
0.37
0.41
0.38
0.42
0.41
0.41
0.46
0.52
0.43
0.45
0.54
0.51
0.5
0.39
0.39
0.37
0.40
0.34
0.45
0.41
0.46
0.43
0.47
0.51
0.43
1.1
0.55
0.42
0.44
0.41
0.42
0.42
0.39
0.47
0.48
0.45
0.46
0.44
0.44
ND
0.38
0.44
0.39
0.41
0.39
0.35
0.62
0.38
0.64
0.44
0.44
0.37
0.62
0.41
0.41
0.41
0.37
0.42
0.42
0.32
0.4
0.39
0.44
0.38
0.4
0.34
0.37
0.44
0.41
0.61
0.57
0.45
0.47
0.42
0.46
0.5
0.34
0.46
0.38
0.63
0.50
0.38
0.4
0.34
0.66
0.39
0.42
0.32
0.43
0.34
0.71
0.32
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
PR
PR
PR
PR
PR
PR
SEC
PR
SEC
PR
PR
PR
SEC
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
SEC
PR
PR
PR
PR
PR
PR
PR
PR
SEC
SEC
PR
PR
PR
PR
SEC
PR
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Fever
day
DENV
serotype
5
4
2
3
4
2
5
2
2
5
5
4
5
3
2
4
5
3
4
4
5
3
5
3
4
2
4
4
4
5
4
5
5
1
5
3
5
3
3
5
5
4
4
5
4
3
5
3
4
5
4
2
3
2
3
1
2
1
1
2
2
1
4
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
3
3
3
1
1
1
1
1
2
2
2
2
2
3
Sex Age Disease
F
F
F
F
F
F
M
F
M
M
F
F
F
M
F
F
F
M
M
M
M
M
F
M
M
F
F
M
M
F
M
F
M
M
F
F
F
F
M
M
F
F
F
F
M
M
F
F
NI
NI
NI
NI
M
M
F
F
M
NI
NI
NI
M
F
NI
52
17
29
31
70
15
75
20
15
7
57
15
36
26
40
19
31
12
15
13
46
46
28
16
38
30
24
24
16
22
27
76
10
16
18
37
23
39
16
69
29
NI
NI
NI
8
NI
NI
NI
NI
NI
NI
NI
9
20
36
9
24
NI
NI
NI
57
55
NI
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
UN
DF
DF
UN
UN
DF
DF
UN
DF
DF
UN
DF
UN
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
UN
UN
UN
UN
DHF
DF
DF
UN
DHF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DF
DHF
P: positive; N: negative; PR: primary; SEC: secondary; ND: not determined; UN: unknown; NI: not informed; IgG: cut off 0.53; IgM: cut off 0.50.
J. Med. Virol. DOI 10.1002/jmv
1432
dos Santos et al.
TABLE II. Diagnosis of DENV Infection using
Different Assays
Serum samples
Assay
Positive (%)
Real-time RT-PCR
Conventional RT-PCR
Virus isolation
IgM capture ELISA
104 (92.8)
59 (52.6)
34 (51.5)
17 (15.2)
Negative (%)
8
53
32
95
(7.1)
(47.3)
(48.5)
(84.8)
Total
112
112
66
112
1996], which can interfere with the annealing of
primers. Therefore, it is usually necessary a previous
denaturation step for an optimal cDNA synthesis.
However, our results demonstrated that the secondary
structure of the 50 -end of DENV does not interfere with
the annealing of the primer used in this assay, allowing
the development of a real-time RT-PCR without a
previous denaturation step.
The use of SYBR makes this real-time RT-PCR less
expensive than RT-PCRs that use fluorogenic probes
[Laue et al., 1999; Callagan et al., 2001; Houng et al.,
2001; Warrilow et al., 2002; Johnson et al., 2005; Kong
et al., 2006]. This assay is also simpler than others
similar methods described in the literature [Chutinimitkul et al., 2005; Yong et al., 2007]; it uses a single pair
of primers and is carried out in a one-step format. In the
near future, with the expected popularization of realtime PCR methods and the cost reduction, the assay
described in this study could become a method for
routine use.
