Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil
Rev. Soc. Bras. Med. Trop
; 49(3): 279-285, tab, graf
Artigo
em Inglês
| LILACS
| ID: lil-785796
Biblioteca responsável:
BR1.1
ABSTRACT
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.
Texto completo:
Disponível
Coleções:
Bases de dados internacionais
Contexto em Saúde:
Doenças Negligenciadas
Problema de saúde:
Dengue
Base de dados:
LILACS
Assunto principal:
Infecções por Flavivirus
/
Flavivirus
Tipo de estudo:
Estudo diagnóstico
/
Estudo de avaliação
Limite:
Humanos
País/Região como assunto:
América do Sul
/
Brasil
Idioma:
Inglês
Revista:
Rev. Soc. Bras. Med. Trop
Assunto da revista:
Medicina Tropical
Ano de publicação:
2016
Tipo de documento:
Artigo
País de afiliação:
Brasil
Instituição/País de afiliação:
Universidade de São Paulo/BR