TY - JOUR
T1 - Evaluation and optimization of SYBR green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the flavivirus genus in Brazil
AU - Romeiro, Marilia Farignoli
AU - De Souza, William Marciel
AU - Tolardo, Aline Lavado
AU - Vieira, Luiz Carlos
AU - Colombo, Tatiana Elias
AU - Aquino, Victor Hugo
AU - Nogueira, Maurício Lacerda
AU - Figueiredo, Luiz Tadeu Moraes
N1 - Publisher Copyright:
© 2016, Sociedade Brasileira de Medicina Tropical. All rights reserved.
PY - 2016/5/1
Y1 - 2016/5/1
N2 - 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.
AB - 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.
KW - Arbovirus
KW - Diagnosis
KW - Flavivirus
KW - Real-time RT-PCR
UR - http://www.scopus.com/inward/record.url?scp=84976636815&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84976636815&partnerID=8YFLogxK
U2 - 10.1590/0037-8682-0444-2015
DO - 10.1590/0037-8682-0444-2015
M3 - Article
C2 - 27384823
AN - SCOPUS:84976636815
SN - 0037-8682
VL - 49
SP - 279
EP - 285
JO - Revista da Sociedade Brasileira de Medicina Tropical
JF - Revista da Sociedade Brasileira de Medicina Tropical
IS - 3
ER -