This is the first report of a real-time RT-PCR for
dengue using a single pair of primers designed based on
the 50 -UTR; most of the tests described in the literature
use primers designed based on NS5 protein and 30 -UTR
[Shu et al., 2003b; Chao et al., 2007; Dyer et al., 2007; Lai
et al., 2007].
The assay described in this study was highly sensitive;
it showed a detection limit of 1 10E1 for all DENV
serotypes, and was able to detect as low as 2.55 10E0
PFU/ml in a serum sample. Other SYBR green-based
methods showed similar sensitivity, detecting between
4.1 10E0 and 1 10E1 PFU/ml [Shu et al., 2003b; Lai
et al., 2007]. This high sensitivity would ensure that
Fig. 2. Standard curve constructed using ten-fold serial dilutions of
RNA obtained from the fluid of DENV-3 (H87) infected C6/36 cells
containing 1 106 PFU/ml.
J. Med. Virol. DOI 10.1002/jmv
clinical samples with low viral load would be detected as
dengue positive.
Analysis of correlation between viral load and clinical
data might provide important information about
the pathogenesis of the different dengue-associated
syndromes. In that sense, some studies have shown
the association of viral load with disease severity
[Murgue et al., 2000; Vaughn et al., 2000; Libraty
et al., 2002; Wang et al., 2003]. In the present study, it
was not possible to analyze the association of viral load
with disease severity because only three of the studied
patients developed DHF. However, it has been shown
that the peak of veremia was within the first 3 days after
the onset of the symptoms, which is in agreement
with data reported by other investigators [Vaughn et al.,
2000; Libraty et al., 2002].
Recently, an ELISA was developed for detection of
dengue NS1 antigen during the acute phase of the
disease [Alcon et al., 2002]. A commercial NS1-capture
ELISA was found to be more sensitive than conventional
RT-PCR for diagnosis of acute dengue infection [Kumarasamy et al., 2007a,b]. In the present study, it has been
shown that the real-time RT-PCR was 1000-fold more
sensitive than the conventional RT-PCR. In a future
study in our laboratory, real-time RT-PCR will be
compared the sensitivity of both NS1-capture ELISA
and real-time RT-PCR for diagnosis of dengue virus
infection.
In conclusion, the present study describes the
development of a simple, specific, and highly sensitive
real-time RT-PCR that could serve as an excellent tool
for routine laboratory diagnosis of acute dengue virus
infection, as well as for the study of the association of
viral load with disease severity.
REFERENCES
Alcon A, Talarmin M, Debruyne A, Falconar V, Deubel M. 2002.
Flamand, enzyme-linked immunosorbent assay specific to dengue
virus type 1 nonstructural protein NS1 reveals circulation of the
antigen in the blood during the acute phase of disease in patients
experiencing primary or secondary infections. J Clin Microbiol 40:
376–381.
Aquino VH, Anatriello E, Gonçalves PF, Silva EV, Vasconcelos PFC,
Vieira DS, Batista WC, Bobadilla ML, Vazquez C, Moran M,
Figueiredo LTM. 2006. Molecular epidemiology of dengue type 3
virus in Brazil and Paraguay, 2002–2004. Am J Trop Med Hyg 75:
710–715.
Bronzoni RVM, Baleotti FG, Nogueira RMR, Nunes M, Figueiredo
LTM. 2005. Duplex reverse transcription-PCR followed by nested
PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses. J Clin Microbiol 43:696–702.
Callahan JD, Wu SL, Dion-Schultz A, Mangold BE, Peruski LF, Watts
DM, Porter KR, Murphy GR, Suharyona W, King CC, Hayes CR,
Temeenak JJ. 2001. Development and evaluation of serotype- and
group-specific fluorogenic reverse-transcriptase PCR (TaqMan)
assays for dengue virus. J Clin Microbiol 39:4119–4124.
Chao DY, Davis BS, Chang GJ. 2007. Development of multiplex realtime reverse transcriptase PCR assays for detecting eight medically
important flaviviruses in mosquitoes. J Clin Microbiol 45:584–589.
Chutinimitkul S, Payungporn S, Theamboonlers A, Poovorawan Y.
2005. Dengue typing assay based on real-time PCR using SYBR
Green I. J Virol Methods 129:8–15.
