Abstract
INTRODUCTION: The genus
Flavivirus includes several pathogenic species that cause severe illness in
humans . Therefore, a rapid and accurate molecular
method for
diagnosis and
surveillance of these
viruses would be of great importance. Here, we evaluate and optimize a quantitative real-
time reverse transcription polymerase chain reaction (RT-PCR)
method for the
diagnosis of the
Flavivirus genus.
METHODS: We evaluated different commercial kits that use the SYBR Green system for real-
time RT-PCR with a primer set that amplifies a fragment of the NS5
flavivirus gene . The
specificity and sensitivity of the assay were tested using twelve
flaviviruses and
ribonucleic acid (
RNA ) transcribed from the
yellow fever virus . Additionally, this assay was evaluated using the sera of 410
patients from different regions of
Brazil with acute febrile illness and a negative
diagnosis for the
dengue virus .
RESULTS: The real-
time RT-PCR amplified all
flaviviruses tested at a
melting temperature of 79.92 to 83.49°C. A
detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected
dengue virus in 4.1% (17/410) of samples from
patients with febrile illness and a supposedly negative
dengue infection diagnosis . The
viral load in
patients ranged from 2.1×107to 3.4×103copies per ml.
CONCLUSIONS: The real-
time RT-PCR
method may be very useful for preliminary
diagnoses in
screenings ,
outbreaks , and other
surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to
measure viral load in
pathogenesis studies.