Deubel V, Laille M, Hugnot JP, Chungue E, Guesdom JL, Drouet MT,
Bassot S, Chevrier D. 1990. Identification of dengue sequences by
genomic amplification: Rapid diagnosis of dengue virus serotypes in
peripheral blood. J Virol Methods 30:41–54.
Real-Time RT-PCR for Diagnosis of DVF
Deubel V, Huerre M, Cathomas G, Drouet MT, Wuscher N, LeGuenno
B, Widmer AF. 1997. Molecular detection and characterization of
yellow fever virus in blood and liver specimens of a non-vaccinated
fatal human case. J Med Virol 53:212–217.
Drosten C, Gottig S, Schilling S, Asper M, Panning M, Schmitz H,
Gunther S. 2002. Rapid detection and quantitation of RNA of Ebola
and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic
fever virus, Rift valley fever virus, dengue virus, and yellow fever
virus by real-time reverse transcription-PCR. J Clin Microbiol
40:2323–2330.
Dyer J, Chisenhall DM, Mores CN. 2007. A multiplexed TaqMan assay
for the detection of arthropod-borne flaviviruses. J Virol Methods
145:9–13.
Figueiredo LTM, Owa A, Carlucci RH, de Oliveira L. 1992. Laboratory
diagnosis and symptoms of dengue, during an outbreak in the
Ribeirao Preto region, SP, Brazil. Rev Inst Med Trop Sao Paulo
March–April 34:121–130.
Figueiredo LTM, Batista WC, Igarashi A. 1997a. A simple reverse
transcription-polymerase chain reaction for dengue type 2 virus
identification. Membr Inst Oswaldo Cruz 92:395–398.
Figueiredo LTM, Batista WC, Igarashi A. 1997b. Detection and
identification of dengue virus isolates from Brazil by a simplified
reverse transcription-polymerase chain reaction (RT-PCR) method.
Rev Inst Med Trop (São Paulo) 39:95–99.
Gibbons RV, Vaughn DW. 2002. Dengue: An escalating problem. Br
Med J 324:1563–1566.
Gubler DJ. 2002. Epidemic dengue/dengue hemorrhagic fever as a
public health, social and economic problem in the 21st century.
Trends Microbiol 10:100–103.
Gubler DJ, Kuno G, Sather GE, Velez M, Oliver A. 1984. Mosquito cell
cultures and specific monoclonal antibodies in surveillance for
dengue viruses. Am J Trop Med Hyg 33:158–165.
Guzman MG, Kouri G. 2002. Dengue: An update. Lancet Infect Dis 2:
33–42.
Halstead SB. 1988. Pathogenesis of dengue: Challenges to molecular
biology. Science 239:476–481.
Henchal EA, Putnak JR. 1990. The dengue viruses. Clin Microbiol Rev
3:376–396.
Houng HH, Chen RCM, Vaughn DW, Kanesa-Thesan N. 2001.
Development of a fluorogenic RT-PCR system for quantitative
identification of dengue virus serotypes1–4 using conserved and
serotype-specific 30 noncoding sequences. J Virol Methods 95:19–
32.
Johnson BW, Russell BJ, Lanciotti RS. 2005. Serotype-specific
detection of dengue viruses in a four plex real-time reverse
transcriptase PCR assay. J Clin Microbiol 43:4977–4983.
Kong YY, Thay CH, Tin TC, Devi. S. 2006. Rapid detection, serotyping
and quantitation of dengue viruses by TaqMan real-time one-step
RT-PCR. J Virol Methods 138:123–130.
Kumarasamy V, Chua SK, Hassan Z, Wahab AH, Chem YK, Mohamad
M, Chua KB. 2007a. Evaluating the sensitivity of a commercial
dengue NS1 antigen-capture ELISA for early diagnosis of acute
dengue virus infection. Singapore Med J 48:669–673.
Kumarasamy V, Wahab AH, Chua SK, Hassan Z, Chem YK, Mohamad
M, Chua KB. 2007b. Evaluation of a commercial dengue NS1
antigen-capture ELISA for laboratory diagnosis of acute dengue
virus infection. J Virol Methods 140:75–79.
Lai YL, Chung YK, Tan HC, Yap HF, Yap G, Ooi EE, Ng. LC. 2007. Cost
effective real-time reverse transcriptase PCR (RT-PCR) to screen
1433
for Dengue virus followed by rapid single-tube multiplex RT-PCR
for serotyping of the virus. J Clin Microbiol 45:935–941.
Lanciotti RS, Calisher CH, Gubler DJ, Chang GL, Vorndam V. 1992.
Rapid detection and typing of dengue viruses from clinical samples
by using reverse transcriptase-polymerase chain reaction. J Clin
Microbiol 30:545–551.
Laue T, Emmerich P, Schmitz H. 1999. Detection of dengue virus RNA
in patients after primary or secondary dengue infection by using the
TaqMan automated amplification system. J Clin Microbiol 37:
2543–2547.
Libraty DH, Endy TP, Houng HSH, Green S, Kalayanarooj S,
Suntayakorn S, Chansiriwongs W, Vaughn DW, Nisalak A, Ennis
FA, Rothman AL. 2002. Differing influence of virus burden and
immune activation on disease severity in secondary dengue-3 virus
infection. J Infect Dis 185:1213–1221.
Monath TP. 1994. Dengue, the risk to developed and developing
countries. Proc Natl Acad Sci U S A 91:2395–2400.
Monath TP, Heinz FX. 1996. Flaviviruses. In: Fields BW, Knipe DM,
Knipe PM, Howley PM, editors. Fields Virology Vol. 1 3rd ed.
Philadelphia: Lippincott-Raven Press. 961–1034.
Murgue B, Roche C, Chungue E, Deparis X. 2000. Prospective study of
the duration of magnitude of viremia in children hospitalized
during the 1996–1997 dengue-2 outbreak in French Polynesia.
J Med Virol 60:432–438.
Shu PY, Chen LK, Chang SF, Yueh YY, Chow L, Chien LJ, Chin C, Lin
TH, Huang JH. 2003a. Comparison of capture immunoglobulin M
(IgM) and IgG enzyme-linked immunosorbent assay (ELISA)
and nonstructural protein NS1 serotype-specific IgG ELISA for
differentiation of primary and secondary dengue virus infections.
Clin Diagn Lab Immunol 10:622–630.
Shu PY, Chang SF, Kuo YC, Yueh YY, Chien LJ, Sue CL, Lin TH,
Huang JH. 2003b. Development of group- and serotype-specific onestep SYBR Green I-based real-time reverse transcription-PCR
assay for dengue virus. J Clin Microbiol 41:2408–2416.
Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S,
Suntayakorn S, Endy TP, Raengsakulrach B, Rothman AL, Ennis
FA, Nisalak A. 2000. Dengue viremia titer, antibody response
pattern, and virus serotype correlate with disease severity. J Infect
Dis 181:2–9.
Vorndam AV, Kuno G. 1997. Laboratory diagnosis of dengue virus
infections. In: Gubler DJ, Kuno G, editors. Dengue and Dengue
Hemorrhagic Fever. New York: CAB International. 313–333.
Wang WK, Chao DY, Kao CL, Wu HC, Liu YC, Li CM, Lin SC, Ho ST,
Huang JH, Kingb CC. 2003. High levels of plasma dengue viral load
during defervescence in patients with dengue hemorrhagic fever:
Implications for pathogenesis. Virology 305:330–338.
Warrilow D, Northill JA, Pyke A, Smith GA. 2002. Single rapid TaqMan
fluorogenic probe-based PCR assay that detects all four dengue
serotypes. J Med Virol 66:524–528.
World Health Organization. 1986. Technical Advisory Committee on
dengue haemorrhagic fever for the Southeast Asian and Western
Pacific regions. Guide for diagnosis, treatment and control of
dengue haemorrhagic fever, Geneva.
World Health Organization (WHO). 1997. Dengue hemorrhagic fever,
diagnosis, treatment and control. 2nd ed. Geneva: WHO, 1997.
Yong YK, Thayan R, Chong HT, Tan CT, Sekaran SD. 2007. Rapid
detection and serotyping of dengue virus by multiplex RT-PCR
and real-time SYBR green RT-PCR. Singapore Med J 48:662–
668.
J. Med. Virol. DOI 10.1002/jmv